WO2000017344A1 - RESPONSE REGULATOR $i(CaSSK1) - Google Patents

RESPONSE REGULATOR $i(CaSSK1) Download PDF

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WO2000017344A1
WO2000017344A1 PCT/US1999/022218 US9922218W WO0017344A1 WO 2000017344 A1 WO2000017344 A1 WO 2000017344A1 US 9922218 W US9922218 W US 9922218W WO 0017344 A1 WO0017344 A1 WO 0017344A1
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cassklp
casskl
cell
albicans
drug
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PCT/US1999/022218
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WO2000017344A8 (en
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Richard A. Calderone
Jose Calera
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Georgetown University Medical Center
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Priority to EP99949857A priority Critical patent/EP1115855A1/en
Priority to AU62636/99A priority patent/AU6263699A/en
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Publication of WO2000017344A8 publication Critical patent/WO2000017344A8/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to nucleic acid molecules encoding response regulator CaSskl protein including mutants, variants, fragments and derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules; methods of using response regulator; method for screening for inhibitors of response regulator; and kits comprising the compositions or polypeptides of the invention.
  • the sensor histidine kinases and their corresponding response regulators which were first described in prokaryotes in phosphotransfer pathways referred to as ' two-component systems ' , enable cells to detect environmental changes and elicit an appropriate response
  • the prototypical prokaryotic two- component regulator system is comprised of two proteins, a histidine protein kinase (or sensor protein) and a response regulator (or effector protein) , which is associated with an internal response.
  • the sensor kinase when activated by an environmental signal, autophosphorylates a histidine residue, which then serves as a phosphodonor to a conserved aspartate residue in the response regulator. This phophorylation modulates the activity of the effector protein so as to elicit an adaptive response to the stimulus .
  • the prokaryotic response regulators are characterized by a conserved domain of approximately 125 amino acids, usually attached via a linker sequence to a domain with an effector function.
  • the effector domain generally acts as a transcription factor.
  • a different type of effector domain is attached to a response regulator (Hoch and
  • a response regulator domain is located at the C-terminus of Slnlp, a transme brane hybrid protein that serves as an osmosensor (Ota and Varshavsky, 1993, Science 262, 566-569) which, after autophosphorylation of a histidine residue in its histidine kinase domain and transfer to an aspartate in its response regulator domain, phosphorylates a histidine residue of Ypdlp (Posas et al . , 1996, supra) . This protein, in turn, phosphorylates Ssklp, the other respnse regulator (Maeda et al .
  • the Mcs4 response regulator from Schizosaccharomyces pombe is an essential component of the Wakl-Wisl-Styl MAP kinase pathway, through which Mcs4 coordinates cell cycle progression with the cellular respnse to environmental stresses such as osmotic and oxidative stress, heat shock and nutritional limitations (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1997, Genes Dev 11, 1008-1022; Shiozaki et al . , 1997, Mol Biol Cell 8, 409-419) .
  • Candida albicans Three hybrid histidine kinase genes have been reported in Candida albicans, including CaSLNl (Nagahashi et al . , 1998, Microbiology 144 , 425-432), CaNIKl/COSl (Nagahashi et al . , 1998, supra; Srikantha et al . , 1998, Microbiology 144, 2715-2729; Alex et al . , 1998, Proc Natl Acad Sci USA 93, 7069-7073) and CaHKl (Calera et al . , 1998, Yeast 14, 665-674).
  • the CaSLNl and CaNIKl encoded proteins, CaSlnlp and CaNiklp, show structural and functional homology with the proteins Slnlp and Nikl from S. cerevisiae and N. crassa , respectively.
  • the CaHKl encoded protein, Cahklp while homologous at its C-terminus with the histidine kinase domains of many sensor proteins, at its N-terminus, only shows homology with a putative histidine kinase from S. pombe (GenBank Accession No. Z98978) with unknown function.
  • Ssklp, Skn7p and Mcs4 have a large N-terminal domain, and the conserved aspartate residues in their response regulators are located towards the C-terminus and are not followed by a domain with an effector function.
  • CaSSKl pathogenic yeast C. albicans
  • CaSsklp encoded protein
  • Candida albicans is the most frequently isolated opportunistic fungal pathogen in humans.
  • Candidiasis is a general- term for a variety of local and systemic processes caused by colonizationor infection of the host by species of the yeast Candida.
  • Candidiasis occurs worldwide; superficial infections of the skin, mouth and other mucus membranes are universal .
  • Invasive systemic disease has become a problem due to the use of high doses of antibiotics that destroy normal bacterial flora, immunosuppressive agents, and agents toxic to bone marrow, e.g. during cancer therapy.
  • Candidiasis is also seen among immunocompromised individuals such as AIDS patients, organ transplant patients, patients receiving parenteral nutrition, and cancer patients receiving parenteral and chemotherapy.
  • disease ranges from aggressive local infections such as peiodontitis, oral ulceration, or esophagitis in HIV-infected patients, to complex and potentially lethal infections of the bloodstream with subsequent dissemination to brain, eye, heart, liver, spleen, kidneys, or bone.
  • the infection typically begins at an epithelial site, evades local defenses, and invades the bloodstream in the face of immunosuppression.
  • disease caused by C. albicans ranges from mild, easily treated, superficial disease (e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water) to more severe, chronic or recurrent infections (e.g., candidal vaginitis) .
  • Candida species occur in two forms that are not temperature- or host-dependent .
  • the usual colonizing forms are yeasts that may assume a pseudomycelial configuration, especialy during tissue invasion. Pseudomyceliae result from sequential budding of yeasts into branching chains of elongated organisms.
  • C. albicans A number of factors have been associated with the virulence properties of C. albicans, such as adherence to host cells and the ability to undergo the transition from yeast to hyphal growth. This switch is induced in vi tro by many environmental conditions including temperature, pH and the presence of serum. Diploid strains of Saccharomyces cerevisiae also switch their pattern of growth from unicellular yeast to chains of elongated cells that remain attached to each other (pseudohyphae) under conditions of nitrogen starvation (Gimeno, et al . , 1992, Cell 68, 1077- 1090) .
  • This switch requires STE20, STEll , STE7 , KSS1 and STE12 , genes of a conserved MAP kinase pathway, but also depends on PHD1 , a gene which functions in a STE12- independent pathway (Banuett, 1998, Mol Biol Rev 62, 249- 274) .
  • the homolog genes CST20 Kelvan and Fink, 1996, Proc Natl Acad Sci USA 93, 13223-13228; Leberer et al .
  • the CPP1 gene which encodes a phosphatase similar to the MAP kinase phosphatase Msg5p of S. cerevisiae, modulates the activity of the CPHl- pathway likely by dephosphorylating Ceklp (Csank et al . , 1997, Mol Biol Cell 8, 2539-2551).
  • the C. albicans genes CaCLA4 (a CLA4 homolog) (Leberer et al . , 1997, Curr Biol 1 , 539-546), EFG1 (a PHD1 homolog) (Lo et al .
  • CaSSKl encodes a protein (CaSsklp) which is a structural homolog of Ssklp and Mcs4 from S. cerevisiae and S. pombe, respectively.
  • Ssklp is a response regulator of the "two-component" cascade that regulates the HOG pathway of S. cerevisiae (Maeda et al .
  • Mcs4 is an element of the Styl pathway of Schizosaccharomyces pombe (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1991 , Genes Dev 11, 1008-1022). Unlike Ssklp which only functions in osmosensing in S. cerevisiae, Mcs4 plays a role in regulating the adaptive responses to different stresses including osmotic and oxidative stress, and coordinates the responses to these environmental stimuli with the cell cycle (Cottarel, 1997, supra; Shieh et al . , 1997, supra) .
  • CaSSKl fails to complement the lack of either SSK1 or mcs4 + in S. cerevisiae and S. pombe, respectively (Calera and Calderone, 1999, supra) . This indicated that CaSSKl may have other functions in C. albicans rather than modulating the response to osmotic or oxidative stress. In this paper, we present data indicating that CaSSKl is involved in the morphogenesis and virulence of C. albicans .
  • CaSSKl a response regulator two- component gene from Candida albicans
  • the CaSSKl gene was cloned following a PCR based approach as described below.
  • CaSSKl has an open reading frame of 2022 base pairs (bp) .
  • bp base pairs
  • STRE stress response elements
  • CaSSKl is located on chromosome 1 and is expressed in either yeast or mycelial phases of C. albicans .
  • CaSSKl encodes a 674 amino acid protein (CaSsklp) with an estimated molecular mass of 73.5 kDa and a basic isoelectric point (pi 9.5). It has a tripeptide (NKA) located in its C-terminus, which resembles the peroxisomal signalling target type 1 sequence (PSTl) of most of the peroxisomal matrix proteins .
  • a homology search of CaSsklp with other proteins in databases showed that the C-terminus of CaSsklp exhibits the greatest similarity with the C-terminus of Ssklp and Mcs4 from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively.
  • the response regulator domain of CaSsklp contains the motifs that are characteristic of all response regulators, including the conserved aspartate and lysine residues as well as the putative aspartate, which is phophorylated by a phosphohistidine residue. Finally, in spite of the structural similarities among CaSsklp, Ssklp and Mcs4, CaSsklp does not seem to exhibit functional homology with these proteins .
  • the Accession No. for the described sequence is AF084608, as filed in the EMBL/GenBank/DDBJ database.
  • DNA fragment of about 3058 bp (SEQ ID NO:l) containing a 5' upstream promotor region of 486 bp, an open reading frame of 2022 bp encoding CaSsklp, and a 3' downstream non-coding region.
  • the DNA fragment is useful as a diagnostic agent, an agent for preparation of CaSsklp, and a therapeutic agent.
  • the inhibition can be at the DNA level by introducing mutations into the gene encoding CaSSKl , by inhibiting transcription of the gene, by inhibiting translation of the RNA encoding CaSsklp, or by inhibiting the function of CaSsklp.
  • Candida albicans said method comprising administering to an individual in need of such treatment an effective amount of an inhibitor of CaSsklp expression or function in a pharmaceutically acceptable diluent or excipient.
  • FIG. 1A and IB (A) Northern blot of total RNA. RNA samples obtained from cultures grown in medium 199 for 3 hours at 28'C (pH 3.5) (lane Y) and at 37 °C (pH 7.5) (lane M) , to induce yeast and hyphal growth, respectively. (B) CHEF gel analysis. It was performed with duplicate samples . The chromosomal bands were visualized with ethidium bromide (left panel) and a Southern blot of the gel (right panel) was hybridized with a 1.85 kb EcoRI-Bglll fragment of the CaSSKl gene as a probe. C. albicans chromosomal designations (on the left side) are those proposed by Wickes et al . (1991, Infect . Immun . 59, 1762-1771).
  • FIG. 2A and 2B (A) Complementation analysis of a S. pombe mcs4-13 cdc25-22 double mutant with CaSSKl (pBREP42-CaSSKl) . This S. pombe strain was also transformed with the plasmids pREP42-mcs4 as a positive control and the plasmids pREP42 and pBREP42 as negative controls . Transformants were grown in EMM minimal medium at either 25°C or 30°C for four days. (B) Complementation analysis of a S. cerevisiae SSKl SLN1 double mutant with CaSSKl (pBRS415-CaSSKl) .
  • This strain was also transformed with the plasmids pRS415- SSK1 as a positive control and the plasmids pBRS415 and pBRS415-p/t as negative controls. Transformants (1.5 X 10 4 cells per spot) were grown in presence of either galactose or glucose for two days at 30°C.
  • FIG. 3A, 3B, and 3C (a) Schematic representation of the construction of the cassette used to disrupt CaSSKl and, (b) the cassette CaSSKl -URA3 -hi sG used to reintroduce one wild type CaSSKl allele.
  • (c) is shown the corresponding southern blot analyses of strains CSSKll-1, CSSK12-1, CSSK21-1 and CSSK22-1 obtained during the disruption process and a revertant strain (CSSK23-1) . Genomic DNA from these strains was Bglll digested, and hybridized with a 1.4 kb Nsil-EcoRI fragment located at the 5 ' -end of the CaSSKl gene as a probe. The exact size and genotype of the expected hybridizing DNA fragments are indicated on the right .
  • Figure 4. Broth cultures of Ura3 + Acasskl mutants
  • strains CSSKll-1, CSSK21-1 and CSSK23-1 Stationary-phase cells from all strains were inoculated (10 7 cells/ml) in M199 (pH 7.5), grown at 37°C for 3 hours and photographed.
  • the CSSK21 null strains flocculated extensively forming clumps of cells that sedimented rapidly to the bottom of the tubes after the cell suspension was shaken. The formation of clumps was not observed in the heterozygote and reconstituted strains.
  • FIG. 7 Survival of mice following infection with C. albicans Acasskl mutants. Only the survival curves of mice infected with CAF2 , CSSKll-1, CSSK21-1 and CSSK23-1 are shown. Similar results were observed with mice infected with strains CSSK21-2 and CSSK23-2.
  • FIG. 8 Hypothetical scheme of the location of CaSsklp in a putative two-component pathway of C. albicans based on the phenotypic characterization of the Acahkl , Acasskl and Acanikl/cosl mutants.
  • the dashed arrows indicate the putative transduction of the signal by CaNiklp/Coslp and the solid arrows the transduction of the signal by Cahklp.
  • the present invention relates to a DNA or cDNA segment which encodes CaSSKlp from Candida albicans .
  • a PCR reaction using an appropriate pair of degenerate primers was performed and a 447 bp PCR product was identified as a partial DNA sequence of CaSSKl .
  • This PCR fragment was then used to isolate the entire CaSSKl gene.
  • the CaSSKl DNA fragment has an open reading frame of 2022 bp in addition to the 5' promoter region and 3' nontranslated region.
  • the complete 3058 bp CaSSKl DNA fragment is specified in SEQ ID NO:l.
  • one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from (a) a nucleotide sequence comprising a sequence encoding a full length CaSsklp polypeptide having the sequence specified in SEQ ID NO:l, (b) a nucleotide sequence which encodes the complete amino acid sequence in SEQ ID NO: 2, or the complete amino acid sequence encoded by the cloned DNA in GenBank accession no. AF084608.
  • isolated nucleic acid molecules of the invention include DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode CaSsklp or fragments thereof.
  • the genetic code and species-specific codon preferences are well known in the art.
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mR ⁇ A, or in the form of D ⁇ A, including, for instance, cD ⁇ A and genomic D ⁇ A obtained by cloning or produced synthetically.
  • the D ⁇ A may be double-stranded or single-stranded.
  • Single-stranded D ⁇ A or R ⁇ A may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the antisense strand.
  • isolated nucleic acid molecule is intended a nucleic acid molecule, D ⁇ A or R ⁇ A, which has been removed from its native environment.
  • recombinant D ⁇ A molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated D ⁇ A molecules include recombinant D ⁇ A molecules maintained in heterologous host cells or purified (partially or substantially) D ⁇ A molecules in solution.
  • Isolated R ⁇ A molecules include in vivo or in vitro R ⁇ A transcripts of the D ⁇ A molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • the present invention is further directed to nucleic acid molecules encoding portions or fragments of the nucleotide sequences described herein.
  • Fragments include portions of the nucleotide sequence of SEQ ID NO:l or at least 10 contiguous nucleotides in length selected from any two integers , one of which representing a 5' nucleotide position and a second of which representing a 3' nucleotide position, where the first nucleotide for each nucleotide sequence is position 1. That is, every combination of a 5 ' and 3' nucleotide position that a fragment at least 10 contiguous nucleotide bases in length or any interger between 10 and the length of an entire nucleotide sequence of CaSSKl minus 1.
  • the invention includes polynucleotides comprising fragments specified by size, in nucleotides, rather than by nucleotide positions.
  • the invention includes any fragment size, in contiguous nucleotides, selected from intergers between 1- and the entire length of an entire nucleotide sequence minus 1.
  • Preferred sizes include 20-50 nucleotides, 50-300 nucleotides useful as primers and probes.
  • Regions from which typical sequences may be derived include but are not limited to, for example, regions encoding specific epitopes or domains within said sequence, such as the region comprising nucleotides 1599-2115 of SEQ ID NO: 1.
  • the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridize under stringent hybridization conditions to a polynucleotide sequence of the present invention described above, or a specified fragment thereof.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6) , 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0. IX SSC at about 65°C.
  • sequences encoding the polypeptides of the present invention or portions thereof may be fused to other sequences which provide additional functions known in the art such as a marker sequence, or a sequence encoding a peptide which facilitates purification of the fused polypeptide, peptides having antigenic determinants known to provide helper T-cell stimulation, peptides encoding sites for post- tranlational modifications, or amino acid sequences which target the fusion protein to a desired location, e.g. a heterologous leader sequence.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the CaSsklp polypeptides.
  • Variant may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus of a chromosome of an organism.
  • Non-naturally occuring variants may be produced by known mutagenesis techniques.
  • Such variants include those produced by nucleotide substitution, deletion, or addition of one or more nucleotides in the coding or noncoding regions or both. Alterations in the coding regions may produce conservative or nonconservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions which do not alter the properties and activities of CaSsklp polypeptides disclosed herein or portions thereof . Also preferred in this regard are conservative substitutions .
  • Nucleic acid molecules with at least 90-99% identity to a nucleic acid shown in SEQ ID NO:l is another aspect of the present invention. These nucleic acids are included irrespective of whether they encode a polypeptide having CaSsklp activity.
  • a polypeptide having CaSsklp activity is intended polypeptides exhibiting activity similar, but not identical, to an activity of the CaSsklp of the invention, as measured in the assays described below.
  • the biological acitivity or function of the polypeptides of the present invention are expected to be similar or identical to polypeptides from other organisms that share a high degree of structural identity/similarity.
  • the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above.
  • the vector can take the form of a plasmid, phage, cosmid, YAC, eukaryotic expression vector such as a DNA vector, Pichia pastoris, or a virus vector such as for example, baculovirus vectors, retroviral vectors or adenoviral vectors, and others known in the art.
  • the cloned gene may optionally be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences , or sequences which may be inducible and/or cell type-specific.
  • Suitable promoters will be known to a person with ordinary skill in the art.
  • the expression construct will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the vectors preferred for use include pGEX-YT3 , YPB11-ADH to name a few.
  • the construct into the host cell can be effected by calcium phosphate transfection, electroporation, infection, and other methods known in the art and described in standard laboratory manuals such as Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. or Sherman et al . , 1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York. All documents cited herein supra and infra are hereby incorporated in their entirety by referece thereto.
  • the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs.
  • the host cell can be prokaryotic (for example, bacterial) , lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to rat and human) . Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites.
  • Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers . These markers may be used to obtain successful transformants by selection. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods .
  • the DNA sequence can be present in the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of CaSsklp, such as glutathione S- transferase, or a series of histidine residues also known as a histidine tag.
  • the recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carlsbergensis , and Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts. Control sequences for yeast vectors are known in the art.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , such as HEK293 cells , and NIH 3T3 cells, to name a few.
  • Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV) , adenovirus (ADV) , bovine papilloma virus (BPV) , and cytomegalovirus (CMV) .
  • suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate.
  • Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequences are known in the art.
  • the transformed or transfected host cells can be used as a source of DNA sequences described above. When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein described below.
  • the present invention relates to a CaSsklp protein having an amino acid sequence corresponding to SEQ ID NO: 2 (GenBank accession no. AF084608) and encompassing 674 amino acids or any allelic variation thereof or biologically active derivative thereof .
  • a polypeptide or amino acid sequence derived from the amino acid sequences mentioned above refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 amino acids, and more preferably at least 8- 10 amino acids, and even more preferably at least 11- 15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence .
  • a "biologically active derivative thereof” is a CaSsklp that is modified by amino acid deletion, addition, substitution, or truncation, or that has been chemically derivatized, but that nonetheless functions in the same manner as the protein of SEQ ID NO: 2.
  • substitutions of aliphatic amino acids such as alanine, valine, and isoleucine with other aliphatic amino acids can often be made without altering the structure or function of a protein.
  • substitution of aspartic acid for glutamic acid, in regions other than the active site of an enzyme are likely to have no appreciable affect on protein structure or function.
  • fragment is meant to refer to any polypeptide subset. Fragments can be prepared by subjecting C.
  • albicans proteins to the action of any one of a number of commonly available proteases, such as trypsin, chymotrypsin or pepsin, or to chemical cleavage agents, such as cyanogen bromide.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire CaSSKl or to a fragment thereof .
  • a protein or peptide is said to be 'substantially similar' if both molecules have substantially similar amino acid sequences, preferably greater than about 80% sequence identity, or if the three-dimensional backbone structures of the molecules are superimposable, regardless of the level of identity between the amino acid sequences.
  • two molecules possess similar activity they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequences of amino acid residues are not identical.
  • 'analog' is meant to refer to a protein that differs structurally from the wild type CaSsklp, but possesses similar activity.
  • a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system.
  • the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants for example.
  • the methods of the present invention are suitable for production of any polypeptide of any length, via insertion of the above- described nucleic acid molecules or vectors into a host cell and expression of the nucleotide sequence encoding the polypeptide of interest by the host cell.
  • Introduction of the nucleic acid molecules or vectors into a host cell to produce a transformed host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods .
  • Such methods are described in many standard laboratory manuals, such as Davis et al . , Basic Methods In Molecular Biology (1986) .
  • Transformations into yeast are typically carried out according to the method of Van Solingen et al . , 1977, J. Bact . , 130, 946 and Hsiao et al . 1979, Proc Natl Acad Sci USA 76, 3829-3833.
  • the cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support host cell growth.
  • Recombinant polypeptide-producing cultivation conditions will vary according to the type of vector used to transform the host cells.
  • certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals or inducing agents to the cell growth medium, to initiate the gene expression resulting in the production of the recombinant polypeptide.
  • the term "recombinant polypeptide-producing conditions," as used herein, is not meant to be limited to any one set of cultivation conditions .
  • Appropriate culture media and conditions for the above-described host cells and vectors are well-known in the art.
  • the polypeptide of interest may be isolated by several techniques. To liberate the polypeptide of interest from the host cells, the cells are lysed or ruptured.
  • This lysis may be accomplished by contacting the cells with a hypotonic solution, by treatment with a cell wall-disrupting enzyme such as lysozyme, by sonication, by treatment with high pressure, or by a combination of the above methods .
  • a cell wall-disrupting enzyme such as lysozyme
  • sonication by treatment with high pressure, or by a combination of the above methods .
  • Other methods of bacterial cell disruption and lysis that are known to one of ordinary skill may also be used.
  • the polypeptide may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The polypeptide may then be purified by well known isolation techniques .
  • Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography.
  • LC liquid chromatography
  • HPLC high performance LC
  • FPLC fast performance LC
  • the recombinant or fusion protein can be used as a diagnostic tool and in a method for producing antibodies against CaSsklp, detectably labeled and unlabeled, or as a bait protein in the yeast 2-hybrid assay to isolate proteins which interact with CaSsklp.
  • the transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit CaSsklp function, such as host proteins or chemically derived agents or natural or synthetic drugs and other proteins which may interact with the cell to down- regulate or alter the expression of CaSsklp, or its cofactors.
  • drugs and agents which inhibit CaSsklp function such as host proteins or chemically derived agents or natural or synthetic drugs and other proteins which may interact with the cell to down- regulate or alter the expression of CaSsklp, or its cofactors.
  • the present invention relates to monoclonal or polyclonal antibodies specific for the above-described recombinant proteins (or polypeptides) .
  • an antibody can be raised against a peptide described above, or against a portion thereof of at least 10 amino acids, perferrably, 11-15 amino acids.
  • Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or polypeptide) of the present invention, or a unique portion thereof .
  • Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986) .
  • the level of expression of CaSsklp can be detected at several levels.
  • assays for the detection and quantitation of CaSSKl RNA can be designed, and include northern hybridization assays, in si tu hybridization assays, and PCR assays, among others. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) , or Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. for general description of methods for nucleic acid hybridization.
  • Polynucleotide probes for the detection of CaSSKl RNA can be designed from the sequence available at accession number AF084608.
  • RNA isolated from samples can be coated onto a surface such as a nitrocellulose membrane and prepared for northern hybridization.
  • tissue sample can be prepared for hybridization by standard methods known in the art and hybridized with polynucleotide sequences which specifically recognize CaSSKl RNA .
  • the presence of a hybrid formed between the sample RNA and the polynucleotide can be detected by any method known in the art such as radiochemistry, or immunochemistry, to name a few.
  • probes are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. In other cases, it may be desirable to use two sets of probes simultaneously, each to a different region of the gene. While the exact length of any probe employed is not critical, typical probe sequences are no greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.
  • the D ⁇ A sequence of CaSSKl can be used to design primers for use in the detection of CaSSKl using the polymerase chain reaction (PCR) or reverse transciption PCR (RT-PCR) .
  • the primers can specifically bind to the CaSSKl cD ⁇ A produced by reverse transcription of CaSSKl R ⁇ A, for the purpose of detecting the presence, absence, or quantifying the amount of CaSSKl R ⁇ A by comparison to a standard.
  • the primers can be any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18-25 nucleotides homologous or complementary to a region of the CaSSKl sequence. Reagents and controls necessary for PCR or RT-PCR reactions are well known in the art.
  • the amplified products can then be analyzed for the presence or absence of CaSSKl sequences, for example by gel fractionation, by radiochemistry, and immunochemical techniques. This method is advantageous since it requires a small number of cells.
  • a determination whether the cell is overexpressing or underexpressing CaSSKl can be made by comparison to the results obtained from a normal cell using the same method.
  • Decreased CaSSKl may be an indication of reduced virulence of the infecting yeast, or an indication that tissue-specific or site- specific expression of the gene is reduced.
  • the present invention relates to a diagnostic kit for the detection of
  • kits comprising a package unit having one or more containers of CaSSKl oligonucleotide primers for detection of CaSSKl by PCR or RT-PCR or CaSSKl polynucleotides for the detection of CaSSKl RNA in cells by in si tu hybridization or northern analysis, and in some kits including containers of various reagents used for the method desired.
  • the kit may also contain one or more of the following items: polymerization enzymes, buffers, instructions, controls, detection labels.
  • Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods .
  • the present invention provides a method for identifying and quantifying the level of CaSSKl present in a particular biological sample. Any of a variety of methods which are capable of identifying (or quantifying) the level of CaSSKl in a sample can be used for this purpose.
  • Diagnostic assays to detect CaSSKl may comprise a biopsy or in si tu assay of cells from an organ or tissue sections, as well as an aspirate of cells from a tumour or normal tissue.
  • assays may be conducted upon cellular extracts from organs, tissues, cells, urine, or serum or blood or any other body fluid or extract.
  • the assay will comprise, contacting the sample to be assayed with a CaSsklp ligand or substrate, natural or synthetic, or an antibody, polyclonal or monoclonal, which recognizes CaSsklp, or antiserum capable of detecting CaSsklp, and detecting the complex formed between CaSsklp present in the sample and the CaSsklp ligand, substrate, or antibody added.
  • CaSsklp ligands or substrates include for example, a downstream component in the CaSSKl pathway, a substrate for phosphorylation by CaSsklp, or an CaSsklp interacting protein, in addition to natural and synthetic classes of ligands and their derivatives which can be derived from natural sources such as animal or plant extracts.
  • CaSsklp ligands or anti-CaSsklp antibodies, or fragments of ligand and antibodies capable of detecting CaSsklp may be labeled using any of a variety of labels and methods of labeling for use in diagnosis and prognosis of disease associated with candidiasis.
  • types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non- radioactive isotopic labels, and chemiluminescent labels .
  • suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha- glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc .
  • radioisotopic labels examples include 3 H, ni ln, 125 I, 32 P, 35 S, 14 C, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 21 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, X1 C, 19 F, 123 I, etc.
  • suitable non-radioactive isotopic labels include 157 Gd, 5 Mn, 162 Dy, 52 Tr, 46 Fe, etc.
  • suitable fluorescent labels include a 152 Eu label, a fluorescein label, an isothiocyanate label , a rhodamine label , a phycoerythrin label , a phycodyanin label, an allophycocyanin label, a fluorescamine label, etc.
  • chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, etc.
  • suitable labels which may be employed in accordance with the present invention.
  • the binding of these labels to ligands and to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H. , et al . , 1976 ( Clin . Chim. Acta 70, 1-31), and Schurs, A. H. W.
  • the detection of the antibodies (or fragments of antibodies) of the present invention can be improved through the use of carriers.
  • Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides , agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to CaSsklp.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • the ligands or antibodies, or fragments of antibodies or ligands of CaSsklp discussed above may be used to quantitatively or qualitatively detect the presence of CaSsklp. Such detection may be accomplished using any of a variety of immunoassays known to persons of ordinary skill in the art such as radioimmunoassays, immunometic assays, etc. Using standard methodology well known in the art, a diagnostic assay can be constucted by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocelluolose membrane) , antibodies specific for CaSsklp or a portion of CaSsklp, and contacting it with a sample from a person suspected of having a
  • CaSSKl related disease The presence of a resulting complex formed between CaSsklp in the sample and antibodies specific therefor can be detected by any of the known detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry.
  • detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry.
  • a good description of a radioimmune assay may be found in Laboratory Techniques and Biochemistry in Molecular
  • Sandwich assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.
  • the diagnostic methods of this invention are predictive of patients suffering from candidiasis, periodontitis, oral ulceration, or esophagitis in HIV- infected patients, candidal vaginitis, and bloodstream candidiasis .
  • the protein can be used to identify inhibitors of CaSsklp activity. Using a phophorylation asssay such as those known in the art, natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of CaSsklp autophosphorylation or phosphorylation activity. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a decrease in the activity of CaSsklp is detected.
  • Inhibitors may include agents or drugs which either bind or sequester CaSsklp substrate (s) or cofactor (s), or inhibit the CaSsklp itself, directly, for example by irreversible binding of the agent or drug to CaSsklp, or indirectly, for example by introducing an agent which binds the CaSsklp substrate. Agents or drugs related to this invention may result in partial or complete inhibition of CaSsklp activity.
  • Inhibitors of CaSsklp may be used in the treatment or amelioration of candidiasis and diseases associated with candidiasis
  • Agents which decrease CaSSKl RNA include, but are not limited to, one or more ribozymes capable of digesting CaSSKl RNA, or antisense oligonucleotides capable of hybridizing to CaSSKl RNA such that the translation of CaSSKl RNA is inhibited or reduced resulting in a decrease in the level of CaSSKlp .
  • These antisense oligonucleotides can be administered as DNA, as DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Kanoda, Y. et al . , 1989, Science 243, 375) or as part of a vector which can be expressed in the target cell such that the antisense DNA or RNA is made.
  • the DNA can be injected along with a carrier.
  • a carrier can be a protein such as a cytokine, for example interleukin 2, or polylysine-glycoprotein carrier.
  • cytokine for example interleukin 2, or polylysine-glycoprotein carrier.
  • carrier proteins and vectors and methods of using same are known in the art.
  • the DNA could be coated onto tiny gold beads and said beads introduced into the skin with, for example, a gene gun (Ulmer, J. B. et al . , 1993, Science 259, 1745).
  • antibodies, or compounds capable of reducing or inhibiting CaSsklp, that is reducing or inhibiting either the expression, production or activity of CaSsklp, such as antagonists can be provided as an isolated and substantially purified protein, or as part of an expression vector capable of being expressed in the target cell such that the
  • CaSsklp-reducing or inhibiting agent is produced.
  • co-factors such as various ions, i.e. Ca2 + or factors which affect the stability of the enzyme can be administered to modulate the expression and function of CaSsklp.
  • These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, oral, rectal, or parenteral (e.g. intravenous , subcutaneous , or intramuscular) route.
  • CaSsklp-inhibiting compounds may be incorporated into biodegradable polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the CaSsklp-inhibiting compound is slowly released systemically.
  • the biodegradable polymers and their use are described, for example, in detail in Brem et al . (1991) J. Neurosurg. 74, 441-446. These compounds are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of CaSsklp.
  • agents which are capable of negatively affecting the expression, production, stability or function of CaSsklp are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of CaSsklp.
  • An amount is said to be sufficient to "effect" the inhibition or induction of CaSsklp if the dosage, route of administration, etc. of the agent are sufficient to influence such a response .
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient .
  • Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • a pharmaceutically acceptable carrier vehicle e.g., water, alcohol, and water.
  • suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin are described, for example, in Remington's Pharmaceutical Sciences [16th ed. , Osol, A. ed., Mack Easton PA. (1980)].
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.
  • Control release preparations may be achieved through the use of polymers to complex or absorb the compounds .
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) - microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres , microemulsions, nanoparticles , and nanocapsules or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres , microemulsions, nanoparticles , and nanocapsules or in macroemulsions .
  • kits for use in the diagnostic or therapeutic methods described above.
  • Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention.
  • kits of the invention may comprise one or more of the following components, one or more compounds or compositions of the invention, and one or more excipient, diluent, or adjuvant.
  • the present invention describes a C. albicans strain which contains a deletion of CaSSKl response regulator gene.
  • the strain ⁇ casskl is avirulent and does not form hyphae on solid inducing media.
  • ⁇ casskl similar to wild type, is able to form hyphae in liquid media, however, unlike wild type, the hyphae flocculate.
  • the ⁇ casskl strain may function as a gene or gene product delivery sytem.
  • an antigen of interest could be delivered to an organ which is naturally invaded by ⁇ casskl in a patient, animal or plant where the antigen can provide benefit .
  • the antigen can be introduced into ⁇ casskl in a second plasmid.
  • a second plasmid could be used to provide a source of vaccine antigen for pathogens found in organs naturally invaded by C. albicans such as a systemic invasion, or kidney, lung, central nervous system, eye, to name a few.
  • expression of spermicides by ⁇ casskl transformed with a desired gene could provide a cheap and infrequent method of contraception.
  • ⁇ casskl represents a safe delivery vehicle and is advantageous because it can carry one or more compounds and can be genetically engineered to carry- one or more nucleic acid molecules capable of effecting gene therapy and/or of encoding one or more proteins and/or RNA molecules.
  • the compound of interest can be carried by ⁇ casskl within the yeast cell, on the yeast membrane surface, spanning the yeast membrane, withing the yeast periplasm, and combinations thereof . At least some of the compound of interest remains associated with the yeast at least until the yeast reaches its target, or site of action (e.g. the bloodstream, interstitial tissue, or a cell) , at which point it is also possible that a compound carried by the yeast may be released.
  • a compound capable of protecting an animal or plant from disease is a compound that when administered to an animal or plant can prevent a disease from occuring and/or cure or alleviate disease symptoms or cause.
  • diseases from which to protect an animal or plant include, but are not limited to, infections, genetic defects and other metabolic disorders .
  • Such classes of diseases can lead to abnormal cell growth (e.g., benign or malignant neoplasia, hyperplastic syndromes) , degenerative processes, and/or immunological defects as well as to a number of other disorders .
  • ⁇ casskl vehicles of the present invention preferably include compounds capable of stimulating an immune response, compounds capable of suppressing an immune respone, toxic compounds, compounds capable of inhibiting transcription of a gene, compounds capable of inhibiting translation of a gene, compounds capable of inhibiting the ability of an infectious agent to produce progeny, compounds capable of replacing a defective gene, compounds capable of replacing a defective protein (including nucleic acid molecules capable of encoding such proteins and mimetopes of such proteins) and/or biological response modifiers (e.g., cytokines, such as lymphokines and monokines, as well as other growth modulating factors), and mixtures thereof.
  • cytokines such as lymphokines and monokines, as well as other growth modulating factors
  • Such compounds include, but are not limited to, antibiotics, antibodies, antifungal compounds, antigens, antiparasite compounds, antisense compounds, antiviral compounds, chemotherapeutic agents , cytokines, growth modulating factors (including both growth stimulants and suppressants), herbicides, hormones, immunosuppressants, nucleic acid-based drugs (e.g., DNA- or RNA-based drugs), nucleic acid molecules comprising coding regions, nucleic acid molecules comprising regulatory sequences, nucleoside analogs, other oligonucleotides, peptide analogs, peptides, pesticides, prodrugs (e.g., compounds that are activated at the site of action), other proteins, ribozymes, steroids, toxins, and/or vitamins.
  • nucleic acid-based drugs e.g., DNA- or RNA-based drugs
  • nucleic acid molecules comprising coding regions
  • nucleic acid molecules comprising regulatory sequences, nucleoside analogs, other oligonu
  • Cell types naturally targeted by ⁇ casskl include, but are not limited to, cells of granulocytic- monocytic lineage, B lymphocytes and T lymphocytes as well as cells in the epithelial regions of an animal .
  • the ⁇ casskl can be engineered such that it targets specific cell types by positioning a cell- binding element (e.g., antibodies, receptors, and receptor ligands) in the membrane of ⁇ casskl.
  • a cell- binding element e.g., antibodies, receptors, and receptor ligands
  • Such an element is preferably absent from, or found in only small amounts, on cell types that are not targeted for delivery.
  • the present invention includes the delivery of a composition comprising ⁇ casskl of the present invention to an animal or to cell in culture.
  • Such compositions can be delivered to an animal either in vivo or ex vivo, or can be delivered to cells in vitro .
  • Such administration can be systemic, mucosal, and/or proximal to the location of the targeted cell type.
  • routes to administer yeast in vivo include aural, bronchial, genital, inhalatory, nasal, ocular, oral, parenteral, rectal, topical, transdermal , and urethral routes .
  • Ex vivo delivery refers to a method that includes the steps of contacting a population of cells removed from an animal with a composition comprising ⁇ casskl of the present invention under conditions such that the yeast is adsorbed by targeted cell types and returning the contacted cells to the animal .
  • a delivery method is particularly useful in the treatment of cells involved in hematopoiesis and the immune response as well as in the treatment of tumors .
  • In vitro delivery refers to the delivery of ⁇ casskl of the present invention to a population of cells (which can also include tissues or organs) in culture .
  • a preferred single dose of a yeast vehicle of the present invention is from about 1 x 10 5 to about 5 x 10 7 yeast cell equivalents per kilogram body weight of the organism being administered the composition.
  • the ⁇ casskl strain of the invention can be used directly in vaccine formulations, or lyophilized, as desired, using lyophilization protocols well known to the artisan. Lyophilized compositions will typically be maintained at about 4°C. When ready for use the lyophilized composition is reconstituted in a stabilizing solution, e.g., saline or comprising Mg ++ and HEPES, with or without adjuvant, as further described below.
  • ⁇ casskl vaccine of the invention contains as an active ingredient an immunogenically effective amount of a nonvirulent C. albicans as described herein.
  • the ⁇ casskl may be introduced into a host, particularly humans, with a physiologically acceptable carrier and/or adjuvant.
  • Useful carriers are well known in the art, and include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration, as mentioned above.
  • the compositions may contain pharmaceutically accepatable auxilliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.
  • Administration of the ⁇ casskl disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection) , by in ovo injection in birds, orally and by topical application of the yeast (typically carried in the pharmaceutical formulation) to an airway surface.
  • Topical application of the yeast to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally) .
  • Topical application of the yeast strain to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the yeast as an aerosol suspension, and then causing the subject to inhale the respirable particles.
  • respirable particles of a pharmaceutical formulation including both solid particles and liquid particles
  • Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.
  • the host becomes at least partially or completely immune to C. albicans infection, or resistant to developing moderate or severe C. albicans infection.
  • the vaccine composition containing the ⁇ casskl of the invention are administered to a person susceptible to or otherwise at risk of C. albicans infection to enhance the individual ' s own immune response capabilities .
  • Such an amount is defined to be a "immunogenically effective dose".
  • the precise amount again depends on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1 x 10 5 to about 5 x 10 7 yeast cell equivalents per kilogram body weight of the organism being administered the composition.
  • the vaccine formulations should provide a quantity of ⁇ casskl of the invention sufficient to effetively protect the patient against serious or life- threatening C. albicans infection.
  • ⁇ casskl vaccines of the invention may be desirable to combine with vaccines which induce protective responses to other agents .
  • Single or multiple administration of the vaccine compositions of the invention can be carried out. Multiple administration may be required to elicit sufficient levels of immunity. Levels of induced immunity can be monitored by measuring amount of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection. It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.
  • Candida albicans (strain SC5314) was routinely grown in YPD complex medium (1% yeast extract, 2% peptone, 2% glucose) , at 28 °C with shaking at 250 rpm for 14 hours. Cells were harvested from liquid medium by centrifugation at 4,000 x g for 10 minutes at 4°C. For RNA isolation, the same strain was grown in medium 199 containing Earle's salts and glutamine but lacking sodium bicarbonate (Gibco BRL) and buffered with 155 mM Tris-HCl either at pH 7.5 or 3.5.
  • YPD complex medium 1% yeast extract, 2% peptone, 2% glucose
  • Cultures were inoculated to a density of 10 7 cells ml -1 into prewarmed medium and incubated at 28 °C or 37 °C with vigorous agitation for 3 hours. In all cases, stationary-phase cells grown at 28 °C in YPD were used as an inoculum.
  • Schizosaccharomyces pombe strain JM1303 (mcs4-13 cdc25-22 ura4-Dl8) (Shieh et al . , 1997, Genes Dev. 11, 1008-1022) was routinely grown in YE (3% dextrose, 0.5% yeast extract, 2% agar) at 25°C or in EMM minimal medium
  • Saccharomyces cerevisiae strain TM187 (MATa. Ieu2 ura3 trpl slnl : :hisG sskl : : TRPl + pSSP25) (Maeda et al . , 1994) was routinely grown in CAD (0.67% YNB, 0.5% casamino acids, 2% dextrose) at 30°C.
  • Escherichia coli strain DH5 ⁇ which was used for transformation and plasmid amplification, was grown at 37 °C in LB broth supplemented with 0.1 mg of ampicillin per ml of medium when necessary.
  • E. coli strain LE392 was used for propagation of bacteriophage ⁇ EMBL3.
  • the Candida albicans strains used in this work are listed in Table 1. All strains were routinely grown either in YPD complex medium (1% yeast extract, 2% peptone and 2% dextrose) or SD minimal medium (0.67% yeast nitrogen base, 2% dextrose) at 28°C.
  • strains were also grown on solid Spider medium (1% nutrient broth, 1% mannitol, 0.2% K 2 HP0 4 ) , solid synthetic low-ammonium- dextrose (SLAD) medium (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 2% dextrose and 50 ⁇ M ammonium sulfate as sole nitrogen source) , serum [10% fetal bovine serum (Gibco BRL)] and Medium 199 (M199) containing Earle's salts and glutamine but lacking sodium bicarbonate (Gibco BRL) and buffered with 155 mM Tris-HCl at pH 7.5 or pH 4.0. All liquid media were sterilized by filtration.
  • SLAD solid synthetic low-ammonium- dextrose
  • M199 Medium 199
  • Solid media were prepared by adding 1.5% agar as a final concentration (2% for SLAD plates) at 50°C after autoclaving. Prewarmed liquid media were inoculated to a density of 10 7 cells/ml and incubated either at 28°C or 37°C with vigorous agitation. On solid media, the appropriate dilutions of cells were plated to obtain approximately 60-80 colonies per plate. In all cases, stationary-phase cells grown at 28°C in YPD were used as an inoculum. Table 1
  • Plasmid DNA was extracted from E. coli DH5 ⁇ cells according to the manufacturer's instructions (Plasmid
  • albicans strains was obtained according to the method described by Sherman et al . (1986, supra) .
  • For southern blot analyses 4 ⁇ g of DNA per lane was loaded in a 0.8% agarose gel and transferred by capillarity to positively-charged nylon membranes (Hybond-N+ , Amersham) by standard protocols (Sambrook et al . , 1989, supra) .
  • Probes were Digoxigenin- labelled by random priming (DIG DNA Labeling Kit, Boehringer Mannheim) , and hybridized and detected according to the chemiluminescent method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim) . Construction of the CaSSKl probe and screening of a ⁇ EMBL3 Candida albicans genomic library
  • a fragment of the CaSSKl gene was isolated by PCR amplification with the degenerate primers, 5'-AA(T/C) GT(A/G/T) TT(G/A) AT(A/T) GT(A/G/T) GAA GA(T/C) AA-3' and 5 ' -CAT (A/G)CA (A/T)CC CCA TTC (T/A/G)GT (T/A)AT TTT-3 ' .
  • These primers were designed according to a pair of amino acid sequences conserved between Ssklp from S. cerevisiae and Mcs4 from S. pombe. These sequences, VLIVEDN (SEQ ID NO: 3) and KITE GCM
  • SEQ ID NO: 4 are located at positions 505-512 and 642- 649 in Ssklp and at positions 363-370 and 500-507 in Mcs4, respectively, being separated in both cases by 145 amino acids .
  • PCR amplification was performed with genomic DNA from C. albicans strain SC5314 as template. Thirty-two cycles of PCR were performed with the following steps: 45 seconds at 94'C; 1 minute at 50 °C and 2.5 minutes at 72 °C. The reaction yielded specifically a fragment with the predicted size of approximately 435 bp.
  • the PCR product was subcloned into pCR2.1 (Invitrogen) to generate pSSKl, sequenced and the 447 bp DNA fragment identified as a partial C.
  • albicans DNA homolog of SSKl and mcs4 ⁇ The PCR product was excised from pSSKl by BcoRI digestion, digoxigenin- labelled by non-radioactive random priming (DIG DNA Labelling Kit, Boehringer Mannheim) and, in turn, used as probe to screen a ⁇ EMBL3 C. albicans genomic library (Monod et al . , 1994, Mol . Microbiol . 13, 557-368), according to the protocol described by Sambrook et al . (1989, supra) . The detection of the positive clones was done according to the colorimetric method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim).
  • the entire ORF of CaSSKl was amplified from pER-CaSSKl by PCR using the 5' oligonucleotide 5'- AAATAGGATCCCATGAATTTTCTCTATAAC-3 ' (SEQ ID NO: 5) and the 3' oligonucleotide 5 ' -CGTTGGATCCTTTCAAGCTTTGTTTAATCTTCG -3' (SEQ ID NO: 6), both of them containing the BamHl site (bolded) and confirmed by DNA sequencing.
  • the resulting 2.04 kb product was digested with BamHI and cloned into the BamHI site of pBREP42 to generate pBREP42 -CaSSKl .
  • the pBREP42 plasmid is a low copy number version of the pREP42 plasmid (Maundrell, 1993, Gene 123, 127-130), which was constructed by introducing the 5.50 kb PvuII-PvuII cassette [ura4 + -nmt promoter -MCS-nmt poly.A signal -arsl] from pREP42 into the pBR322 digested with EcoRV/PshAI .
  • the pREP42-mcs4 plasmid (Shieh et al . ,
  • TM187 strain To ensure a correct level of expression of CaSSKl in S. cerevisiae, we first constructed a low copy version of the pRS415 plasmid (Sikorski and Hieter, 1989, Genetics 122, 19-27) (which we named as pBRS415) and then, both the promotor and terminator of the SSKl gene from S. cerevisiae were introduced into pBRS415 to generate pBRS415-p/t.
  • the pBRS415 plasmid was constructed by introducing a 4.17 kb Seal-Sail fragment which contains the cassette [LEU2-APSH4-CEN6] from pRS415 (Stratagene) in the pBR322 digested with Seal/ Sail .
  • the pBRS415-p/t plasmid was generated by introducing sequentially a 0.72 kb fragment that contains the promotor of SSKl and the 0.36 kb terminator sequence of SSKl , linked by a BamHI restriction site.
  • the promotor sequence was obtained by PCR using the oligonucleotides 5 ' -AGGTCGACCCACTGCTGGATC-3 ' (SEQ ID NO: 7) and 5 ' -AGCGGATCCTTCCACAGTAACGC-3 ' (SEQ ID NO: 8).
  • the terminator sequence was also obtained by PCR using the oligonucleotides 5 ' -TTTGGATCCACCGTGTAGAGGACATTATG- 3 ' (SEQ ID NO: 9 ) and 5 ' -ATAGGTACCCAGCTGGTG AATCCAAGCCCG- 3' (SEQ ID NO: 10) .
  • the 2.04 kb PCR product that contains the entire ORF of CaSSKl was digested with BamHI and cloned into the only BamHI site of pBRS415-p/t to generate pBRS415- CaSSKl .
  • the pSSK1222 plasmid (Maeda et al . , 1994, Nature, 369, 242-245) was used as a control.
  • Each of these plasmids (pSSK1222, pBRS415, pBRS415-p/t and pBRS415-CaSSKl ) was used to transform S. cerevisiae strain TM187. Southern and Northern blot analysis Genomic DNA from C.
  • albicans was obtained according to the method described by Sherman et al . (1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York) . Four micrograms of DNA per lane were typically loaded for Southern analyses. DNA was transferred by capillarity to positively-charged nylon membranes (Hybond-N+, Amersham) by standard protocols (Sambrook et al . , 1989, supra) and hybridized with the same probe that was used for screening the genomic library.
  • RNA ladder 0.24-9.5 kb RNA ladder (Gibco-BRL) was used for sizing in formaldehyde agarose gels.
  • Chromosomal DNA was resolved by pulsed-field gel electrophoresis according to the protocol described by Wickes et al . (1991, Infect . Immun. 59, 1762-1771), in a Bio-Rad CHEF DRII unit with a linear ramp switch time of 300 seconds to 1400 seconds for 144 hours at 13 °C.
  • Gels were stained for 30 minutes with ethidium bromide (0.5 ⁇ g ml "1 in water) , destained in distilled water for 30 minutes and then photographed. The gels were blotted onto nylon membranes by alkaline transfer for 16 hours.
  • the 1.85 kb EcoRI-Bglll fragment was radioactively labelled by random priming and used as a probe. Hybridization was carried out as described above.
  • Homology searches, sequence analysis and multiple augments Homology searches were performed using the BLAST network service (Altschul et al . , 1990, J " . Mol . Biol . 215, 403-410). Motif searches were performed using the PROSITE and BLOCKS network databases . The computer analysis of sequences was performed using the DNA Strider 1.2. Sequences were aligned by the CLUSTAL program (Higgings and Sharp, 1988, Gene 73, 237-244).
  • plasmids used for C. albicans transformation were constructed, which carries a cassette designed to delete most of the ORF of CaSSKl .
  • a 3.80 kb Stul-Bglll DNA fragment from pMB7 (Fonzi, and irwin, 1993, Genetics 134, 717-728), which contains the hisG-URA3-hisG cassette, was used to replace most of the ORF of CaSSKl (Fig. 3A) .
  • the pBRl plasmid was linearized by digestion with Aatll which cuts once in the plasmid outside of the cassette, and approximately 2 ⁇ g of
  • DNA was used to transform the Ura ⁇ C. albicans strain CAI4 by electroporation (Thompson, et al . , 1998, Yeast 14, 565- 571) . Electroporation was chosen for transformation experiments since the most commonly used LiAc procedure (Gietz, et al . , 1991, Nucleic Acids Res . 20, 1425) consistently failed to yield transformants, even when a high amount of DNA (up to 30 ⁇ g) was used.
  • Transformed cells were selected as Ura + on SD minimal medium and spontaneous Ura " ⁇ derivates from a Ura + independent clone were selected on SD medium containing 5 ' -fluoroorotic acid (1 mg/ml) and uridine (25 ⁇ g/ml) .
  • the transformants were then used to delete the second allele of CaSSKl .
  • the pBR2 vector was designed.
  • a 2.37 kb Xbal-Bglll DNA fragment from pMB7 that contains the URA3-hisG cassette was introduced into the only Spel/BamHI sites of pBR- CaSSKl, which are located downstream of CaSSKl , generating the CaSSKl -URA3-hisG cassette (Fig. 3B) .
  • the pBR2 plasmid was linearized and used to transform a
  • Ura Acasskl null strain as described above.
  • the construction of the expression plasmids pLJ19 and pCCal (kindly provided by Dr. D. Harcus) , which carry the CPH1 and CPP1 genes of C. albicans respectively under the control of the ADHl promoter, was previously described (Csank et al . , 1997, Mol . Biol . Cell 8, 2539-2551; Csank et al, 1998, Infect . Immun . 66, 2713-2721) .
  • the encoding region of CaHOGl flanked by Bglll sites was amplified by PCR with the 5' oligonucleotide 5 ' -GCAGATCTGAAAATGTCTGCAGATGGAG-3 ' (SEQ ID NO: 11) and the 3' oligonucleotide 5'- TTAGATCTTTGAAGATTAAGCTCCGTTGGC-3' (SEQ ID NO: 12) using the pBSK8 plasmid containing the CaHOGl gene as a template .
  • flanking regions of the PCR product were confirmed by sequencing, digested with Bglll and inserted into the Bglll site of the plasmid pYPBl-ADHpt (kindly provided by D. Harcus) containing the ADHl promoter, the C. albicans URA3 as a selectable marker, and an autonomously replicating sequence (Csank et al . , 1997, 1998, supra; Leberer et al . , 1997, Curr. Biol . 7, 539-546). Determination of generation time. Preinoculum cultures of each strain were always prepared in YPD medium for 24 h at 28° C.
  • the optical density of precultures was determined at 600 nm (OD 600 ) , and 250 ml flasks containing 50 ml of prewarmed fresh medium (YPD or M199 pH 4.0) was inoculated to a final OD 600 of 0.1 with the appropriate preinoculum and incubated at 28° C and 200 rpm. The OD 600 was measured every 1.5 hours until the stationary phase of the growth curve was reached.
  • the generation times calculated for each strain are the averages of three independent experiments .
  • the C. albicans strains used in these experiments included a parental control with only one functional URA3 allele (strain CAF2 ) and five strains in which either one (CSSKll-1, CSSK23-1 and CSSK23-2) or both alleles (CSSK21-1 and CSSK21-2) have been deleted.
  • CSSK23 strains were included in all experiments to ensure that all phenotypic traits observed with the CSSK21 strains were due solely to the CaSSKl mutation rather than to unrelated mutations that may have occurred during construction of the Acasskl null strains.
  • Example 1 Isolation of a putative response regulator gene of C. albicans (CaSSKl)
  • the CaSSKl gene was cloned following a PCR-based approach as described in the Material and Methods section. A PCR reaction using an appropriate pair of degenerate primers was performed and a 447 bp PCR product was identified as a partial DNA sequence of CaSSKl . This PCR fragment was then used to isolate the entire CaSSKl gene.
  • 6 clones were selected for further analysis. These ⁇ EMBL3 clones revealed that they each carried the same C. albicans DNA fragment as we observed upon restriction analysis with Bg-lII. Also, each one contained the two expected hybridizing fragments as shown by Southern analysis. After subcloning and sequencing both fragments from one of the clones, we obtained the entire open reading frame (ORF) of CaSSKl .
  • Example 2 Structure of the C. albicans CaSSKl gene and its encoded protein CaSSKl has a 2022 bp ORF.
  • the 5 ' noncoding region upstream from the ATG codon ending at position -89, there is a long AT-rich sequence (84% A+T) in which two putative TATAAA elements were found between positions -17 to -22 and -28 to -33, upstream from the start codon, respectively.
  • two CAAT motifs were found at -309 and -329 positions, respectively.
  • the 2022 bp ORF of CaSSKl encodes a protein of 674 amino acids with a predicted molecular mass of 73.5 kDa and a pi of 9.5.
  • the codon usage frequency in CaSSKl corresponds to the low expressed genes in C. albicans (Lloyd and Sharp, 1992, Nucleic Acids Res . 20, 5289-5295).
  • the PTSl motifs have been involved in targeting proteins to peroxisomes and are located in the extreme C-termini of the majority of the peroxisomal matrix proteins. Their specificity is remarkable considering the small size and relaxed consensus sequence.
  • the C-terminal PTSl has been studied by extensive mutational analyses (Elgersma et al . , 1996, supra) . These studies revealed a high degeneracy of the PTSl consensus sequence and suggests that additional domains in these proteins may be of importance to determine whether or not a certain PTSl is recognized by the components of the peroxisomal import machinery (Elgersma et al . , 1996, supra) . However, whether or not this motif is used as a PTSl sequence in CaSsklp remains to be elucidated.
  • the C-terminal sequences from both prokaryote and eukaryote response regulators show highly conserved residues.
  • these residues include a pair of aspartates near the N- terminus (one aspartate in eukaryotes) , an aspartate motif which accepts a phosphate and a motif near the C- terminus which contains a key lysine (Parkinson, 1993, Cell 73, 857-871) .
  • the Asp 556 should be the predicted site of phosphorylation.
  • the Asp 513 should be one of the pair of aspartates that are conserved among prokaryotic response regulators but apparently not in eukaryotic cells, which appears as ED instead of DD.
  • the Lys 638 should be the other conserved residue.
  • Example 3 Complementation analysis of a S. pombe mcs4 mutant with CaSSKl
  • Mcs4 functions in adapting cells to oxidative, heat, and nutritional stresses by coordinating environmental responses with meiosis and mitosis (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1997, supra; Shiozaki et al . , 1997, Mol . Biol . Cell 8, 409-419). Since Mcs4 and CaSsklp share structural similarity, it could be also possible that CaSsklp plays a similar function in C. albicans .
  • Example 4 Complementation analysis of a S. cerevisiae SSKl mutant with CaSSKl It has been reported that Ssklp acts downstream of the Slnlp-Ypdlp osmosensor pathway of S. cerevisiae (Posas et al . , 1996, Cell 86, 865-875) . Thus, considering the high similarity that CaSsklp shares with the C-terminus of Ssklp, and that homologues of Slnlp and Ypdlp from S. cerevisiae also exist in C.
  • CaSsklp is a component of the putative homologous CaSlnlp-CaYpdlp- CaSsklp pathway of C. albicans , which also could be involved in osmoregulation.
  • CaSSKl was expressed in a ssklA S. cerevisiae strain. However, since the deletion of SSKl in S.
  • Example 5 Chromosomal deletion of CaSSKl It was previously shown that CaSSKl was unable to rescue the lack of SSKl or mcs4 + in S. cerevisiae or S. pombe respectively, which suggested that despite their structural homology, they may not be functional homologs (Calera and Calderone, 1999, Yeast 15) . Thus, to further investigate the function of CaSSKl in the growth, morphogenesis and virulence of C. albicans, we used the urablaster technique (Fonzi and Irwin, 1993, supra) to obtain Acasskl mutants.
  • the hisG- URA3-hisG cassette was used to replace a 1.47 kb fragment of CaSSKl that includes the encoding region for the conserved putative aspartate residue which is phosphorylated (Fig. 3A) .
  • Ura + transformants were selected on SD minimal medium and several isolates were tested by southern blot to confirm this replacement. DNA from a CSSK11 representative isolate exhibited two hybridizing bands, a
  • CSSK12-1 and CSSK12-2 A representative CSSK12 intrachromosomal recombinant strain is shown by southern blot (Fig. 3C) .
  • Example 6 CaSSKl is not involved in a response to osmotic or oxidative stress.
  • CaSSKl does not function in regulating the response to either osmotic or oxidative stress (Calera and
  • Example 7 CaSSKl is required for hyphal formation on solid media.
  • the Acasskl null and heterozygote Ura3 + strains were grown in several media (solid and liquid) that induce this morphological switch, including Spider, serum and M199 (pH 7.5) medium (Fig. 5) .
  • Colonies from the wild type (CAF2) and the heterozygote strains (CSSK11) developed radial filaments emerging from the edge of the colonies after two days in M199 (pH 7.5) and after three days in Spider medium.
  • the Acasskl null mutants when grown in agar with 10% serum showed a severe reduction in hyphal development in comparison with the CAF2 , forming irregular smooth colonies with yeast growth in the center of the colonies from which some filaments emerged.
  • the heterozygote strains formed colonies intermediate between those of the Acasskl null strains and the wild type (Fig. 5) .
  • Example 8 Avirulence of Acasskl mutants .
  • the ability of Acasskl C. albicans null strains to establish infection in a murine model of hematogenously disseminated candidiasis was investigated.
  • two factors that may also affect the virulence of the mutants such as their generation time and orotidine 5 ' -monophosphate (OMP) decarboxylase activity.
  • OMP orotidine 5 ' -monophosphate
  • survival of mice infected with CSSK21, CSSK11 and CSSK23 was greater than that of mice infected with CAF2.
  • mice succumbed to CAF2 infection by 72 h.
  • the avirulence of the Acasskl null strains indicate that CaSSKl is required for the pathogenesis of C. albicans . Furthermore, several experiments indicated that the virulence of the CSSKll and CSSK23 strains was due to the presence of one functional allele, while the avirulence of the CSSK21 strains was due to the absence of any CaSSKl allele: 1) The same results in survival and tissue counts were obtained with two independent Acasskl null strains (CSSK21-1 and CSSK21-2) . 2) The virulence of the CSSK21 strains was restored by the reintroduction of a parental copy of CaSSKl in them both (CSSK23-1 and CSSK23-2) .
  • albicans homologues have been identified, including CST20, HST7 , CEK1 and CPH1 (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . , 1996, supra; Liu et al . , 1994, supra) , which function in a C. albicans STE12-homolog MAPK cascade, as well as EFGl (Lo et al . , 1997, Cell 90, 939-949; Stoldt et al . , 1997, EMBO J. 16, 1982-1991) which, like its S.
  • CaHKl Calera and Calderone, 1999, Microbiol . 145; Calera et al . , 1998, Yeast 14, 665-674) and CaNIKl/COSl (Alex et al . , 1998, Proc . Natl . Acad . Sci . USA 95, 7069-7073).
  • CaSSKl the only response regulator kinase gene described so far in C. albicans (Calera and Calderone, 1999, Yeast 15) .
  • CaSSKl does not restore the normal growth of either Asskl or Amcs strains of S. cerevisiae or S. pombe respectively, suggesting that CaSSKl may have another role in C. albicans rather than in adaptation of cells to stress (Calera and Calderone, 1999, Yeast 15, supra) .
  • the phenotypic characterization of Acasskl mutants supports our previous observation and confirms that CaSSKl is not functionally related to either SSKl or mcs4 + .
  • CaSSKl is essential for hyphal formation on solid inducing media and virulence, even though it is not required for formation of hyphae in liquid media.
  • Efglp must be the final element of a morphogenesis pathway (Lo et al . , 1997, supra; Stoldt et al, 1997 , supra) .
  • the Aefgl mutants are unable to form true hyphae either on solid or liquid media (Lo et al . , 1997, supra) .
  • the Aefg mutants grow as a yeast in liquid media even in the presence of serum (Lo et al . , 1997 , supra) .
  • the phenotype of the Acasskl null strains resembles the defect in hyphal formation on solid media observed for mutants in genes of the CPHl filamentation pathway but, in contrast, the mutants in genes of the CPHl pathway are able to form hyphae on solid serum, are not totally avirulent, and fail to hyper-invade the agar under low nitrogen availability (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . 1996, supra; Liu et al . , 1994, supra) .
  • the hyper-invasive phenotype of the Acasskl null strains resembles that of the Acppl mutants, the Acasskl null strains show this phenotype only under low nitrogen while the Acppl mutants show derepressed hyper- invasion of agar under both normally noninducing and inducing conditions in a wide variety of rich and defined solid media (Csank et al . , 1997, supra) .
  • CaSSKl affects the activity of the CPHl pathway
  • the overproduction of downstream components of the CPHl pathway in a Acasskl background should rescue the lack of CaSSKl .
  • in preliminary experiments we were unable to rescue the normal filamentous growth of a Acasskl null mutant by overexpressing either CHP1 or CPP1 . Similar results were previously described by others in a
  • the flocculation displayed by the Acasskl null strains under conditions of germ tube formation occurs in a manner similar to the Acahkl mutants (Calera and Calderone, 1999, Microbiology 145) . Both the Acasskl and Acahkl strains show similar growth rates and are totally avirulent in a murine model of hematogenously disseminated candidiasis (Calera et al . , 1999, Infect . Immun. 67, 4280-4284).
  • CaSsklp should function as a response regulator in this putative cascade, similar to other response regulators (Banuett 1998, supra) , it must lie downstream of a sensor histidine kinase component, such as Cahklp or CaNiklp/Coslp.
  • albicans i.e., the morphological transition from yeast to hyphae and changes in the expression of a hyphal surface compound (s) that occur during hyphal growth.
  • a "two-component" cascade may have been adopted by C. albicans to modulate its morphological switching and that other undiscovered elements or pathways must exist that regulate the transition from yeast to hyphae in liquid media in C. albicans .
  • two-component signal tranduction cascades have not been found in mammalian cells which emphasize the interest of both CaSslp and Cahklp as targets for the development of antifungals .

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Abstract

The present invention relates to nucleic acids encoding a response regulator, CaSsk1p, and methods for use, as well as describes the construction and characterization and use of a Δcassk1 mutant of C. albicans and methods of use.

Description

TITLE OF THE INVENTION
Response Regulator CaSSKl
Field of the Invention
The present invention relates to nucleic acid molecules encoding response regulator CaSskl protein including mutants, variants, fragments and derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules; methods of using response regulator; method for screening for inhibitors of response regulator; and kits comprising the compositions or polypeptides of the invention.
Background of the Invention
The sensor histidine kinases and their corresponding response regulators, which were first described in prokaryotes in phosphotransfer pathways referred to as ' two-component systems ' , enable cells to detect environmental changes and elicit an appropriate response
(Parkinson, 1993, Cell 73, 857-871; Hoch and Silhavy (eds), 1995, Two component Signal Transduetion. ASM Press, Washington, DC) . The prototypical prokaryotic two- component regulator system is comprised of two proteins, a histidine protein kinase (or sensor protein) and a response regulator (or effector protein) , which is associated with an internal response. The sensor kinase, when activated by an environmental signal, autophosphorylates a histidine residue, which then serves as a phosphodonor to a conserved aspartate residue in the response regulator. This phophorylation modulates the activity of the effector protein so as to elicit an adaptive response to the stimulus . The prokaryotic response regulators are characterized by a conserved domain of approximately 125 amino acids, usually attached via a linker sequence to a domain with an effector function. The effector domain generally acts as a transcription factor. However, there are several cases in which a different type of effector domain is attached to a response regulator (Hoch and
Silhavy, 1995, supra) . Also, although the general sequence of events and the number of proteins involved is similar in prokaryotes, each pathway exhibits some variation on the basic scheme and, in the most complex phosphorelays , four phosphorylation events occur in sequence, creating a four- step His-Asp-His-Asp phophorelay (Appleby et al . , 1996, Cell 86, 845-848) .
More recently, homologues of both sensor and response regulator genes have been demonstrated in yeast, filamentous fungi and plants (Loomis et al . , 1997, J Cell Science 110, 1141-1145) . In S. cerevisiae, three proteins with domains which show homology with bacterial response regulators have been described. One is the Skn7p protein (Brown et al . , 1994, EMBO J 13, 5186-5194), whose function is associated with the response to oxidative stress in S. cerevisiae (Morgan et al . , 1997, EMBO J 16, 1035-1044). Also, it has recently been reported that the Skn7p activity is modulated by the Slnlp-Ypdlp osmosensor and contributes to regulation of the HOG pathway, regulating the cellular response to hyperosmotic conditions (Posas et al . , 1996,
Cell 86, 865-875) . A response regulator domain is located at the C-terminus of Slnlp, a transme brane hybrid protein that serves as an osmosensor (Ota and Varshavsky, 1993, Science 262, 566-569) which, after autophosphorylation of a histidine residue in its histidine kinase domain and transfer to an aspartate in its response regulator domain, phosphorylates a histidine residue of Ypdlp (Posas et al . , 1996, supra) . This protein, in turn, phosphorylates Ssklp, the other respnse regulator (Maeda et al . , 1994, Nature 369, 242-245) . The activation of a downstream MAP kinase cascade is dependent upon the phosphorylation state of Ssklp (Posas et al . , 1996, supra) . In Dictyostelium discoideum, three different hybrid histidine kinases (DokA, DhkA, and DhkB) , which show response regulator domains at their C-terminus, have been described. DokA is involved in the osmoregulatory pathway (Schuster et al . , 1996, EMBO J 15, 3880-3889) while DhkA (Wang et al . , 1996, EMBO J 15, 3890-3898) and DhkB (Zinda and Singleton, 1998, Dev Biol 196, 171-183), as well as the Reg A response regulator (Thomason et al . , 1998, EMBO J 17, 2838-2845), play a role in the develoopment of D. discoideum . In Neurospora crassa, a hybrid histidine kinase (Nik-1) , which is involved in hyphal development and osmosensing (Alex et al . , 1996, Proc Natl Acad Sci USA 93, 3416-3421) has been described. Also, a link between environmental stress and growth in an eukaryotic microorganism has also been established. Thus, the Mcs4 response regulator from Schizosaccharomyces pombe is an essential component of the Wakl-Wisl-Styl MAP kinase pathway, through which Mcs4 coordinates cell cycle progression with the cellular respnse to environmental stresses such as osmotic and oxidative stress, heat shock and nutritional limitations (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1997, Genes Dev 11, 1008-1022; Shiozaki et al . , 1997, Mol Biol Cell 8, 409-419) . Three hybrid histidine kinase genes have been reported in Candida albicans, including CaSLNl (Nagahashi et al . , 1998, Microbiology 144 , 425-432), CaNIKl/COSl (Nagahashi et al . , 1998, supra; Srikantha et al . , 1998, Microbiology 144, 2715-2729; Alex et al . , 1998, Proc Natl Acad Sci USA 93, 7069-7073) and CaHKl (Calera et al . , 1998, Yeast 14, 665-674). The CaSLNl and CaNIKl encoded proteins, CaSlnlp and CaNiklp, show structural and functional homology with the proteins Slnlp and Nikl from S. cerevisiae and N. crassa , respectively. The CaHKl encoded protein, Cahklp, while homologous at its C-terminus with the histidine kinase domains of many sensor proteins, at its N-terminus, only shows homology with a putative histidine kinase from S. pombe (GenBank Accession No. Z98978) with unknown function. However, dcahkl strains flocculate extensively when they form hyphae, indicating a putative role for Cahklp in the regulation of cell wall development in hyphae (Calera and Calderone, 1999, Microbiology 145, 1431-1442). Finally, the S. cerevisiae YPD1 homologous gene from C. albicans ( CaYPDl) has recently been partially sequenced through the C. albicans genome sequencing project.
In summary, although in prokaryotes many response regulators have been studied at the molecular level, in fungi only four response regulators are well characterized thus far, including Ssklp and Skn7p from S. cerevisiae
(Posas and Saito, 1998, EMBO J 17, 1385-1394; Ketela et al, 1998, Mol Gen Genet 259, 372-378), RegA from D. discoideum (Thomason et al . , 1998, supra) and Mcs4 from S. Po be (Cottarel, 1997, supra; Shieh et al . , 1997, supra) . In addition, even though the response regulator domains are similar to those of prokaryotes, only RegA shows a domain with an effector function (phosphodiesterase) at its C- terminus , which is attached to the response regulator domain by a linker sequence. Conversely, Ssklp, Skn7p and Mcs4 have a large N-terminal domain, and the conserved aspartate residues in their response regulators are located towards the C-terminus and are not followed by a domain with an effector function. In this application we describe the first response regulator homologue gene in the pathogenic yeast C. albicans ( CaSSKl) , whose encoded protein (CaSsklp) shows structural homology with both Ssklp and Mcs4 response regulators .
Candida albicans is the most frequently isolated opportunistic fungal pathogen in humans. Candidiasis is a general- term for a variety of local and systemic processes caused by colonizationor infection of the host by species of the yeast Candida. Candidiasis occurs worldwide; superficial infections of the skin, mouth and other mucus membranes are universal . Invasive systemic disease has become a problem due to the use of high doses of antibiotics that destroy normal bacterial flora, immunosuppressive agents, and agents toxic to bone marrow, e.g. during cancer therapy. Candidiasis is also seen among immunocompromised individuals such as AIDS patients, organ transplant patients, patients receiving parenteral nutrition, and cancer patients receiving parenteral and chemotherapy. In this population, disease ranges from aggressive local infections such as peiodontitis, oral ulceration, or esophagitis in HIV-infected patients, to complex and potentially lethal infections of the bloodstream with subsequent dissemination to brain, eye, heart, liver, spleen, kidneys, or bone. The infection typically begins at an epithelial site, evades local defenses, and invades the bloodstream in the face of immunosuppression. In normal hosts, disease caused by C. albicans ranges from mild, easily treated, superficial disease (e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water) to more severe, chronic or recurrent infections (e.g., candidal vaginitis) . Candida species occur in two forms that are not temperature- or host-dependent . The usual colonizing forms are yeasts that may assume a pseudomycelial configuration, especialy during tissue invasion. Pseudomyceliae result from sequential budding of yeasts into branching chains of elongated organisms.
A number of factors have been associated with the virulence properties of C. albicans, such as adherence to host cells and the ability to undergo the transition from yeast to hyphal growth. This switch is induced in vi tro by many environmental conditions including temperature, pH and the presence of serum. Diploid strains of Saccharomyces cerevisiae also switch their pattern of growth from unicellular yeast to chains of elongated cells that remain attached to each other (pseudohyphae) under conditions of nitrogen starvation (Gimeno, et al . , 1992, Cell 68, 1077- 1090) . This switch requires STE20, STEll , STE7 , KSS1 and STE12 , genes of a conserved MAP kinase pathway, but also depends on PHD1 , a gene which functions in a STE12- independent pathway (Banuett, 1998, Mol Biol Rev 62, 249- 274) . Based upon functional complementation studies of the STE20 , STE7, KSS1 and STE12 genes of S. cerevisiae, the homolog genes CST20 (Kohler and Fink, 1996, Proc Natl Acad Sci USA 93, 13223-13228; Leberer et al . , 1996, Proc Natl Acad Sci USA 93, 13217-13222), HST7 (Kohler and Fink, 1996, supra; Leberer et al, 1996, supra) , CEK1 (Csank et al . , 1998, Infect I-nun 66, 2713-2721) and CPH1 (Liu, et al . , 1994, Science 266, 1723-1726) of C. albicans respectively, have been identified. Mutants of C. albicans in each of these genes are unable to undergo the transition from yeast to hyphae on solid media, except when serum is included.
In the same way, it was reported that the CPP1 gene, which encodes a phosphatase similar to the MAP kinase phosphatase Msg5p of S. cerevisiae, modulates the activity of the CPHl- pathway likely by dephosphorylating Ceklp (Csank et al . , 1997, Mol Biol Cell 8, 2539-2551). Following a similar experimental approach, the C. albicans genes CaCLA4 (a CLA4 homolog) (Leberer et al . , 1997, Curr Biol 1 , 539-546), EFG1 (a PHD1 homolog) (Lo et al . , 1997, Cell 90, 939-949; Stoldt et al . , 1997, EMBO J 16, 1982-1991), CaTUPl (a TUPl homolog) (Braun and Johnson, 1997, Science 277, 105-109), and CaRSRl (a RSR1 homolog) (Yaar et al . , 1997, Microbiology 143, 3033-3044) were also studied. The virulence of each single mutant described above (except for CaTUPl mutants with which virulence studies were not performed) was reduced in a murine model of hematogenously disseminated candidiasis (Csank et al . , 1997, supra; Csank et al . , 1998, supra; Leberer et al . , 1996, supra; Leberer et al . , 1997, supra; Lo et al . , 1997, supra; Yaar et al . , 1997, supra) but a Acphl/Aefgl double mutant was shown to be avirulent (Lo et al . , 1997, supra) . However, recent studies indicate that in fact the Acphl/Aefgl double mutant can still form hyphae when colonizing tissue, indicating that an additional EFG1- and CPHl-independent pathway may exist that regulates morphogenesis in C. albicans (Riggle et al . , 1999, Infect Immun 67, 3649-3652).
More recently, it has been demonstrated that the CaHOGl gene, in addition to its role in cytokinesis and response to osmostress in C. albicans, also functions in morphogenesis (Alonso-Monge et al . , 1999, J Bacteriol 181, 3058-3068) . Interestingly, its HOG1 homolog of S. cerevisiae does not function in the filamentation-invasion pathway of S. cerevisiae but in the HOG pathway, which is in part regulated by a "two-component" cascade whose functional proteins have domains similar to the sensor histidine kinases (Slnlp, Ypdl) and response regulators (Slnlp, Ssklp) of prokaryota (Banuett, 1998, supra) . In this regard, in C. albicans the two-component sensor histidine kinase gene CaHKl (Calera et al . , 1998, supra) , seems to regulate the expression of hyphal surface components (Calera and Calderone, 1999, supra) and perhaps also virulence factors since a Acahkl null mutant is avirulent (Calera et al . , 1999, Infect Immun 67, 4280- 4284), while the CaNIKl/COSl gene (Nagahashi et al, 1998, Microbiology 144, 425-432; Srikantha et al . , 1998, supra) is required for hyphal development on solid media (Alex et al . , 1998, supra) .
We have identified the putative CaSSKl response regulator gene of C. albicans (Calera and Calderone, 1999, supra) . CaSSKl encodes a protein (CaSsklp) which is a structural homolog of Ssklp and Mcs4 from S. cerevisiae and S. pombe, respectively. Ssklp is a response regulator of the "two-component" cascade that regulates the HOG pathway of S. cerevisiae (Maeda et al . , 1994, Nature 369, 242-245), and Mcs4 is an element of the Styl pathway of Schizosaccharomyces pombe (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1991 , Genes Dev 11, 1008-1022). Unlike Ssklp which only functions in osmosensing in S. cerevisiae, Mcs4 plays a role in regulating the adaptive responses to different stresses including osmotic and oxidative stress, and coordinates the responses to these environmental stimuli with the cell cycle (Cottarel, 1997, supra; Shieh et al . , 1997, supra) . However, we have shown that CaSSKl fails to complement the lack of either SSK1 or mcs4+ in S. cerevisiae and S. pombe, respectively (Calera and Calderone, 1999, supra) . This indicated that CaSSKl may have other functions in C. albicans rather than modulating the response to osmotic or oxidative stress. In this paper, we present data indicating that CaSSKl is involved in the morphogenesis and virulence of C. albicans .
Summary of the Invention We have isolated a response regulator two- component gene from Candida albicans, CaSSKl . The CaSSKl gene was cloned following a PCR based approach as described below. CaSSKl has an open reading frame of 2022 base pairs (bp) . In the promotor region of CaSSKl a short sequence is found that matches the consensus sequence of the stress response elements (STRE) from Saccharomyces cerevisiae . CaSSKl is located on chromosome 1 and is expressed in either yeast or mycelial phases of C. albicans . CaSSKl encodes a 674 amino acid protein (CaSsklp) with an estimated molecular mass of 73.5 kDa and a basic isoelectric point (pi 9.5). It has a tripeptide (NKA) located in its C-terminus, which resembles the peroxisomal signalling target type 1 sequence (PSTl) of most of the peroxisomal matrix proteins . A homology search of CaSsklp with other proteins in databases showed that the C-terminus of CaSsklp exhibits the greatest similarity with the C-terminus of Ssklp and Mcs4 from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The response regulator domain of CaSsklp contains the motifs that are characteristic of all response regulators, including the conserved aspartate and lysine residues as well as the putative aspartate, which is phophorylated by a phosphohistidine residue. Finally, in spite of the structural similarities among CaSsklp, Ssklp and Mcs4, CaSsklp does not seem to exhibit functional homology with these proteins . The Accession No. for the described sequence is AF084608, as filed in the EMBL/GenBank/DDBJ database.
In addition, we have constructed and phenotypically characterized the Acasskl mutants of C. albicans . The results confirmed that CaSSKl , unlike SSK1 or MCS4+, does not regulate cellular responses to either osmotic or oxidative stress. Instead, the Acasskl null strains are severely reduced in hyphal formation on serum agar and are totaly defective in hyphal development on other solid media, such as Medium 199 (pH 7.5) and Spider medium. In contrast, under conditions of low nitrogen availability on solid media, the Acasskl null strains dramatically hyper- invaded the agar. However, while forming germ tubes and hyphae in liquid media similar to the wild type, in contrast, the Acasskl null strains flocculated in a manner similar to the Acahkl two-component histidine kinase mutants, as we have previously described. Finally, virulence studies indicated that CaSSKl is essential for the pathogenesis of C. albicans, which suggests that the CaSsklp response regulator could be a good target for antifungal therapy.
Therefore, it is an object of the present invention to provide a DNA fragment of about 3058 bp (SEQ ID NO:l) containing a 5' upstream promotor region of 486 bp, an open reading frame of 2022 bp encoding CaSsklp, and a 3' downstream non-coding region. The DNA fragment is useful as a diagnostic agent, an agent for preparation of CaSsklp, and a therapeutic agent. It is another object of the invention to provide an amino acid sequence for CaSsklp encoding 674 amino acids (SEQ ID NO: 2) .
It is another object of the invention to provide a recombinant vector comprising a vector and the above described DNA fragments .
It is a further object of the present invention to provide a host cell transformed with the above- described recombinant DNA construct.
It is another object of the present invention to provide a method for producing CaSsklp which comprises culturing a host cell under conditions such that the above-described DNA fragment is expressed and CaSsklp is thereby produced, and isolating CaSsklp for use as a reagent, for example for screening of drugs and inhibitors of CaSsklp.
It is a further object of the present invention to provide an antibody to the above-described recombinant CaSsklp protein.
It is yet another object of the present invention to provide a method for detecting CaSsklp in a sample comprising:
(i) contacting a sample with antibodies which recognize CaSsklp; and (ii) detecting the presence or absence of a complex formed between CaSsklp and antibodies specific therefor .
It is a further object of the present invention to provide a diagnostic kit comprising an antibody against CaSsklp and ancillary reagents suitable for use in detecting the presence of CaSsklp in cells, tissue or serum from, mammals including humans, animals, birds, fish, plants and fungi. It is yet another object of the present invention to provide a method for the detection of CaSsklp from a sample using the polymerase chain reaction.
It is a further object of the present invention to provide a diagnostic kit comprising primers or oligonucleotides specific for CaSSKl RNA or cDNA suitable for hybridization to CaSSKl RNA or cDNA and/or amplification of CaSSKl sequences and ancillary reagents suitable for use in detecting CaSSKl RNA/cDNA in mammalian tissue. It is yet another object of the present invention to provide a method for the detection of CaSSKl in a sample which comprises assaying for the presence or absence of CaSSKl RNA or cDNA in a sample by hybridization assays . It is yet another object of the present invention to provide a method for reducing Candida albicans virulence by inhibiting the expression of CaSSKl in said cell . The inhibition can be at the DNA level by introducing mutations into the gene encoding CaSSKl , by inhibiting transcription of the gene, by inhibiting translation of the RNA encoding CaSsklp, or by inhibiting the function of CaSsklp.
It is still another object of the present invention to provide a method for inhibiting hyphae formation in Candida albicans by providing into the cell a antisense RNA which hybridizes to CaSSKl RNA or DΝA or a portion of CaSSKl RΝA or DΝA.
It is still another object of the invention to provide a therapeutic method for the treatment or amelioration of diseases resulting from virulent
Candida albicans, said method comprising administering to an individual in need of such treatment an effective amount of an inhibitor of CaSsklp expression or function in a pharmaceutically acceptable diluent or excipient.
It is another object of the present invention to provide a delivery molecule comprising a nonvirulent strain of Candida albicans such as Δcasskl and a nucleic acid or protein of interest. Such a delivery molecule can be used to delivery a desired antigen to a tissue naturally invaded by Candida albicans. It is still another object of the present invention to provide a method for identifying downstream components or interacting proteins important in C. albicans morphogenesis or virulence activated by CaSsklp, by identifying genes expressed in the wild type Candida albicans but not the mutant strain Δcasskl.
It is yet another object of the present invention to provide a method and composition to elicit C. albicans specific immune response in an individual comprising administering to the individual Δcasskl in an amount sufficient to induce such a response. It is yet a further object of the present invention to provide a cDΝA sequence encoding CaSsklp and vectors incorporating all or a fragment of said sequence, and cells, prokaryotic and eukaryotic, transformed or transfected with said vectors, for use in screening agents and drugs which inhibit expression or function of CaSsklp in such cells. BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims, and accompanying drawings where:
Figure 1A and IB. (A) Northern blot of total RNA. RNA samples obtained from cultures grown in medium 199 for 3 hours at 28'C (pH 3.5) (lane Y) and at 37 °C (pH 7.5) (lane M) , to induce yeast and hyphal growth, respectively. (B) CHEF gel analysis. It was performed with duplicate samples . The chromosomal bands were visualized with ethidium bromide (left panel) and a Southern blot of the gel (right panel) was hybridized with a 1.85 kb EcoRI-Bglll fragment of the CaSSKl gene as a probe. C. albicans chromosomal designations (on the left side) are those proposed by Wickes et al . (1991, Infect . Immun . 59, 1762-1771).
Figure 2A and 2B. (A) Complementation analysis of a S. pombe mcs4-13 cdc25-22 double mutant with CaSSKl (pBREP42-CaSSKl) . This S. pombe strain was also transformed with the plasmids pREP42-mcs4 as a positive control and the plasmids pREP42 and pBREP42 as negative controls . Transformants were grown in EMM minimal medium at either 25°C or 30°C for four days. (B) Complementation analysis of a S. cerevisiae SSKl SLN1 double mutant with CaSSKl (pBRS415-CaSSKl) . This strain was also transformed with the plasmids pRS415- SSK1 as a positive control and the plasmids pBRS415 and pBRS415-p/t as negative controls. Transformants (1.5 X 104 cells per spot) were grown in presence of either galactose or glucose for two days at 30°C.
Figure 3A, 3B, and 3C. (a) Schematic representation of the construction of the cassette used to disrupt CaSSKl and, (b) the cassette CaSSKl -URA3 -hi sG used to reintroduce one wild type CaSSKl allele. In (c) is shown the corresponding southern blot analyses of strains CSSKll-1, CSSK12-1, CSSK21-1 and CSSK22-1 obtained during the disruption process and a revertant strain (CSSK23-1) . Genomic DNA from these strains was Bglll digested, and hybridized with a 1.4 kb Nsil-EcoRI fragment located at the 5 ' -end of the CaSSKl gene as a probe. The exact size and genotype of the expected hybridizing DNA fragments are indicated on the right . Figure 4. Broth cultures of Ura3+ Acasskl mutants
(strains CSSKll-1, CSSK21-1 and CSSK23-1) . Stationary-phase cells from all strains were inoculated (107 cells/ml) in M199 (pH 7.5), grown at 37°C for 3 hours and photographed. The CSSK21 null strains flocculated extensively forming clumps of cells that sedimented rapidly to the bottom of the tubes after the cell suspension was shaken. The formation of clumps was not observed in the heterozygote and reconstituted strains.
Figure 5. Phenotype of the Acasskl mutants (strains CSSKll-1, CSSK21-1 and CSSK23-1) grown on solid media which induce hyphal development . Plates were incubated for 5 days at 37°C. The Spider plates were incubated up to 8 days at 37°C, and the same CSSK21 colony was photographed again (small inserted panel) to show the formation of short filaments emerging from the edge of the colony. Bars, 1 mm. Figure 6. Phenotypes of the CAF2 and Acasskl mutants (strains CSSKll-1, CSSK21-1 and CSSK23-1) grown under conditions of low nitrogen availability on solid SLAD medium. Plates were incubated for 8 days at 30° C. Invasion of the agar initially was observed after 5 days of incubation and increased with time. The colonies of the top panel (UW, unwashed) show darker spots more clearly observed in the thinner areas of the colonies , which correspond to those cells of the colonies that have invaded the agar. In the lower panel (W, washed) , the same colonies are shown after the non-invading cells were washed off the surface of the agar. Bar, 1 mm.
Figure 7. Survival of mice following infection with C. albicans Acasskl mutants. Only the survival curves of mice infected with CAF2 , CSSKll-1, CSSK21-1 and CSSK23-1 are shown. Similar results were observed with mice infected with strains CSSK21-2 and CSSK23-2.
Figure 8. Hypothetical scheme of the location of CaSsklp in a putative two-component pathway of C. albicans based on the phenotypic characterization of the Acahkl , Acasskl and Acanikl/cosl mutants. The dashed arrows indicate the putative transduction of the signal by CaNiklp/Coslp and the solid arrows the transduction of the signal by Cahklp.
DETAILED DESCRIPTION In one embodiment, the present invention relates to a DNA or cDNA segment which encodes CaSSKlp from Candida albicans . A PCR reaction using an appropriate pair of degenerate primers was performed and a 447 bp PCR product was identified as a partial DNA sequence of CaSSKl . This PCR fragment was then used to isolate the entire CaSSKl gene. The CaSSKl DNA fragment has an open reading frame of 2022 bp in addition to the 5' promoter region and 3' nontranslated region. The complete 3058 bp CaSSKl DNA fragment is specified in SEQ ID NO:l.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from (a) a nucleotide sequence comprising a sequence encoding a full length CaSsklp polypeptide having the sequence specified in SEQ ID NO:l, (b) a nucleotide sequence which encodes the complete amino acid sequence in SEQ ID NO: 2, or the complete amino acid sequence encoded by the cloned DNA in GenBank accession no. AF084608.
In addition, isolated nucleic acid molecules of the invention include DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode CaSsklp or fragments thereof. Of course, the genetic code and species-specific codon preferences are well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) . Nucleic acid molecules of the present invention may be in the form of RNA, such as mRΝA, or in the form of DΝA, including, for instance, cDΝA and genomic DΝA obtained by cloning or produced synthetically. The DΝA may be double-stranded or single-stranded. Single-stranded DΝA or RΝA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the antisense strand.
By "isolated" nucleic acid molecule (s) is intended a nucleic acid molecule, DΝA or RΝA, which has been removed from its native environment. For example, recombinant DΝA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DΝA molecules include recombinant DΝA molecules maintained in heterologous host cells or purified (partially or substantially) DΝA molecules in solution. Isolated RΝA molecules include in vivo or in vitro RΝA transcripts of the DΝA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
The present invention is further directed to nucleic acid molecules encoding portions or fragments of the nucleotide sequences described herein.
Fragments include portions of the nucleotide sequence of SEQ ID NO:l or at least 10 contiguous nucleotides in length selected from any two integers , one of which representing a 5' nucleotide position and a second of which representing a 3' nucleotide position, where the first nucleotide for each nucleotide sequence is position 1. That is, every combination of a 5 ' and 3' nucleotide position that a fragment at least 10 contiguous nucleotide bases in length or any interger between 10 and the length of an entire nucleotide sequence of CaSSKl minus 1.
Further, the invention includes polynucleotides comprising fragments specified by size, in nucleotides, rather than by nucleotide positions. The invention includes any fragment size, in contiguous nucleotides, selected from intergers between 1- and the entire length of an entire nucleotide sequence minus 1. Preferred sizes include 20-50 nucleotides, 50-300 nucleotides useful as primers and probes. Regions from which typical sequences may be derived include but are not limited to, for example, regions encoding specific epitopes or domains within said sequence, such as the region comprising nucleotides 1599-2115 of SEQ ID NO: 1. In another aspect, the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridize under stringent hybridization conditions to a polynucleotide sequence of the present invention described above, or a specified fragment thereof. By "stringent hybridization conditions" is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6) , 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0. IX SSC at about 65°C.
The sequences encoding the polypeptides of the present invention or portions thereof may be fused to other sequences which provide additional functions known in the art such as a marker sequence, or a sequence encoding a peptide which facilitates purification of the fused polypeptide, peptides having antigenic determinants known to provide helper T-cell stimulation, peptides encoding sites for post- tranlational modifications, or amino acid sequences which target the fusion protein to a desired location, e.g. a heterologous leader sequence.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the CaSsklp polypeptides. Variant may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus of a chromosome of an organism. Non-naturally occuring variants may be produced by known mutagenesis techniques. Such variants include those produced by nucleotide substitution, deletion, or addition of one or more nucleotides in the coding or noncoding regions or both. Alterations in the coding regions may produce conservative or nonconservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions which do not alter the properties and activities of CaSsklp polypeptides disclosed herein or portions thereof . Also preferred in this regard are conservative substitutions .
Nucleic acid molecules with at least 90-99% identity to a nucleic acid shown in SEQ ID NO:l is another aspect of the present invention. These nucleic acids are included irrespective of whether they encode a polypeptide having CaSsklp activity. By "a polypeptide having CaSsklp activity" is intended polypeptides exhibiting activity similar, but not identical, to an activity of the CaSsklp of the invention, as measured in the assays described below. The biological acitivity or function of the polypeptides of the present invention are expected to be similar or identical to polypeptides from other organisms that share a high degree of structural identity/similarity.
In another embodiment, the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above. The vector can take the form of a plasmid, phage, cosmid, YAC, eukaryotic expression vector such as a DNA vector, Pichia pastoris, or a virus vector such as for example, baculovirus vectors, retroviral vectors or adenoviral vectors, and others known in the art. The cloned gene may optionally be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences , or sequences which may be inducible and/or cell type-specific. Suitable promoters will be known to a person with ordinary skill in the art. The expression construct will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. Among the vectors preferred for use include pGEX-YT3 , YPB11-ADH to name a few.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, electroporation, infection, and other methods known in the art and described in standard laboratory manuals such as Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. or Sherman et al . , 1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York. All documents cited herein supra and infra are hereby incorporated in their entirety by referece thereto. In a further embodiment, the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs. The host cell can be prokaryotic (for example, bacterial) , lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to rat and human) . Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers . These markers may be used to obtain successful transformants by selection. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods . The DNA sequence can be present in the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of CaSsklp, such as glutathione S- transferase, or a series of histidine residues also known as a histidine tag. The recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carlsbergensis , and Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts. Control sequences for yeast vectors are known in the art. Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , such as HEK293 cells , and NIH 3T3 cells, to name a few. Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV) , adenovirus (ADV) , bovine papilloma virus (BPV) , and cytomegalovirus (CMV) . Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate. Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequences are known in the art. The transformed or transfected host cells can be used as a source of DNA sequences described above. When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein described below.
In another embodiment, the present invention relates to a CaSsklp protein having an amino acid sequence corresponding to SEQ ID NO: 2 (GenBank accession no. AF084608) and encompassing 674 amino acids or any allelic variation thereof or biologically active derivative thereof .
A polypeptide or amino acid sequence derived from the amino acid sequences mentioned above, refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 amino acids, and more preferably at least 8- 10 amino acids, and even more preferably at least 11- 15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence .
A "biologically active derivative thereof" is a CaSsklp that is modified by amino acid deletion, addition, substitution, or truncation, or that has been chemically derivatized, but that nonetheless functions in the same manner as the protein of SEQ ID NO: 2. For example, it is known that substitutions of aliphatic amino acids such as alanine, valine, and isoleucine with other aliphatic amino acids can often be made without altering the structure or function of a protein. Similarly, substitution of aspartic acid for glutamic acid, in regions other than the active site of an enzyme, are likely to have no appreciable affect on protein structure or function. The term "fragment" is meant to refer to any polypeptide subset. Fragments can be prepared by subjecting C. albicans proteins to the action of any one of a number of commonly available proteases, such as trypsin, chymotrypsin or pepsin, or to chemical cleavage agents, such as cyanogen bromide. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire CaSSKl or to a fragment thereof . A protein or peptide is said to be 'substantially similar' if both molecules have substantially similar amino acid sequences, preferably greater than about 80% sequence identity, or if the three-dimensional backbone structures of the molecules are superimposable, regardless of the level of identity between the amino acid sequences. Thus, provided that two molecules possess similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequences of amino acid residues are not identical. The term
'analog' is meant to refer to a protein that differs structurally from the wild type CaSsklp, but possesses similar activity.
A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system. In addition the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants for example.
As noted above, the methods of the present invention are suitable for production of any polypeptide of any length, via insertion of the above- described nucleic acid molecules or vectors into a host cell and expression of the nucleotide sequence encoding the polypeptide of interest by the host cell. Introduction of the nucleic acid molecules or vectors into a host cell to produce a transformed host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods . Such methods are described in many standard laboratory manuals, such as Davis et al . , Basic Methods In Molecular Biology (1986) . Transformations into yeast are typically carried out according to the method of Van Solingen et al . , 1977, J. Bact . , 130, 946 and Hsiao et al . 1979, Proc Natl Acad Sci USA 76, 3829-3833. Once transformed host cells have been obtained, the cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support host cell growth. Recombinant polypeptide-producing cultivation conditions will vary according to the type of vector used to transform the host cells. For example, certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals or inducing agents to the cell growth medium, to initiate the gene expression resulting in the production of the recombinant polypeptide. Thus, the term "recombinant polypeptide-producing conditions," as used herein, is not meant to be limited to any one set of cultivation conditions . Appropriate culture media and conditions for the above-described host cells and vectors are well-known in the art. Following its production in the host cells, the polypeptide of interest may be isolated by several techniques. To liberate the polypeptide of interest from the host cells, the cells are lysed or ruptured. This lysis may be accomplished by contacting the cells with a hypotonic solution, by treatment with a cell wall-disrupting enzyme such as lysozyme, by sonication, by treatment with high pressure, or by a combination of the above methods . Other methods of bacterial cell disruption and lysis that are known to one of ordinary skill may also be used. Following disruption, the polypeptide may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The polypeptide may then be purified by well known isolation techniques . Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography.
The recombinant or fusion protein can be used as a diagnostic tool and in a method for producing antibodies against CaSsklp, detectably labeled and unlabeled, or as a bait protein in the yeast 2-hybrid assay to isolate proteins which interact with CaSsklp.
The transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit CaSsklp function, such as host proteins or chemically derived agents or natural or synthetic drugs and other proteins which may interact with the cell to down- regulate or alter the expression of CaSsklp, or its cofactors.
In another embodiment, the present invention relates to monoclonal or polyclonal antibodies specific for the above-described recombinant proteins (or polypeptides) . For instance, an antibody can be raised against a peptide described above, or against a portion thereof of at least 10 amino acids, perferrably, 11-15 amino acids. Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or polypeptide) of the present invention, or a unique portion thereof . Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986) .
The level of expression of CaSsklp, can be detected at several levels. Using standard methodology well known in the art, assays for the detection and quantitation of CaSSKl RNA can be designed, and include northern hybridization assays, in si tu hybridization assays, and PCR assays, among others. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) , or Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. for general description of methods for nucleic acid hybridization. Polynucleotide probes for the detection of CaSSKl RNA can be designed from the sequence available at accession number AF084608. For example, RNA isolated from samples can be coated onto a surface such as a nitrocellulose membrane and prepared for northern hybridization. In the case of in situ hybridization of biopsy samples for example, the tissue sample can be prepared for hybridization by standard methods known in the art and hybridized with polynucleotide sequences which specifically recognize CaSSKl RNA . The presence of a hybrid formed between the sample RNA and the polynucleotide can be detected by any method known in the art such as radiochemistry, or immunochemistry, to name a few.
One of skill in the art may find it desirable to prepare probes that are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. In other cases, it may be desirable to use two sets of probes simultaneously, each to a different region of the gene. While the exact length of any probe employed is not critical, typical probe sequences are no greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.
The DΝA sequence of CaSSKl can be used to design primers for use in the detection of CaSSKl using the polymerase chain reaction (PCR) or reverse transciption PCR (RT-PCR) . The primers can specifically bind to the CaSSKl cDΝA produced by reverse transcription of CaSSKl RΝA, for the purpose of detecting the presence, absence, or quantifying the amount of CaSSKl RΝA by comparison to a standard. The primers can be any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18-25 nucleotides homologous or complementary to a region of the CaSSKl sequence. Reagents and controls necessary for PCR or RT-PCR reactions are well known in the art. The amplified products can then be analyzed for the presence or absence of CaSSKl sequences, for example by gel fractionation, by radiochemistry, and immunochemical techniques. This method is advantageous since it requires a small number of cells. Once CaSSKl is detected, a determination whether the cell is overexpressing or underexpressing CaSSKl can be made by comparison to the results obtained from a normal cell using the same method. Decreased CaSSKl may be an indication of reduced virulence of the infecting yeast, or an indication that tissue-specific or site- specific expression of the gene is reduced.
In another embodiment, the present invention relates to a diagnostic kit for the detection of
CaSSKl RNA in cells, said kit comprising a package unit having one or more containers of CaSSKl oligonucleotide primers for detection of CaSSKl by PCR or RT-PCR or CaSSKl polynucleotides for the detection of CaSSKl RNA in cells by in si tu hybridization or northern analysis, and in some kits including containers of various reagents used for the method desired. The kit may also contain one or more of the following items: polymerization enzymes, buffers, instructions, controls, detection labels. Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods .
In a further embodiment, the present invention provides a method for identifying and quantifying the level of CaSSKl present in a particular biological sample. Any of a variety of methods which are capable of identifying (or quantifying) the level of CaSSKl in a sample can be used for this purpose.
Diagnostic assays to detect CaSSKl may comprise a biopsy or in si tu assay of cells from an organ or tissue sections, as well as an aspirate of cells from a tumour or normal tissue. In addition, assays may be conducted upon cellular extracts from organs, tissues, cells, urine, or serum or blood or any other body fluid or extract.
When assaying a biopsy, the assay will comprise, contacting the sample to be assayed with a CaSsklp ligand or substrate, natural or synthetic, or an antibody, polyclonal or monoclonal, which recognizes CaSsklp, or antiserum capable of detecting CaSsklp, and detecting the complex formed between CaSsklp present in the sample and the CaSsklp ligand, substrate, or antibody added.
CaSsklp ligands or substrates include for example, a downstream component in the CaSSKl pathway, a substrate for phosphorylation by CaSsklp, or an CaSsklp interacting protein, in addition to natural and synthetic classes of ligands and their derivatives which can be derived from natural sources such as animal or plant extracts.
CaSsklp ligands or anti-CaSsklp antibodies, or fragments of ligand and antibodies capable of detecting CaSsklp may be labeled using any of a variety of labels and methods of labeling for use in diagnosis and prognosis of disease associated with candidiasis. Examples of types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non- radioactive isotopic labels, and chemiluminescent labels .
Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha- glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc . Examples of suitable radioisotopic labels include 3H, niln, 125I, 32P, 35S, 14C, 57To, 58Co, 59Fe, 75Se, 152Eu, 90Y, 67Cu, 21Ci, 211At, 212Pb, 47Sc, 109Pd, X1C, 19F, 123I, etc.
Examples of suitable non-radioactive isotopic labels include 157Gd, 5Mn, 162Dy, 52Tr, 46Fe, etc. Examples of suitable fluorescent labels include a 152Eu label, a fluorescein label, an isothiocyanate label , a rhodamine label , a phycoerythrin label , a phycodyanin label, an allophycocyanin label, a fluorescamine label, etc. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, etc. Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to ligands and to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H. , et al . , 1976 ( Clin . Chim. Acta 70, 1-31), and Schurs, A. H. W. M., et al . 1977 ( Clin . Chim Acta 81, 1-40). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, and others, all of which are incorporated by reference herein.
The detection of the antibodies (or fragments of antibodies) of the present invention can be improved through the use of carriers. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides , agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to CaSsklp. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody, or will be able to ascertain the same by use of routine experimentation.
The ligands or antibodies, or fragments of antibodies or ligands of CaSsklp discussed above may be used to quantitatively or qualitatively detect the presence of CaSsklp. Such detection may be accomplished using any of a variety of immunoassays known to persons of ordinary skill in the art such as radioimmunoassays, immunometic assays, etc. Using standard methodology well known in the art, a diagnostic assay can be constucted by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocelluolose membrane) , antibodies specific for CaSsklp or a portion of CaSsklp, and contacting it with a sample from a person suspected of having a
CaSSKl related disease. The presence of a resulting complex formed between CaSsklp in the sample and antibodies specific therefor can be detected by any of the known detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry. A good description of a radioimmune assay may be found in Laboratory Techniques and Biochemistry in Molecular
Biology, by Work, T.S., et al . North Holland Publishing Company, N.Y. (1978) , incorporated by reference herein. Sandwich assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.
The diagnostic methods of this invention are predictive of patients suffering from candidiasis, periodontitis, oral ulceration, or esophagitis in HIV- infected patients, candidal vaginitis, and bloodstream candidiasis .
The protein can be used to identify inhibitors of CaSsklp activity. Using a phophorylation asssay such as those known in the art, natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of CaSsklp autophosphorylation or phosphorylation activity. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a decrease in the activity of CaSsklp is detected. Inhibitors may include agents or drugs which either bind or sequester CaSsklp substrate (s) or cofactor (s), or inhibit the CaSsklp itself, directly, for example by irreversible binding of the agent or drug to CaSsklp, or indirectly, for example by introducing an agent which binds the CaSsklp substrate. Agents or drugs related to this invention may result in partial or complete inhibition of CaSsklp activity. Inhibitors of CaSsklp may be used in the treatment or amelioration of candidiasis and diseases associated with candidiasis.
Agents which decrease CaSSKl RNA include, but are not limited to, one or more ribozymes capable of digesting CaSSKl RNA, or antisense oligonucleotides capable of hybridizing to CaSSKl RNA such that the translation of CaSSKl RNA is inhibited or reduced resulting in a decrease in the level of CaSSKlp . These antisense oligonucleotides can be administered as DNA, as DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Kanoda, Y. et al . , 1989, Science 243, 375) or as part of a vector which can be expressed in the target cell such that the antisense DNA or RNA is made. Vectors which are expressed in particular cell types are known in the art, for example, for the mammary gland, please see Furth, (1997) (J". Mammary Gland Biol . Neopl . 2 , 373) for examples of conditional control of gene expression in the mammary gland. Alternatively, the DNA can be injected along with a carrier. A carrier can be a protein such as a cytokine, for example interleukin 2, or polylysine-glycoprotein carrier. Such carrier proteins and vectors and methods of using same are known in the art. In addition, the DNA could be coated onto tiny gold beads and said beads introduced into the skin with, for example, a gene gun (Ulmer, J. B. et al . , 1993, Science 259, 1745).
Alternatively, antibodies, or compounds capable of reducing or inhibiting CaSsklp, that is reducing or inhibiting either the expression, production or activity of CaSsklp, such as antagonists, can be provided as an isolated and substantially purified protein, or as part of an expression vector capable of being expressed in the target cell such that the
CaSsklp-reducing or inhibiting agent is produced. In addition, co-factors such as various ions, i.e. Ca2+ or factors which affect the stability of the enzyme can be administered to modulate the expression and function of CaSsklp. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, oral, rectal, or parenteral (e.g. intravenous , subcutaneous , or intramuscular) route. In addition, CaSsklp-inhibiting compounds may be incorporated into biodegradable polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the CaSsklp-inhibiting compound is slowly released systemically. The biodegradable polymers and their use are described, for example, in detail in Brem et al . (1991) J. Neurosurg. 74, 441-446. These compounds are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of CaSsklp.
Similarly, agents which are capable of negatively affecting the expression, production, stability or function of CaSsklp, are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of CaSsklp. An amount is said to be sufficient to "effect" the inhibition or induction of CaSsklp if the dosage, route of administration, etc. of the agent are sufficient to influence such a response . In providing a patient with agents which modulate the expression or function of CaSsklp to a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.
A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient . Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences [16th ed. , Osol, A. ed., Mack Easton PA. (1980)]. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.
Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the compounds . The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) - microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres , microemulsions, nanoparticles , and nanocapsules or in macroemulsions . Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) .
The present invention also provides kits for use in the diagnostic or therapeutic methods described above. Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention.
The kits of the invention may comprise one or more of the following components, one or more compounds or compositions of the invention, and one or more excipient, diluent, or adjuvant.
In another embodiment, the present invention describes a C. albicans strain which contains a deletion of CaSSKl response regulator gene. The strain Δcasskl is avirulent and does not form hyphae on solid inducing media. Δcasskl, similar to wild type, is able to form hyphae in liquid media, however, unlike wild type, the hyphae flocculate. The Δcasskl strain may function as a gene or gene product delivery sytem. For example, it is envisioned that an antigen of interest could be delivered to an organ which is naturally invaded by Δcasskl in a patient, animal or plant where the antigen can provide benefit . The antigen can be introduced into Δcasskl in a second plasmid. Alternatively, a second plasmid could be used to provide a source of vaccine antigen for pathogens found in organs naturally invaded by C. albicans such as a systemic invasion, or kidney, lung, central nervous system, eye, to name a few. In the genitourinary tract, expression of spermicides by Δcasskl transformed with a desired gene could provide a cheap and infrequent method of contraception.
Δcasskl represents a safe delivery vehicle and is advantageous because it can carry one or more compounds and can be genetically engineered to carry- one or more nucleic acid molecules capable of effecting gene therapy and/or of encoding one or more proteins and/or RNA molecules. The compound of interest can be carried by Δcasskl within the yeast cell, on the yeast membrane surface, spanning the yeast membrane, withing the yeast periplasm, and combinations thereof . At least some of the compound of interest remains associated with the yeast at least until the yeast reaches its target, or site of action (e.g. the bloodstream, interstitial tissue, or a cell) , at which point it is also possible that a compound carried by the yeast may be released. As used herein, a compound capable of protecting an animal or plant from disease is a compound that when administered to an animal or plant can prevent a disease from occuring and/or cure or alleviate disease symptoms or cause. Examples of diseases from which to protect an animal or plant include, but are not limited to, infections, genetic defects and other metabolic disorders . Such classes of diseases can lead to abnormal cell growth (e.g., benign or malignant neoplasia, hyperplastic syndromes) , degenerative processes, and/or immunological defects as well as to a number of other disorders .
In accordance with the present invention, compounds included in Δcasskl vehicles can have a variety of functions . Δcasskl vehicles of the present invention preferably include compounds capable of stimulating an immune response, compounds capable of suppressing an immune respone, toxic compounds, compounds capable of inhibiting transcription of a gene, compounds capable of inhibiting translation of a gene, compounds capable of inhibiting the ability of an infectious agent to produce progeny, compounds capable of replacing a defective gene, compounds capable of replacing a defective protein (including nucleic acid molecules capable of encoding such proteins and mimetopes of such proteins) and/or biological response modifiers (e.g., cytokines, such as lymphokines and monokines, as well as other growth modulating factors), and mixtures thereof. Examples of such compounds include, but are not limited to, antibiotics, antibodies, antifungal compounds, antigens, antiparasite compounds, antisense compounds, antiviral compounds, chemotherapeutic agents , cytokines, growth modulating factors (including both growth stimulants and suppressants), herbicides, hormones, immunosuppressants, nucleic acid-based drugs (e.g., DNA- or RNA-based drugs), nucleic acid molecules comprising coding regions, nucleic acid molecules comprising regulatory sequences, nucleoside analogs, other oligonucleotides, peptide analogs, peptides, pesticides, prodrugs (e.g., compounds that are activated at the site of action), other proteins, ribozymes, steroids, toxins, and/or vitamins.
Cell types naturally targeted by Δcasskl include, but are not limited to, cells of granulocytic- monocytic lineage, B lymphocytes and T lymphocytes as well as cells in the epithelial regions of an animal . Alternatively, the Δcasskl can be engineered such that it targets specific cell types by positioning a cell- binding element (e.g., antibodies, receptors, and receptor ligands) in the membrane of Δcasskl. Such an element is preferably absent from, or found in only small amounts, on cell types that are not targeted for delivery.
The present invention includes the delivery of a composition comprising Δcasskl of the present invention to an animal or to cell in culture. Such compositions can be delivered to an animal either in vivo or ex vivo, or can be delivered to cells in vitro . Such administration can be systemic, mucosal, and/or proximal to the location of the targeted cell type. Examples of routes to administer yeast in vivo include aural, bronchial, genital, inhalatory, nasal, ocular, oral, parenteral, rectal, topical, transdermal , and urethral routes .
Ex vivo delivery refers to a method that includes the steps of contacting a population of cells removed from an animal with a composition comprising Δcasskl of the present invention under conditions such that the yeast is adsorbed by targeted cell types and returning the contacted cells to the animal . Such a delivery method is particularly useful in the treatment of cells involved in hematopoiesis and the immune response as well as in the treatment of tumors .
In vitro delivery refers to the delivery of Δcasskl of the present invention to a population of cells (which can also include tissues or organs) in culture .
Methods to prepare and administer compositions via these routes are well known to those skilled in the art. A preferred single dose of a yeast vehicle of the present invention is from about 1 x 105 to about 5 x 107 yeast cell equivalents per kilogram body weight of the organism being administered the composition.
For vaccine use, the Δcasskl strain of the invention can be used directly in vaccine formulations, or lyophilized, as desired, using lyophilization protocols well known to the artisan. Lyophilized compositions will typically be maintained at about 4°C. When ready for use the lyophilized composition is reconstituted in a stabilizing solution, e.g., saline or comprising Mg++ and HEPES, with or without adjuvant, as further described below. Thus, Δcasskl vaccine of the invention contains as an active ingredient an immunogenically effective amount of a nonvirulent C. albicans as described herein. The Δcasskl may be introduced into a host, particularly humans, with a physiologically acceptable carrier and/or adjuvant. Useful carriers are well known in the art, and include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration, as mentioned above. The compositions may contain pharmaceutically accepatable auxilliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.
Administration of the Δcasskl disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection) , by in ovo injection in birds, orally and by topical application of the yeast (typically carried in the pharmaceutical formulation) to an airway surface. Topical application of the yeast to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally) . Topical application of the yeast strain to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the yeast as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. As a result of the vaccination the host becomes at least partially or completely immune to C. albicans infection, or resistant to developing moderate or severe C. albicans infection.
The vaccine composition containing the Δcasskl of the invention are administered to a person susceptible to or otherwise at risk of C. albicans infection to enhance the individual ' s own immune response capabilities . Such an amount is defined to be a "immunogenically effective dose". In this use, the precise amount again depends on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1 x 105 to about 5 x 107 yeast cell equivalents per kilogram body weight of the organism being administered the composition. In any event, the vaccine formulations should provide a quantity of Δcasskl of the invention sufficient to effetively protect the patient against serious or life- threatening C. albicans infection.
In some instances it may be desirable to combine the Δcasskl vaccines of the invention with vaccines which induce protective responses to other agents .
Single or multiple administration of the vaccine compositions of the invention can be carried out. Multiple administration may be required to elicit sufficient levels of immunity. Levels of induced immunity can be monitored by measuring amount of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection. It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.
The following Materials and Methods were used in the Examples desribed below.
MATERIALS AND METHODS
Strains and growth media Candida albicans (strain SC5314) was routinely grown in YPD complex medium (1% yeast extract, 2% peptone, 2% glucose) , at 28 °C with shaking at 250 rpm for 14 hours. Cells were harvested from liquid medium by centrifugation at 4,000 x g for 10 minutes at 4°C. For RNA isolation, the same strain was grown in medium 199 containing Earle's salts and glutamine but lacking sodium bicarbonate (Gibco BRL) and buffered with 155 mM Tris-HCl either at pH 7.5 or 3.5. Cultures were inoculated to a density of 107 cells ml-1 into prewarmed medium and incubated at 28 °C or 37 °C with vigorous agitation for 3 hours. In all cases, stationary-phase cells grown at 28 °C in YPD were used as an inoculum.
Schizosaccharomyces pombe strain JM1303 (mcs4-13 cdc25-22 ura4-Dl8) (Shieh et al . , 1997, Genes Dev. 11, 1008-1022) was routinely grown in YE (3% dextrose, 0.5% yeast extract, 2% agar) at 25°C or in EMM minimal medium
(BIO101) for specific experiments at either 25° or 30°C. Saccharomyces cerevisiae strain TM187 (MATa. Ieu2 ura3 trpl slnl : :hisG sskl : : TRPl + pSSP25) (Maeda et al . , 1994) was routinely grown in CAD (0.67% YNB, 0.5% casamino acids, 2% dextrose) at 30°C. For specific experiments, it was grown in SD (0.67% YNB, 0.067% CSM- LEU-URA (BIO101) , 2% dextrose, 2% agar) or SG (0.67% YNB, 0.067% CSM-LEU-URA, 2% galactose [glucose-free grade, Sigma] , 2% agar) .
Escherichia coli strain DH5α, which was used for transformation and plasmid amplification, was grown at 37 °C in LB broth supplemented with 0.1 mg of ampicillin per ml of medium when necessary. E. coli strain LE392 was used for propagation of bacteriophage λEMBL3. The Candida albicans strains used in this work are listed in Table 1. All strains were routinely grown either in YPD complex medium (1% yeast extract, 2% peptone and 2% dextrose) or SD minimal medium (0.67% yeast nitrogen base, 2% dextrose) at 28°C. For specific experiments, strains were also grown on solid Spider medium (1% nutrient broth, 1% mannitol, 0.2% K2HP04) , solid synthetic low-ammonium- dextrose (SLAD) medium (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 2% dextrose and 50 μM ammonium sulfate as sole nitrogen source) , serum [10% fetal bovine serum (Gibco BRL)] and Medium 199 (M199) containing Earle's salts and glutamine but lacking sodium bicarbonate (Gibco BRL) and buffered with 155 mM Tris-HCl at pH 7.5 or pH 4.0. All liquid media were sterilized by filtration. Solid media were prepared by adding 1.5% agar as a final concentration (2% for SLAD plates) at 50°C after autoclaving. Prewarmed liquid media were inoculated to a density of 107 cells/ml and incubated either at 28°C or 37°C with vigorous agitation. On solid media, the appropriate dilutions of cells were plated to obtain approximately 60-80 colonies per plate. In all cases, stationary-phase cells grown at 28°C in YPD were used as an inoculum. Table 1
Strain Relevant Generation genotype time" (μ, hours) Source
CAF2 Δura3 ιmm434/ΔURA3 1 10+001 (12)
CAI4 Δura3 ιmm434/ΔURA3 ιmm434 ndh (12)
CSSKU-l Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hιsG-URA3-hιsG/Cα55 i ; 1 14±004 This work
CSSK'i-2 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hιsG-URA3-hιsG/Ca55A; 1 16±002 This work
CSSK12-1 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl isG/CaSSKl nd This work
CSSK12-2 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl
Figure imgf000047_0001
nd This work
CSSK21-1 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hisG/Δcasskl hιsG-URA3-hιsG 1 36±O 01 This work
CSSK21-2 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hisG/Δcasskl hιsG-URA3-hιsG 1 1 3333±±000022 This work
CSSK22-1 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hisG/Δcasskl hisG nd This work
CSSK22-2 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hisG/Δcasskl hisG nd This work
CSSK23-1 Δura3 ιmm434/ΔURA3 ιmm434 Δcassk 1 hisG/ΔCαSSΛ:./ URA3-hιsG 1 12±002 This work
CSSK23-2 Δura3 ιmm434/ΔURA3 ιmm434 Δcasskl hisG/ΔCαSS/ URA3-hιsG 1 15±003 This work
a Calculated in YPD Average of three independent experiments ±standard deviations b Not determined
(12) Fonzi and Irwm 1993, Genetics 134, 717-728
DNA manipulations
Plasmid DNA was extracted from E. coli DH5α cells according to the manufacturer's instructions (Plasmid
Midi Kit, Qiagen) . Restriction enzymes and T4 DNA ligase were obtained from Gibco-BRL and used with buffers provided by the supplier under the recommended conditions. Agarose gel electrophoresis of both DNA and RNA was performed according to standard protocols
(Sambrook et al . , 1989, Molecular Cloning: A Laboratory- Manual , 2nd edn. Cold Spring Harbor Laboratory Press, New York) . DNA samples were obtained from λEMBL3 clones according to the manufacturer's instructions (Lambda Mini Kit, Qiagen) . Construction of recombinant plasmids and selection of transformants were done by standard techniques (Sambrook et al . , 1989, supra) . Sequencing was carried out by the dideoxy chain termination method using an automatic sequencer (373A DNA sequencer, Applied Biosystems) . Standard molecular biology procedures for DNA manipulation were used (Sambrook et al . , 1989, supra) . Genomic DNA from all C. albicans strains was obtained according to the method described by Sherman et al . (1986, supra) . For southern blot analyses 4 μg of DNA per lane was loaded in a 0.8% agarose gel and transferred by capillarity to positively-charged nylon membranes (Hybond-N+ , Amersham) by standard protocols (Sambrook et al . , 1989, supra) . Probes were Digoxigenin- labelled by random priming (DIG DNA Labeling Kit, Boehringer Mannheim) , and hybridized and detected according to the chemiluminescent method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim) . Construction of the CaSSKl probe and screening of a λEMBL3 Candida albicans genomic library
A fragment of the CaSSKl gene was isolated by PCR amplification with the degenerate primers, 5'-AA(T/C) GT(A/G/T) TT(G/A) AT(A/T) GT(A/G/T) GAA GA(T/C) AA-3' and 5 ' -CAT (A/G)CA (A/T)CC CCA TTC (T/A/G)GT (T/A)AT TTT-3 ' . These primers were designed according to a pair of amino acid sequences conserved between Ssklp from S. cerevisiae and Mcs4 from S. pombe. These sequences, VLIVEDN (SEQ ID NO: 3) and KITE GCM
(SEQ ID NO: 4) are located at positions 505-512 and 642- 649 in Ssklp and at positions 363-370 and 500-507 in Mcs4, respectively, being separated in both cases by 145 amino acids . PCR amplification was performed with genomic DNA from C. albicans strain SC5314 as template. Thirty-two cycles of PCR were performed with the following steps: 45 seconds at 94'C; 1 minute at 50 °C and 2.5 minutes at 72 °C. The reaction yielded specifically a fragment with the predicted size of approximately 435 bp. The PCR product was subcloned into pCR2.1 (Invitrogen) to generate pSSKl, sequenced and the 447 bp DNA fragment identified as a partial C. albicans DNA homolog of SSKl and mcs4\ The PCR product was excised from pSSKl by BcoRI digestion, digoxigenin- labelled by non-radioactive random priming (DIG DNA Labelling Kit, Boehringer Mannheim) and, in turn, used as probe to screen a λEMBL3 C. albicans genomic library (Monod et al . , 1994, Mol . Microbiol . 13, 557-368), according to the protocol described by Sambrook et al . (1989, supra) . The detection of the positive clones was done according to the colorimetric method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim). Finally, a 2.77 kb EcoRI-Xbal fragment containing the entire ORF of CaSSKl was subcloned into pBR322 (Gibco-BRL) to generate pBR- CaSSKl . The low copy number pBR322 plasmid was used instead a higher copy number E. coli plasmid due to the toxicity of the C. albicans DNA CaSSKl-containing fragment when it is highly replicated in bacteria. Overexpression of CaSSKl in the S. pombe JM1303 strain The entire ORF of CaSSKl was amplified from pER-CaSSKl by PCR using the 5' oligonucleotide 5'- AAATAGGATCCCATGAATTTTCTCTATAAC-3 ' (SEQ ID NO: 5) and the 3' oligonucleotide 5 ' -CGTTGGATCCTTTCAAGCTTTGTTTAATCTTCG -3' (SEQ ID NO: 6), both of them containing the BamHl site (bolded) and confirmed by DNA sequencing. The resulting 2.04 kb product was digested with BamHI and cloned into the BamHI site of pBREP42 to generate pBREP42 -CaSSKl . The pBREP42 plasmid is a low copy number version of the pREP42 plasmid (Maundrell, 1993, Gene 123, 127-130), which was constructed by introducing the 5.50 kb PvuII-PvuII cassette [ura4+-nmtpromoter-MCS-nmtpoly.A signal -arsl] from pREP42 into the pBR322 digested with EcoRV/PshAI . The pREP42-mcs4 plasmid (Shieh et al . ,
1997 , supra) was used as a control. Each of these plasmids (pBREP42, pBREP42 -CaSSKl , pREP42 and pREP42- mcs4) was used to transform S. pombe strain JM1303 by electroporation (Prentice, 1991, Nucleic Acids Res . 20,
621) .
Expression of CaSSKl in the S. cerevisiae
TM187 strain To ensure a correct level of expression of CaSSKl in S. cerevisiae, we first constructed a low copy version of the pRS415 plasmid (Sikorski and Hieter, 1989, Genetics 122, 19-27) (which we named as pBRS415) and then, both the promotor and terminator of the SSKl gene from S. cerevisiae were introduced into pBRS415 to generate pBRS415-p/t. The pBRS415 plasmid was constructed by introducing a 4.17 kb Seal-Sail fragment which contains the cassette [LEU2-APSH4-CEN6] from pRS415 (Stratagene) in the pBR322 digested with Seal/ Sail . Next, the pBRS415-p/t plasmid was generated by introducing sequentially a 0.72 kb fragment that contains the promotor of SSKl and the 0.36 kb terminator sequence of SSKl , linked by a BamHI restriction site. The promotor sequence was obtained by PCR using the oligonucleotides 5 ' -AGGTCGACCCACTGCTGGATC-3 ' (SEQ ID NO: 7) and 5 ' -AGCGGATCCTTCCACAGTAACGC-3 ' (SEQ ID NO: 8). The terminator sequence was also obtained by PCR using the oligonucleotides 5 ' -TTTGGATCCACCGTGTAGAGGACATTATG- 3 ' (SEQ ID NO: 9 ) and 5 ' -ATAGGTACCCAGCTGGTG AATCCAAGCCCG- 3' (SEQ ID NO: 10) . Finally, the 2.04 kb PCR product that contains the entire ORF of CaSSKl was digested with BamHI and cloned into the only BamHI site of pBRS415-p/t to generate pBRS415- CaSSKl . The pSSK1222 plasmid (Maeda et al . , 1994, Nature, 369, 242-245) was used as a control. Each of these plasmids (pSSK1222, pBRS415, pBRS415-p/t and pBRS415-CaSSKl ) was used to transform S. cerevisiae strain TM187. Southern and Northern blot analysis Genomic DNA from C. albicans was obtained according to the method described by Sherman et al . (1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York) . Four micrograms of DNA per lane were typically loaded for Southern analyses. DNA was transferred by capillarity to positively-charged nylon membranes (Hybond-N+, Amersham) by standard protocols (Sambrook et al . , 1989, supra) and hybridized with the same probe that was used for screening the genomic library.
However, for Southern blots, the detection was carried out according to the chemiluminescent method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim) . Total RNA was isolated according to the protocol described by Sherman et al . (1986, supra) . Fifteen micrograms of total RNA per lane were loaded onto 1% formaldehyde agarose gels and transferred by capillarity to nylon membranes (Hybond-N, Amersham) using 20 x SSC by standard protocols (Sambrook et al . , 1989, supra) . For Northern analysis, a 1.85 kb EcoRI-Bglll fragment, which contains most of the ORF of CaSSKl , was labelled by random priming using the DNA Labelling Beads (-dCTP) kit and [32P] -dCTP (3000 Ci mmol-1) (Pharmacia Biotech). Hybridization was carried out at 68 °C in a solution containing 0.5 M NaH2P04 (pH 7.2), 7% SDS and 1 mM EDTA. After hybridization, the blots were washed twice with 2 x SSC containing 0.2% SDS at 68 °C for 20 minutes. The
0.24-9.5 kb RNA ladder (Gibco-BRL) was used for sizing in formaldehyde agarose gels.
Pulsed-field gel electrophoresis
Chromosomal DNA was resolved by pulsed-field gel electrophoresis according to the protocol described by Wickes et al . (1991, Infect . Immun. 59, 1762-1771), in a Bio-Rad CHEF DRII unit with a linear ramp switch time of 300 seconds to 1400 seconds for 144 hours at 13 °C. Gels were stained for 30 minutes with ethidium bromide (0.5 μg ml"1 in water) , destained in distilled water for 30 minutes and then photographed. The gels were blotted onto nylon membranes by alkaline transfer for 16 hours. The 1.85 kb EcoRI-Bglll fragment was radioactively labelled by random priming and used as a probe. Hybridization was carried out as described above.
Homology searches, sequence analysis and multiple augments Homology searches were performed using the BLAST network service (Altschul et al . , 1990, J". Mol . Biol . 215, 403-410). Motif searches were performed using the PROSITE and BLOCKS network databases . The computer analysis of sequences was performed using the DNA Strider 1.2. Sequences were aligned by the CLUSTAL program (Higgings and Sharp, 1988, Gene 73, 237-244).
Construction of plasmids used for C. albicans transformation. To obtain Acasskl mutants, the pBRl plasmid was constructed, which carries a cassette designed to delete most of the ORF of CaSSKl . To construct this plasmid, a 3.80 kb Stul-Bglll DNA fragment from pMB7 (Fonzi, and irwin, 1993, Genetics 134, 717-728), which contains the hisG-URA3-hisG cassette, was used to replace most of the ORF of CaSSKl (Fig. 3A) . The pBRl plasmid was linearized by digestion with Aatll which cuts once in the plasmid outside of the cassette, and approximately 2 μg of
DNA was used to transform the Ura~ C. albicans strain CAI4 by electroporation (Thompson, et al . , 1998, Yeast 14, 565- 571) . Electroporation was chosen for transformation experiments since the most commonly used LiAc procedure (Gietz, et al . , 1991, Nucleic Acids Res . 20, 1425) consistently failed to yield transformants, even when a high amount of DNA (up to 30 μg) was used. Transformed cells were selected as Ura+ on SD minimal medium and spontaneous Ura"~ derivates from a Ura+ independent clone were selected on SD medium containing 5 ' -fluoroorotic acid (1 mg/ml) and uridine (25 μg/ml) . The transformants were then used to delete the second allele of CaSSKl . In order to obtain a reconstituted strain with one CaSSKl allele, the pBR2 vector was designed. To construct this plasmid, a 2.37 kb Xbal-Bglll DNA fragment from pMB7 that contains the URA3-hisG cassette was introduced into the only Spel/BamHI sites of pBR- CaSSKl, which are located downstream of CaSSKl , generating the CaSSKl -URA3-hisG cassette (Fig. 3B) . The pBR2 plasmid was linearized and used to transform a
Ura" Acasskl null strain as described above. The construction of the expression plasmids pLJ19 and pCCal (kindly provided by Dr. D. Harcus) , which carry the CPH1 and CPP1 genes of C. albicans respectively under the control of the ADHl promoter, was previously described (Csank et al . , 1997, Mol . Biol . Cell 8, 2539-2551; Csank et al, 1998, Infect . Immun . 66, 2713-2721) . To construct the plasmid pYPBl-ADHpt-HOGl, the encoding region of CaHOGl flanked by Bglll sites was amplified by PCR with the 5' oligonucleotide 5 ' -GCAGATCTGAAAATGTCTGCAGATGGAG-3 ' (SEQ ID NO: 11) and the 3' oligonucleotide 5'- TTAGATCTTTGAAGATTAAGCTCCGTTGGC-3' (SEQ ID NO: 12) using the pBSK8 plasmid containing the CaHOGl gene as a template . The flanking regions of the PCR product were confirmed by sequencing, digested with Bglll and inserted into the Bglll site of the plasmid pYPBl-ADHpt (kindly provided by D. Harcus) containing the ADHl promoter, the C. albicans URA3 as a selectable marker, and an autonomously replicating sequence (Csank et al . , 1997, 1998, supra; Leberer et al . , 1997, Curr. Biol . 7, 539-546). Determination of generation time. Preinoculum cultures of each strain were always prepared in YPD medium for 24 h at 28° C. The optical density of precultures was determined at 600 nm (OD600) , and 250 ml flasks containing 50 ml of prewarmed fresh medium (YPD or M199 pH 4.0) was inoculated to a final OD600 of 0.1 with the appropriate preinoculum and incubated at 28° C and 200 rpm. The OD600 was measured every 1.5 hours until the stationary phase of the growth curve was reached. The generation time (μ) during the log phase (exponential growth) was determined by the formula μ = (t£ - t0) /n, where n is the number of generations calculated from the formula n = (logNf- logN0) /log2 in which N£ is the number of cells at the end of the time period (t£) and N0 is the number of cells at the beginning of the time period (t0) . The generation times calculated for each strain are the averages of three independent experiments .
Animal model of hematogenously disseminated candidiasis. The C. albicans strains used in these experiments included a parental control with only one functional URA3 allele (strain CAF2 ) and five strains in which either one (CSSKll-1, CSSK23-1 and CSSK23-2) or both alleles (CSSK21-1 and CSSK21-2) have been deleted. CSSK23 strains were included in all experiments to ensure that all phenotypic traits observed with the CSSK21 strains were due solely to the CaSSKl mutation rather than to unrelated mutations that may have occurred during construction of the Acasskl null strains. All strains were grown, harvested, resuspended to a density of 2 x 106 cells per ml in PBS (pH 7.5), and 0.5 ml (106 cells) of this cell suspension was injected intravenously per mice as previously described (8) . Concomitantly, each C. albicans strain was used to inoculate an additional 15 mice. Five members from each group were sacrificed after 24, 48, and 72 h postinfection to quantitate the CFU/g of tissue and for histological examination as was already described (Calera et al . , 1999, Infect . Immun . 67, 4280-4284) .
Example 1 Isolation of a putative response regulator gene of C. albicans (CaSSKl)
The CaSSKl gene was cloned following a PCR-based approach as described in the Material and Methods section. A PCR reaction using an appropriate pair of degenerate primers was performed and a 447 bp PCR product was identified as a partial DNA sequence of CaSSKl . This PCR fragment was then used to isolate the entire CaSSKl gene.
In order to identify the CaSSKl gene to a specific restriction fragment of genomic DNA from C. albicans, a Southern blot analysis at high stringency was performed after digestion of genomic DNA with several restriction enzymes, including digestion with Bglll as a reference, since it cleaves the 447 bp PCR fragment at 147 bp from its 3 '-end. Using this PCR product as a probe, we detected two differentially stained Bglll-Bglll fragments of 3.45 kb and 1.35 kb from genomic DNA. We expected differences in the intensity of each band since a different amount of the labelled probe hybridized with each (Data not shown) . The differential staining indicated that the 5 ' -end of the gene should be located in the 3.45 kb fragment (larger hybridizing probe and stronger signal) and the 3 ' -end in the 1.35 kb fragment (smaller hybridizing probe and weaker signal) . After screening a λEMBL3 C. albicans genomic library, 6 clones were selected for further analysis. These λEMBL3 clones revealed that they each carried the same C. albicans DNA fragment as we observed upon restriction analysis with Bg-lII. Also, each one contained the two expected hybridizing fragments as shown by Southern analysis. After subcloning and sequencing both fragments from one of the clones, we obtained the entire open reading frame (ORF) of CaSSKl . Finally, in order to obtain the entire ORF of CaSSKl in one piece, a 2.77 kb EcoRl-Xbal fragment from of one the λEMBL3 clones was subcloned into pBR322. The 3.0 kb DNA sequence contains the 591 bp 5' upstream promotor region, the 2022 bp ORF and the 497 bp 3 ' downstream noncoding region.
In order to corroborate the size of the gene as well as its expression in both yeast and hyphae, we carried out a Northern blot analysis using RNA obtained from C. albicans grown in medium 199 (pH 3.5) at 28°C to induce yeast-like growth and in medium 199 (pH 7.5) at
37 °C to induce germ tube formation. We detected a 2.3 kb mRNA (approximately) which matches the size of the gene. In addition, CaSSKl was expressed under both growth conditions. This indicates that the expression of the gene is not dependent on the growth morphology (Figure
1A) . Also, we mapped CaSSKl to chromosome 1 (Figure IB) .
Example 2 Structure of the C. albicans CaSSKl gene and its encoded protein CaSSKl has a 2022 bp ORF. In the 5 ' noncoding region, upstream from the ATG codon ending at position -89, there is a long AT-rich sequence (84% A+T) in which two putative TATAAA elements were found between positions -17 to -22 and -28 to -33, upstream from the start codon, respectively. Also two CAAT motifs were found at -309 and -329 positions, respectively. In addition, upstream of the AT-rich sequence between positions -166 and -173, we found a sequence (5'- TGAGGGGG-3 ' ) that matches the consensus motif (5'- TNAGGGGG-3 ' ) of the stress response elements (STRE) from yeast (Shϋller et al . , 1994). However, although CaSSKl could function under stress conditions, as it was inferred from its similarity with other response regulators such as Mcs4 (Shieh et al . , 1997, supra) , we do not know whether the STRE-like sequence modulates the expression of CaSSKl .
In the 3 ' noncoding region, there is a putative AATAAA polyadenylation signal between positions 2071 to 2076 downstream from the stop codon. Within the encoding region, no introns were identified, similar to most of the C. albicans genes characterized thus far. The 2022 bp ORF of CaSSKl encodes a protein of 674 amino acids with a predicted molecular mass of 73.5 kDa and a pi of 9.5. The codon usage frequency in CaSSKl corresponds to the low expressed genes in C. albicans (Lloyd and Sharp, 1992, Nucleic Acids Res . 20, 5289-5295). It also shows four CUG codons that are presumably translated as Ser instead Leu (Santos and Tuite, 1995, Nucleic Acids Res . 23, 1481-1486). Of relevance is the high content of asparagine residues of CaSsklp (13.3% of its molecular weight) . In addition, most of the asparagine residues appear clustered in twos or in short stretches of asparagines along the entire length of the protein (Data not shown) . Similarly, stretches of asparagine residues have also been found in all of the hybrid histidine kinases from D. discoideum described thus far; however their function remains unknown (Wang et al . , 1996, EMBO J. 15, 3890-3898; Schuster et al . , 1996, EMBO J. 15, 3880-3889; Zinda et al . , 1998, Dev. Biol . 196, 171-183). Analysis using Kyte-Doolittle and Goldman algorithms showed no extensive hydrophobic stretch of amino acids which indicates that CaSsklp is not a membrane-bound protein, similarly to all other eukaryotic and prokaryotic response regulators described thus far. Finally, in the C-terminus of CaSsklp, there is a tripeptide NKA which resembles the peroxisomal targeting signal type 1 (PTSl) consensus sequence (Elgersma et al . , 1996, J. Biol . Chem . 271, 26375-26382). The PTSl motifs have been involved in targeting proteins to peroxisomes and are located in the extreme C-termini of the majority of the peroxisomal matrix proteins. Their specificity is remarkable considering the small size and relaxed consensus sequence. The C-terminal PTSl has been studied by extensive mutational analyses (Elgersma et al . , 1996, supra) . These studies revealed a high degeneracy of the PTSl consensus sequence and suggests that additional domains in these proteins may be of importance to determine whether or not a certain PTSl is recognized by the components of the peroxisomal import machinery (Elgersma et al . , 1996, supra) . However, whether or not this motif is used as a PTSl sequence in CaSsklp remains to be elucidated.
Example 3 Homology of the amino acid sequence of CaSsklp with other related response regulators A computer search in the GenBank database using the BLASTP program (Altschul et al . , 1990, supra) revealed that the CaSsklp C-terminal end shares some homology with many prokaryotic response regulators and the response regulator domains of many eukaryotic hybrid histidine kinases, but the highest similarity is shared with the C-terminus of the eukaryotic Ssklp and Mcs4 response regulators from S. cerevisiae [P(N), 3 X 10"53; 61.3% identity and 72.8% similarity] and S. pombe [P(N), 8 X
10"51; 59.5% identity and 71.6% similarity], respectively. However, CaSsklp does not show similarity at its N-terminus with Ssklp, but a significant similarity [P(N), 5 x 10~6; 28.7% identity and 50.0% similarity] is shared with the N-terminus of Mcs4. Thus, the highest overall similarity of CaSsklp was shared with Mcs4 (31% identity) rather than with Ssklp (22.5% identity) .
The C-terminal sequences from both prokaryote and eukaryote response regulators show highly conserved residues. In the response regulator domains, these residues include a pair of aspartates near the N- terminus (one aspartate in eukaryotes) , an aspartate motif which accepts a phosphate and a motif near the C- terminus which contains a key lysine (Parkinson, 1993, Cell 73, 857-871) . In the response regulator domain of CaSsklp (residues 502-674) , and by inference from sequence similarities, the Asp556 should be the predicted site of phosphorylation. The Asp513 should be one of the pair of aspartates that are conserved among prokaryotic response regulators but apparently not in eukaryotic cells, which appears as ED instead of DD. The Lys638 should be the other conserved residue.
Example 3 Complementation analysis of a S. pombe mcs4 mutant with CaSSKl In S. pombe Mcs4 functions in adapting cells to oxidative, heat, and nutritional stresses by coordinating environmental responses with meiosis and mitosis (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1997, supra; Shiozaki et al . , 1997, Mol . Biol . Cell 8, 409-419). Since Mcs4 and CaSsklp share structural similarity, it could be also possible that CaSsklp plays a similar function in C. albicans . Thus, to determine whether CaSSKl can complement the absence of the mcs4+ functional gene in S. pombe, CaSSKl was overexpressed in an S. pombe mcs4 mutant. To perform this experiment we took into consideration the previously reported observation that the S. pombe mcs4-13 cdc25-22 double mutant cells undergo cell cycle arrest at 30°C but not at 25°C.
However, at both temperatures the mcs4 and cdc25 single mutants were able to proliferate normally at 30°C and the overexpression of mcs4* in the S. pombe mcs4-13 cdc25-22 double mutant restored the normal growth of this strain at 30°C (Shieh et al . , 1997, supra) . Thus, if CaSSKl was able to complement the function of mcs4+ , the overexpression of CaSSKl in the S. pombe JM1303 strain will allow the cells to grow normally at 30°C. The results showed that CaSSKl does not complement the lack of mcs4+ in the S. pombe mcs4-13 cdc25-22 double mutant since it was not able to grow at 30°C (Figure
2A) .
Example 4 Complementation analysis of a S. cerevisiae SSKl mutant with CaSSKl It has been reported that Ssklp acts downstream of the Slnlp-Ypdlp osmosensor pathway of S. cerevisiae (Posas et al . , 1996, Cell 86, 865-875) . Thus, considering the high similarity that CaSsklp shares with the C-terminus of Ssklp, and that homologues of Slnlp and Ypdlp from S. cerevisiae also exist in C. albicans, it could be that CaSsklp is a component of the putative homologous CaSlnlp-CaYpdlp- CaSsklp pathway of C. albicans , which also could be involved in osmoregulation. To test this hypothesis, CaSSKl was expressed in a ssklA S. cerevisiae strain. However, since the deletion of SSKl in S. cerevisiae has no effect on growth, we used as a recipient an slnlA ssklA double mutant (strain TM187) which is able to overexpress the PTP2 tyrosine phosphatase under the control of the Ε>αAL1 promotor (Maeda et al . , 1994, supra) . Thus, upon expression of a SSKl functional gene in this strain, the overexpression of PTP2 in a medium containing galactose will keep certain proteins of the HOG pathway dephosphorylated turning off the synthesis of glycerol (Maeda et al . , 1994, supra) . However, in a medium containing glucose, the overexpression of PTP2 is repressed and the constitutive activation of the HOG pathway causes lethality (Maeda et al . , 1994, supra). Thus, if CaSSKl was able to complement the function of SSKl , the expression of CaSSKl in the S. cerevisiae TM187 strain will allow the cells to grow normally in the presence of galactose but not in presence of glucose. The results showed that CaSSKl does not complement the lack of SSKl in a S. cerevisiae SSKl mutant since it was able to grow in medium containing glucose (Figure 2B) . This result indicates that CaSSKl may have another function in C. albicans rather than osmoregulation, but this does not preclude the possibility that CaSsklp is a component of the putative phosphorelay pathway CaSlnlp-CaYpdlp-CaSsklp of C. albicans . This result is also consistent with the observation that a caslnlA C. albicans strain, contrary to a slnlA S. cerevisiae strain in which the disruption of S N1 is lethal, grows normally (but slowly) under osmostressing conditions (Nagahashi et al . , 1998, Microbiology 144, 425-532) .
Finally, even though a positive complementation could indicate that CaSSKl has a function similar to mcs4+ or SSKl , the non-complementation observed in both cases does not absolutely rule out a similar function for CaSSKl in C. alJbicans. In this context, we can not preclude that other factors, such as the differences in the codon frequency usage between S. pombe or S. cerevisiae and C. albicans, as well as the presence in the CaSSKl gene of four CUG codons, presumably translated as serine by C. albicans but as leucine by S. pombe and S. cerevisiae, could affect the level and efficiency of translation of a functional CaSsklp protein in these yeast.
Example 5 Chromosomal deletion of CaSSKl . It was previously shown that CaSSKl was unable to rescue the lack of SSKl or mcs4+ in S. cerevisiae or S. pombe respectively, which suggested that despite their structural homology, they may not be functional homologs (Calera and Calderone, 1999, Yeast 15) . Thus, to further investigate the function of CaSSKl in the growth, morphogenesis and virulence of C. albicans, we used the urablaster technique (Fonzi and Irwin, 1993, supra) to obtain Acasskl mutants. The hisG- URA3-hisG cassette was used to replace a 1.47 kb fragment of CaSSKl that includes the encoding region for the conserved putative aspartate residue which is phosphorylated (Fig. 3A) . After the first round of transformation, Ura+ transformants were selected on SD minimal medium and several isolates were tested by southern blot to confirm this replacement. DNA from a CSSK11 representative isolate exhibited two hybridizing bands, a
3.46 kb Bglll-Bglll fragment characteristic of the parental strain and an additional fragment of 5.80 kb, consistent with the replacement of one allele of CaSSKl with the hisG- URA3-hisG cassette (Fig. 3C) . Two independent Ura+ transformants (CSSKll-1 and CSSK11-2) were used as parentals to obtain two independent Ura- segregants
(CSSK12-1 and CSSK12-2) . A representative CSSK12 intrachromosomal recombinant strain is shown by southern blot (Fig. 3C) . The 5.80 kb Bglll-Bglll fragment seen in the CSSK11 strains, which had contained the Acahkl : : hisG-
URA3-hisG disruption, was absent and a new 3.07 kb hybridizing fragment was present in the CSSK12 strains. The size of this fragment is consistent with the desired event, the loss of URA3 and one copy of hisG. Following the same protocol, a second round of transformation was performed and the remaining allele was disrupted to generate the strains CSSK21-1 and CSSK21-2. In Fig. 3C a representative
CSSK21 Ura+ isolate and a CSSK22 Ura" segregant are shown. Two independent strains reconstituted for one allele
(CSSK23-1 and CSSK23-2) were also constructed to ensure that the resulting phenotype was not due to extraneous mutations that could happen during transformation. To do that, both strains CSSK22-1 and CSSK22-2 were transformed with the cassette CaSSKl -URA3-hisG. This cassette allowed the integration event to occur between the remaining 5 ' -end ORF of CaSSKl and the hisG that had replaced 1.47 kb of the CaSSKl sequence in the CSSK22 strains (Fig. 3B) . Ura+ transformants were selected on SD minimal medium, and the integration of the transforming DNA in the Acahkl : :hisG locus was verified by southern blot analysis from several isolates. Southern blot analysis of one CSSK23 representative isolate is shown in Fig. 3C. The 3.46 kb Bglll-Bglll fragment that was detected is consistent with the replacement of one disrupted allele (Acahkl : :hisG) in the CSSK22 strains with the CaSSKl-URA3-hisG cassette, restoring one CaSSKl allele.
Example 6 CaSSKl is not involved in a response to osmotic or oxidative stress. In order to corroborate our previous findings by complementation analyses, which indicated that CaSSKl does not function in regulating the response to either osmotic or oxidative stress (Calera and
Calderone, 1999, Yeast 15, supra) , the Acasskl null strains and the CAF2 strain as a control were grown in M199 (pH
7.5) at 37°C and in M199 (pH 4.0) at 28° C (both solid and liquid) to induce filamentous or yeast growth under conditions of osmotic or oxidative stress, for which media were supplemented with 1 M sorbitol or 2 mM H202, respectively. The results indicated that under these conditions of stress, the ability of the Acasskl null strains to grow either as a yeast or form germ tubes was similar to wild type cells although both wild type and null strains were growth retarded compared to untreated cultures. Thus, the increase observed in either the generation time or initiation of germ tube formation of the Acasskl null strains in the presence of stress, when compared to untreated cultures, did not differ significantly from the increases observed for the wild type strain (P > 0.05) . However, interestingly the Acasskl null mutant flocculated (Fig. 4) in liquid M199 (pH 7.5) medium similar to the Acahkl mutants as previously described (Calera and Calderone, 1999, Microbiology 145, 1431-1442). Also, we observed that the Acasskl null strain was unable to undergo the morphological transition from yeast to hyphae on solid M199 (pH 7.5) medium either in the presence or absence of stress factors. Taken together these results, indicate that CaSSKl does not function in regulating the response to osmotic and oxidative stress in C. albicans, which is consistent with our previous results obtained by complementation analyses (Calera and Calderone, 1999, Yeast 15, supra) . Moreover, these results indicate that CaSSKl could play a role in morphogenesis and also may be associated with changes in the expression of hyphal cell surface components, since its absence results in flocculation.
Example 7 CaSSKl is required for hyphal formation on solid media. To study the role of CaSSKl in the morphological transition from yeast to hyphae, the Acasskl null and heterozygote Ura3+ strains were grown in several media (solid and liquid) that induce this morphological switch, including Spider, serum and M199 (pH 7.5) medium (Fig. 5) . Colonies from the wild type (CAF2) and the heterozygote strains (CSSK11) developed radial filaments emerging from the edge of the colonies after two days in M199 (pH 7.5) and after three days in Spider medium. In both media radial filaments had grown extensively after 5 days, even though the heterozygote strain showed a severely reduced extension of these filaments compared with the wild type (Fig. 5) . In contrast, the Acasskl null strains were suppressed in hyphal formation on M199 (pH 7.5) and Spider medium after five days of culture, as revealed by the observation of smooth colonies (Fig. 5) . However, after eight days of incubation, short filaments emerged from the edge of the colonies on Spider medium (Fig. 5, small insert) . The CSSK23 strains, in which one wild type copy of CaSSKl was reintroduced, regained the ability to form hyphae similar to the CSSK11 heterozygote strains grown under the same conditions. Additionally, the Acasskl null mutants when grown in agar with 10% serum showed a severe reduction in hyphal development in comparison with the CAF2 , forming irregular smooth colonies with yeast growth in the center of the colonies from which some filaments emerged. The heterozygote strains formed colonies intermediate between those of the Acasskl null strains and the wild type (Fig. 5) .
On the other hand, growth of the Acasskl null strains was dramatically influenced by low nitrogen availability in SLAD medium (Fig. 6) . Contrary to the growth of the wild type strain (CAF2) on solid Spider or M199 (pH 7.5), where hyphal formation creates fuzzy colonies, on SLAD agar, hyphae did not irradiate from the edge but formed beneath the colonies growing into the agar in a way typically known as agar invasion. Thus, when these colonies were washed off the agar, only the agar-invasive section of the colonies remained. Interestingly, we observed that the Acasskl null strains invaded the agar much more extensively than the heterozygote strain which, in fact, invaded the agar similar to the wild type strain. The abnormal hyper- invasion of the agar by the Acasskl null strains was completely restored by introduction of one wild type copy of CaSSKl (Fig. 6) , indicating that the hyper-invasive growth was a recesive phenotype of the Acasskl null strains. In addition, when ammonium sulfate as a nitrogen source was supplemented at the final concentration of 40 mM instead 50 μM, the Acasskl null strains did not show hyper- invasive growth, suggesting that the low concentration of the nitrogen source was the critical factor that affected the invasion of the agar by the Acasskl null mutants. Since the yeast to hyphae switch phenotype depends not only on the media and growth conditions but also on the physical state of these media (solid or liquid) (Csank et al . , 1997, supra; Kohler and Fink, 1996, Proc . Natl . Acad. Sci . USA 93, 13223-13228; Leberer et al . , 1996, Proc . Natl . Acad. Sci . USA 93, 13217-13222; Liu et al . , 1994, Science 266, 1723-1726), we analyzed the ability of the Acasskl mutants to form hyphae in liquid media. Thus, in all media tested, the Acasskl null strains developed hyphae identical to the wild type and no differences in the pattern and timing of germ tube formation or elongation of hyphae could be observed between CSSK21 and CAF2 strains. Again, the most remarkable finding was that in M199 broth (pH 7.5), cells flocculated (Fig. 5) through interactions of their germ tubes at all cell densities tested (105, 106 or 107) , in the same way as the Acahkl mutants, which we recently reported (Calera and Calderone, 1999, Microbiology 145, supra) .
Example 8 Avirulence of Acasskl mutants . In order to determinate whether CaSSKl was required for virulence, the ability of Acasskl C. albicans null strains to establish infection in a murine model of hematogenously disseminated candidiasis was investigated. Prior to animal studies, we evaluated two factors that may also affect the virulence of the mutants, such as their generation time and orotidine 5 ' -monophosphate (OMP) decarboxylase activity. The generation time of the Acasskl null strains was slighly higher than that calculated for the wild type, heterozygote and revertant strains, whose generation times did not differ significantly from one another ( P > 0.09). The OMP decarboxylase activity of each Acasskl mutant did not differ significantly from the OMP decarboxylase activity of
CAF2 ( P > 0.1) . The data in Fig. 7 show that mice infected with the CSSK21 strains survived throughout the experiment. In contrast, all mice infected with the parental control (CAF2) succumbed to infection within 3 days, and survival times for mice injected with either the heterozygote (CSSK11) or revertant (CSSK23) strains were longer than that observed for mice inoculated with the parental strain (CAF2). Product-limit survival estimates were calculated by the Kaplan Meier method, and the log rank test was employed to examine the homogeneity of survival curves among the four strains . The overall differences in survival among strains were highly statistically significant ( P - 0.0001). Individual comparisons did not vary from the overall pattern: CSSK21 > CSΞK23 « CSSK11 (P = 0.0001), CSSK21 > CAF2 (P = 0.0003), CSSK23 > CAF2 (P = 0.0003), CSSK11 >
CAF2 (P = 0.0005), CSSK23 ~ CHK11 (P = 0.50). Thus, survival of mice infected with CSSK21, CSSK11 and CSSK23 was greater than that of mice infected with CAF2.
Quantitative determinations of the level of each C. albicans strain associated with host tissues suggest that the CSSK21 strains were slowly cleared from kidneys but quickly cleared from liver (Table 2) . Levels of both CSSKll and CSSK23 strains are similar or lower than that observed for the parental control. Both strains persisted in tissues at 72 h. In order to determine the significance of differences observed among strains for each target organ at each of three points in time (24, 48 and 72 h) , a general linear models procedure was used. Tukey's multiple comparison test was employed to hold the Type I error (α) constant at 0.01. In terms of virulence in the liver, several statistically significant differences were seen at
P < 0.01 in mean log10 (CFU/g) at each of the time intervals. At 24 hours, CAF2 > CSSKll > CSSK21; no other comparisons differed from one another. At 48 hours, CAF2 > CSSKll and CSSK21, CSSK23 > CSSKll and CSSK21; no other comparisons differed from one another. At 72 hours, CSSK23 > CSSKll > CHK21. In terms of virulence in the kidney, several statistically significant differences were seen at P < 0.01 in mean log10 (CFU/g) at each of the time intervals. At 24 hours, CAF2 > CSSKll ~ CSSK23 > CSSK21. At
48 hours, CAF2 > CSSK23 > CSSKll > CSSK21. At 72 hours,
CSSK23 and CSSKll > CHK21; CHK23 = CHK11. By 72 h, all mice infected with CAF2 had died. Histological examinations of kidney tissue support these observations (data not shown) . Thus, all strains had formed mycelia in infected tissue but smaller amounts of fungal burden were observed in tissue infected with the CSSK21 strains in comparison to those infected with CSSKll, CSSK23 or CAF2 , probably because a more effective clearing of the CSSK21 strains was performed by murine phagocytes . Table 2
Time postinfection Log^ CFU/g (mean±SD) in:
(h) Strain Kidney Liver
24 CAF2 6.20±0.27 3.88+0.10
CSSKll-1 5.11±0.11 3.42±0.10
CSSK21-1 4.13±0.12 3.28±0.17
CSSK23-1 5.57±0.50 3.53±0.19
48 CAF2 6.66±0.76 3.98±0.34
CSSKll-1 4.74+0.44 2.42±0.24
CSSK21-1 3.69±0.24 2.39±0.25
CSSK23-1 5.80+0.94 3.47±0.52
72 CAF2 a
_
CSSKll-1 4.68±0.21 1.93±0.16
CSSK21-1 3.52±0.24 0.68±0.85
CSSK23-1 5.56±0.50 2.87±0.25
a , all mice succumbed to CAF2 infection by 72 h.
The avirulence of the Acasskl null strains indicate that CaSSKl is required for the pathogenesis of C. albicans . Furthermore, several experiments indicated that the virulence of the CSSKll and CSSK23 strains was due to the presence of one functional allele, while the avirulence of the CSSK21 strains was due to the absence of any CaSSKl allele: 1) The same results in survival and tissue counts were obtained with two independent Acasskl null strains (CSSK21-1 and CSSK21-2) . 2) The virulence of the CSSK21 strains was restored by the reintroduction of a parental copy of CaSSKl in them both (CSSK23-1 and CSSK23-2) . 3) The generation time of the Acasskl mutants was similar to the generation time of the wild type, 4) No differences were observed in the OMP decarboxylase activities of the Acasskl mutants compared to the wild type (CAF2) . DISCUSSION. The morphological switch from yeast to hyphae is one of the most important biological features that enables C. albicans to colonize, invade, and survive in the host tissues during infection. Although S. cerevisiae is rarely an opportunistic human pathogen and does apparently not require this dimorphic transition as a mechanism to survive in tissue, by using functional complementation studies of mutations in genes related to the filamentation-invasion of S. cerevisiae as a model, several C. albicans homologues have been identified, including CST20, HST7 , CEK1 and CPH1 (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . , 1996, supra; Liu et al . , 1994, supra) , which function in a C. albicans STE12-homolog MAPK cascade, as well as EFGl (Lo et al . , 1997, Cell 90, 939-949; Stoldt et al . , 1997, EMBO J. 16, 1982-1991) which, like its S. cerevisiae homolog PHD1 , is probably part of an independently regulated, unknown pathway (Banuett, 1998, Microbiol . Mol . Biol . Rev. 62, 249- 274) . Initially, studies using a Acphl/Aefgl strain of C. albicans indicated that the signal pathways that regulate the activity of these transcription factors constitute the main regulatory mechanisms of the yeast-to-hyphae transition in C. albicans (Lo et al, 1997, supra) . However, the CaHOGl gene, unlike its S. cerevisiae homolog, exhibits functional properties related to morphogenesis in C. albicans (Alonso-Monge et al . , 1999, J". Bacteriol . 181, 3058-3068) . Furthermore, in C. albicans there are genes without a S. cerevisiae counterpart that also play a role in morphogenesis, such as the sensor histidine kinases
CaHKl (Calera and Calderone, 1999, Microbiol . 145; Calera et al . , 1998, Yeast 14, 665-674) and CaNIKl/COSl (Alex et al . , 1998, Proc . Natl . Acad . Sci . USA 95, 7069-7073). In this work, we have reported and discussed the function of CaSSKl , the only response regulator kinase gene described so far in C. albicans (Calera and Calderone, 1999, Yeast 15) .
As we have shown by complementation analyses, CaSSKl does not restore the normal growth of either Asskl or Amcs strains of S. cerevisiae or S. pombe respectively, suggesting that CaSSKl may have another role in C. albicans rather than in adaptation of cells to stress (Calera and Calderone, 1999, Yeast 15, supra) . The phenotypic characterization of Acasskl mutants supports our previous observation and confirms that CaSSKl is not functionally related to either SSKl or mcs4+ . Moreover, CaSSKl is essential for hyphal formation on solid inducing media and virulence, even though it is not required for formation of hyphae in liquid media. It has been proposed that like Cphlp, Efglp must be the final element of a morphogenesis pathway (Lo et al . , 1997, supra; Stoldt et al, 1997 , supra) . Thus, similar to the Acasskl null strains, the Aefgl mutants are unable to form true hyphae either on solid or liquid media (Lo et al . , 1997, supra) . However, in contrast to the Acasskl mutants that form hyphae in liquid media similar to the wild type, the Aefg mutants grow as a yeast in liquid media even in the presence of serum (Lo et al . , 1997 , supra) . This phenotypic difference between the Acasskl and the Aefgl strain makes it unlikely that CaSSKl can influence the activity of the EFGl pathway. Also, it has previously been reported that mutations in the genes CST20, HST7 , CEK1 and CPHl make C. albicans unable to undergo the yeast to hyphal transition on solid media (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . 1996, supra; Liu et al . , 1994, supra) , while the CPP1 mutants hyperfilament under conditions that normally do not induce filamentation (Csank et al . , 1997, supra) . The phenotype of the Acasskl null strains resembles the defect in hyphal formation on solid media observed for mutants in genes of the CPHl filamentation pathway but, in contrast, the mutants in genes of the CPHl pathway are able to form hyphae on solid serum, are not totally avirulent, and fail to hyper-invade the agar under low nitrogen availability (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . 1996, supra; Liu et al . , 1994, supra) . In addition, although the hyper-invasive phenotype of the Acasskl null strains resembles that of the Acppl mutants, the Acasskl null strains show this phenotype only under low nitrogen while the Acppl mutants show derepressed hyper- invasion of agar under both normally noninducing and inducing conditions in a wide variety of rich and defined solid media (Csank et al . , 1997, supra) . In any case, if CaSSKl affects the activity of the CPHl pathway, then the overproduction of downstream components of the CPHl pathway in a Acasskl background should rescue the lack of CaSSKl . However, in preliminary experiments we were unable to rescue the normal filamentous growth of a Acasskl null mutant by overexpressing either CHP1 or CPP1 . Similar results were previously described by others in a
Acanikl l ' cosl background (Alex et al . , 1998, supra) . Overexpression of the HST7 gene did not rescue the normal filamentous growth of a Acosl null strain (Alex et al . , 1998, supra) , suggesting that CaNIKl/COSl may lie in a different pathway or interact with components downstream of HST7 (Alex et al . , 1998, supra) . Interestingly, similar to the Acasskl null strains, the Acosl mutants can not undergo the yeast-to-hyphal transition on Spider agar and have reduced hyphal formation on serum agar (Alex et al . , 1998, supra) . On the other hand, the flocculation displayed by the Acasskl null strains under conditions of germ tube formation occurs in a manner similar to the Acahkl mutants (Calera and Calderone, 1999, Microbiology 145) . Both the Acasskl and Acahkl strains show similar growth rates and are totally avirulent in a murine model of hematogenously disseminated candidiasis (Calera et al . , 1999, Infect . Immun. 67, 4280-4284). These observations, together with the non restored filamentous growth by overexpression in the Acasskl background of either CPP1 or CPHl (which unlike HST7, has been proposed to be the last element of the pathway) , indicate that CaSSKl may also lie in a CPH1- independent "two component" filamentation pathway.
Finally, even though CaSSKl is not functionally related to SSKl , it does not preclude the possibility that a structural homolog "two-component" Slnlp-Ypdl-Ssklp cascade (Banuett, 1998, supra) that affects the activity of a putative homolog HOG MAPK pathway could exist in C. albicans. In this context, some phenotypical properties of the Acasskl mutants resemble those observed for the Acahogl mutants (Alonso-Monge et al . , 1999, supra) , such as both are unable to form hyphae on Spider agar, are more invasive in SLAD medium than the wild type, and are avirulent. However, we were unable to rescue the normal phenotype of a Acasskl null strain by overexpressing CaHOGl in the Acasskl background. In spite of this, it still remains possible that CaSsklp could function in a "two-component" phosphorelay cascade which may or may not influence the activity of CaHoglp. In addition, since CaSsklp should function as a response regulator in this putative cascade, similar to other response regulators (Banuett 1998, supra) , it must lie downstream of a sensor histidine kinase component, such as Cahklp or CaNiklp/Coslp. However, since the Acasskl mutants displayed two unrelated phenotypes that resemble those of the Acahkl and Acanikl/ Acosl mutants, our hypothesis is that two Cahklp- and CaNiklp- independent branches of the same two-component cascade modulate the phosphorylation state of CaSsklp through one or more intermediate Ypdl-like histidine kinases (Fig. 8) . In this regard, we have recently isolated a C. albicans gene which encodes a Ypdlp-like protein (unpublished data) . In summary, our data indicate that CaSSKl links two of the most important aspects of the biology of C. albicans , i.e., the morphological transition from yeast to hyphae and changes in the expression of a hyphal surface compound (s) that occur during hyphal growth. Also, in addition to the STE12-like MAPK cascade that is known to regulate morphogenesis in C. albicans, these results suggest that a "two-component" cascade may have been adopted by C. albicans to modulate its morphological switching and that other undiscovered elements or pathways must exist that regulate the transition from yeast to hyphae in liquid media in C. albicans . Furthermore, two-component signal tranduction cascades have not been found in mammalian cells which emphasize the interest of both CaSslp and Cahklp as targets for the development of antifungals .

Claims

What is claimed is:
1. An isolated CaSSKl DNA fragment or any portion thereof .
2. An isolated and purified DNA fragment which encodes a CaSsklp.
3. An isolated and purified DNA fragment which encodes CaSsklp, said DNA fragment comprising the sequence specified in Genbank Accession no. AF084608 or a polynucleotide fragment of said sequence comprising at least 30 nucleotides.
4. An isolated and purified CaSSKl DNA fragment according to claim 2 which encodes 674 amino acids of CaSsklp or a natural variant or synthetic variant thereof encoding CaSsklp, or a peptide fragment thereof comprising at least 10 amino acids.
5. A recombinant DNA construct comprising:
(i) a vector, and (ii) the CaSSKl DNA fragment of claim 1.
6. A recombinant DNA construct comprising:
(i) a vector, and (ii) the CaSSKl DNA fragment of claim 3.
7. A recombinant DNA construct according to claim 6, wherein said vector is an expression vector.
8. The recombinant DNA construct according to claim 6, wherein said vector is a prokaryotic vector.
9. The recombinant DNA construct according to claim 6, wherein said vector is a eukaryotic vector.
10. A host cell transformed with a recombinant DNA construct according to claim 6.
11. A host cell according to claim 10, wherein said cell is prokaryotic .
12. A host cell according to claim 10, wherein said cell is eukaryotic .
13. A method for producing CaSsklp peptide which comprises culturing the cells according to either claim 11 or 12, under conditions such that said DNA fragment is expressed and said CaSsklp peptide is thereby produced.
14. An isolated recombinant CaSsklp produced by the method of claim 13.
15. A method for screening agents or drugs which reduce or eliminate CaSsklp activity said method comprising detecting a decrease CaSsklp enzyme activity in the presence of said agent or drug by detecting a decrease in phosporylation of a CaSsklp substrate.
16. A method for detecting CaSsklp in a sample comprising
(i) contacting a sample with antibodies which recognize CaSsklp; and (ii) detecting the presence or absence of a complex formed between CaSsklp and antibodies specific therefor .
17. An antibody to a peptide having the amino acid sequence specified in SEQ ID NO: 2, or any portion thereof .
18. A method for detecting agents or drugs which inhibit CaSsklp activity, said method comprising:
(i) delivering a recombinant DNA construct according to claim 5 into a cell such that CaSsklp is produced in said cell;
(ii) adding at least one drug or agent to said cell alone or in combination; and,
(iii) detecting whether or not said drug or agent inhibits CaSsklp activity by measuring CaSsklp- dependent phosphorylation of molecules in said cell and comparing it to a control which did not receive said drug or agent wherein a decrease in the amount of CaSsklp-dependent phosphorylation as compared to control indicates an inhibitory drug or agent .
19. A method for detecting agents or drugs which promote CaSsklp activity, said method comprising:
(i) delivering a recombinant DNA construct according to claim 5 into a cell such that CaSsklp is produced in said cell;
(ii) adding at least one drug or agent to said cell alone or in combination; and,
(iii) detecting whether or not said drug or agent stimulates CaSsklp activity by measuring CaSsklp- dependent phosphorylation in said cell and comparing it to a control which did not receive said drug or agent wherein an increase in the amount of CaSsklp- dependent phosphorylation in said cell as compared to control indicates a stimulatory drug or agent.
20. An agent or drug capable of inhibiting CaSsklp activity.
21. An agent or drug capable of promoting CaSsklp activity.
22. A therapeutic compound comprising said agent or drug according to claim 20 for use in treatment of candidiasis .
23. A method for detecting CaSSKl in a sample using the polymerase chain reaction.
24. A diagnostic kit for detecting CaSSKl RNA/cDNA in a sample comprising primers or oligonucleotides specific for CaSSKl RNA or cDNA suitable for hybridization to CaSSKl RNA or cDNA and/or amplification of CaSSKl sequences and suitable ancillary reagents .
29. A therapeutic method for the treatment or amelioration of diseases resulting from candidiasis, said method comprising providing to an individual in need of such treatment an effective amount of an agent or drug which reduces or eliminates CaSSKl expression or function in a pharmaceutically acceptable diluent.
30. A mutant C. albicans yeast strain with reduced CaSsklp activity.
31. A mutant C. albicans yeast strain devoid of CaSsklp activity.
32. The yeast strain of claim 31 wherein said strain is Δcasskl .
33. A method to elicit an antigen-specific immne response in a mammal, said method comprising administering to said mammal Δcasskl transformed with a heterologous nucleic acid molecule encoding an antigen, wherein said antigen has been expressed by said Δcasskl ; and wherein said which has been expressed by said Δcasskl elicits an antigen-specific immune response.
34. A method to elicit a C. albicans immune response in a mammal, said method comprising administering to said mammal a composition comprising Δcasskl wherein said Δcasskl elicits a C. albicans immune response.
PCT/US1999/022218 1998-09-23 1999-09-23 RESPONSE REGULATOR $i(CaSSK1) WO2000017344A1 (en)

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GB2506178A (en) * 2012-09-25 2014-03-26 Vib Vzw Mutant yeast strain with decreased glycerol production

Non-Patent Citations (3)

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Title
CALERA, JOSE A. (1) ET AL: "Identification of a putative response regulator two-component phosphorelay gene ( CaSSK1 ) from Candida albicans.", YEAST, (SEPT. 15, 1999) VOL. 15, NO. 12, PP. 1243-1254., XP000865799 *
SHIGEHISA NAGAHASHI ET AL.: "Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans", MICROBIOLOGY, vol. 144, February 1998 (1998-02-01), pages 425 - 432, XP002083238 *
TATSUYA MAEDA ET AL.: "A two-component system that regulates an osmosensing MAP kinase cascade in yeast", NATURE, vol. 369, no. 6477, 19 May 1994 (1994-05-19), LONDON GB, pages 242 - 245, XP002129354 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2506178A (en) * 2012-09-25 2014-03-26 Vib Vzw Mutant yeast strain with decreased glycerol production

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