WO2014006025A2 - Marqueur du pouvoir pathogène de salmonella - Google Patents

Marqueur du pouvoir pathogène de salmonella Download PDF

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Publication number
WO2014006025A2
WO2014006025A2 PCT/EP2013/063900 EP2013063900W WO2014006025A2 WO 2014006025 A2 WO2014006025 A2 WO 2014006025A2 EP 2013063900 W EP2013063900 W EP 2013063900W WO 2014006025 A2 WO2014006025 A2 WO 2014006025A2
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Prior art keywords
antitoxin
toxin
salmonella
seq
amino acid
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PCT/EP2013/063900
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English (en)
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WO2014006025A3 (fr
Inventor
Miguel Angel DE LA CRUZ VILLEGAS
Stéphane MERESSE
Jean-Pierre Gorvel
Didier Raoult
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique - Cnrs -
Université D'aix-Marseille
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Publication of WO2014006025A2 publication Critical patent/WO2014006025A2/fr
Publication of WO2014006025A3 publication Critical patent/WO2014006025A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to marker(s) of pathogenicity in Salmonella and the uses thereof.
  • Salmonellae are Gram-negative bacterial pathogens that are capable of infecting a wide range of animals.
  • Salmonella enterica The bacterial genus Salmonella consists of two species: Salmonella enterica and Salmonella bongori. Salmonella are common causes of food poisoning in humans and can also cause more severe disease such as typhoid fever. Most of the Salmonella that cause disease in humans and animals are members of S. enterica. On the other hand, S. bongori is largely associated with reptiles but can cause disease in humans, albeit rarely.
  • the Salmonella genus comprises only two species: bongori and enterica. Whereas all Salmonellae are able to invade intestinal epithelium, Salmonella bongori is unable to survive and replicate intracellularly.
  • Salmonella enterica is able to spread from the intestinal tissue via the lymphatics into the bloodstream and to multiply within the macrophages of the liver and spleen.
  • SPI 2 Salmonella pathogenicity island 2
  • the host specificities and the disease symptoms are caused by some of the thousands of Salmonella subspecies (Haraga et al., 2008).
  • markers will also give lead to develop new drugs capable of impacting the viability of Salmonella but not of other bacteria present for instance in the gut flora.
  • a subject of the present invention relates to a toxin:
  • a subject of the present invention also relates to the antitoxin cognate to the toxin of the invention in the toxin-antitoxin system.
  • the present invention also relates to an antitoxin:
  • Toxin- antitoxin systems are known in many biotechnogical and medical applications (Gerdes et al.,2005). First, toxin- antitoxin systems are used in stabilization or selection systems.
  • the present invention also relates to a cassette of expression comprising a nucleic acid molecule encoding a toxin according to claim 1 operatively linked to a promoter.
  • Toxin- antitoxin system may also provide useful targets for the development of antibacterial drug.
  • the present invention relates to the toxin of the invention for use in the treatment of an Enterobacteriaceae infection.
  • the present invention also relates to an inhibitor of the antitoxin of the invention and it use in the treatment of Salmonella infection. Due to their relation with the pathogenicity of Salmonella, these toxin-antitoxin systems may also be used to detect pathogenic strains of Salmonella in warm blood animals including humans.
  • the present invention also relates to a method for detecting the presence of a pathogenic strain of Salmonella in a sample comprising the step of detecting the presence of a toxin of the invention or an antitoxin of the invention and it related kit.
  • T/AT systems also called herein toxin-antitoxin module, are derived from genes located in the bacterial chromosome (as described in the invention) and in bacterial plasmids involved in plasmid stability. This phenomenon called addiction was described when a daughter cell lacks its plasmid. The fast degradation of the antitoxin leads to the release of the toxin, which causes bacterial growth inhibition (Hayes, 2003).
  • T/AT systems form bicistronic operons, where generally, the first and second genes encode for an antitoxin and a toxin, respectively (Gerdes et al., 2005).
  • T/AT systems Three types of T/AT systems have been described based on the protein or RNA nature of the antitoxin (Blower et al., 2010; Bukowski et al., 2011; Yamaguchi and Inouye, 2011). These toxins might be interf erases or ribonucleases that stop the translation by mRNA cleavage in the presence (Li et al., 2009; Zhang et al., 2009) or absence of ribosomes (Kamada and Hanaoka, 2005; Christensen-Dalsgaard and Gerdes 2008; Prysak et al., 2009), respectively.
  • type II antitoxins are proteins that interact with their toxin counterpart to neutralize their toxic effects and that act as a transcriptional repressor of their own expression (Gerdes et al., 2005).
  • a database is dedicated to Type 2 toxin-antitoxin systems (TADB: http://bioinfo- mmi . s j tu . edu.cn/TADB/) .
  • the present invention relates to a toxin:
  • the present invention also relates to an antitoxin:
  • SehTl -SehAl and SehT2-SehA2 are both homologous to the host inhibition of growth (higBA) toxin/antitoxin system of E.
  • the toxin/antitoxin system SehTl-SehAl comprises a toxin called herein sehTl and an antitoxin called herein sehAl.
  • the toxin/antitoxin system SehT2-SehA2 comprises a toxin called herein sehT2 and an antitoxin called herein sehA2.
  • the percentage of sequence identity refers to comparisons among amino acid sequences, and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage may be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the percentage may be calculated by determining the number of positions at which either the identical amino acid residue occurs in both sequences or an amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.
  • HSPs high scoring sequence pairs
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.
  • the toxin of the invention has the amino acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 7 or an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the amino acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 7.
  • the antitoxin of the invention has the amino acid sequence set forth as SEQ ID NO: 6 or SEQ ID NO: 8 or an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the amino acid sequence set forth as SEQ ID NO: 6 or SEQ ID NO: 8.
  • the toxin has the amino acid sequence set forth as SEQ ID NO: 5.
  • the toxin has the amino acid sequence set forth as SEQ ID NO: 7.
  • the antitoxin has the amino acid sequence set forth as SEQ ID NO: 6.
  • the antitoxin has the amino acid sequence set forth as SEQ ID NO: 8.
  • the present invention also relates to a nucleic acid molecule encoding a toxin of the invention.
  • the nucleic acid molecule has the sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3.
  • nucleic acid molecule has the sequence set forth as SEQ ID NO: 1.
  • the present invention also relates to a nucleic acid molecule encoding an antitoxin of the invention.
  • the nucleic acid molecule has the sequence set forth as SEQ ID NO: 2 or SEQ ID NO: 4.
  • nucleic acid molecule has the sequence set forth as SEQ ID NO: 2.
  • Toxin- antitoxin systems may be used in plasmid stabilization or positive selection vectors.
  • a further object of the invention relates to a cassette of expression comprising a nucleic acid molecule encoding a toxin of the invention operatively linked to a promoter.
  • This cassette of expression may be used as a selection system. Indeed, by introducing unique restriction sites for cloning into toxin gene in a way that the gene still encodes the active toxin, the toxin gene is disrupted when DNA restriction fragment is introduced into the vector and thus the vector can be maintained in host cells that are sensitive to the toxin whereas the vector harbouring a functional toxin gene can not be maintained.
  • the cassette of expression further comprises a nucleic acid molecule encoding an antitoxin of the invention operatively linked to a promoter.
  • the cassette of expression comprises the operon set forth as SEQ ID NO: 9 or SEQ ID NO: 10.
  • the nucleic acid sequence of the operon sehTlAl is set forth as SEQ ID NO: 9.
  • the nucleic acid sequence of the operon sehT2A2 is set forth as SEQ ID NO: 10.
  • the operon is set forth as SEQ ID NO: 9.
  • the vector of the invention may also be used as stabilization vector.
  • toxin/antitoxin genes can efficiently stabilize vector.
  • Toxin/antitoxin genes can be inserted into unstably inherited vectors, and thereby lead to an increased genetic stability of producer strains. Therefore, another object of the invention relates to a vector comprising a cassette of expression of the invention.
  • vector means the vehicle by which a DNA or a RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • the vectors of the invention are plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. Such vectors may comprise regulatory elements, such as an enhancer, a terminator and the like.
  • the cassette of expression encoding the toxin and the cassette of expression encoding the antitoxin may be comprised in different vectors, in a bacterial chromosome or one in a vector the other on a bacterial chromosome.
  • Nucleic acid 2 ATGGATGCAACCAGCGCAAAAAAGATCGTT sequence GATACCTTCAGCGATGCGGTAAAAACCGTCC sehAl CACTGATGGGGGAAGACCGAAATGACAATG
  • Nucleic acid 3 ATGCAATTTATAGAAACGGAACTCTTTACTG sequence AAGATGTTAAAAAACTGCTCGATGATGATGA sehT2 ATACCATAAGCTTCAGGTTTTTATGGCTCAG
  • Nucleic acid 4 ATGGATAAAGTGTTATTTGAGCGATTAACTC sequence AAAGTATGTCTCAAATGAATGAAATCATTGA sehA2 AGGGACCCGTGAACCTTCTCGTACCTTTCAT
  • the present invention also relates to a host cell transformed by a nucleic acid molecule of the invention, a cassette of expression of the invention and/or the vector of the invention.
  • transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence into a host cell, so that the host cell will express the introduced gene or sequence.
  • a host cell that receives and expresses introduced DNA or RNA has been "transformed”.
  • the host cell is preferably a recombinant bacterium, more preferably a recombinant Gram-negative bacterium and most preferably a recombinant Salmonella.
  • the present invention also relates to an inhibitor of the antitoxin of the invention.
  • inhibitor of antitoxin should be understood broadly, it encompasses inhibitors of the antitoxin activity, and inhibitors of the expression of antitoxin.
  • an “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of a gene. Consequently an “inhibitor of antitoxin expression” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for the antitoxin.
  • an “inhibitor of antitoxin expression” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for the antitoxin.
  • the antitoxin inhibitor is a compound that can partially or completely blocks the ability of antitoxin to inhibit it cognate toxin.
  • Agarwal et al. have shown how to design peptides able to perturb PemK-PemI interaction and that lead to bacterial cell death. These peptides were notably 7-8 residues of Peml determined to be in the region necessary for the toxin binding.
  • the inhibitor of the antitoxin inhibits the formation of the complex toxin-antitoxin.
  • the inhibitor of the antitoxin disrupts the toxin-antitoxin complex.
  • the inhibitor of the antitoxin may be a fragment of the antitoxin located in the toxin- antitoxin interface.
  • the inhibitor is a fragment of 5 to 10 amino acids of the antitoxin.
  • the inhibitor of antitoxin may also play a role by modulating the expression of the toxin- antitoxin complex, for example at the promoter level.
  • Another aspect of the invention relates to an inhibitor of antitoxin expression.
  • the antitoxin is quickly degraded by cellular proteases. Therefore, a molecule that binds promoter DNA and inhibits transcription at the TA locus would prevent replenishment of the antitoxin. The non replenishment of the antitoxin would lead to the liberation of the latent toxin and to the killing of the cell.
  • the antitoxin of the invention negatively regulates it own expression.
  • the compound having homology with antitoxin that keeps the ability to bind to the promoter of the antitoxin promoter region with having an antitoxin activity may be efficient inhibitors of the antitoxin.
  • the inhibitors of antitoxin expression for use in the present invention may also be based on anti-sense oligonucleotide constructs.
  • Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of antitoxin mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of antitoxin.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding antitoxin can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs can also function as inhibitors of antitoxin expression for use in the present invention.
  • Antitoxin expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that antitoxin expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • the inhibitor is a compound that prevents the antitoxin to repress the expression of the toxin.
  • the inhibitor of antitoxin is an inhibitor of the antitoxin set forth as SEQ ID NO: 6. The inventors have found that only SehAl plays a role in S. Typhimurium virulence.
  • the inhibitor of antitoxin is an inhibitor of the antitoxin set forth as SEQ ID NO: 6 and SEQ ID NO: 8.
  • SehA2 was able to act as molecular backup of SehAl. It is therefore more efficient to inhibit the two antitoxins.
  • the present invention also relates to a method for screening an inhibitor of an antitoxin of the invention.
  • the method for screening an inhibitor may comprise the step of contacting a test compound with a nucleic acid molecule encoding the antitoxin.
  • a plasmid with an operon toxin- antitoxin where the antitoxin gene is replaced with a fusion of the gene of antitoxin with a reporter gene such as gfp may be used to transform strains.
  • the strains are contacted with a test compound and the level of expression of reporter is measured indicating the level of expression of antitoxin when the strains are in contact with the test compound. If the level of expression of the antitoxin is significantly higher with the test compound than the level of expression in the control without the test compound then the test compound is an inhibitor of the antitoxin.
  • the method for screening an inhibitor may comprise the step of contacting a test compound with said antitoxin.
  • the antitoxin activity of the antitoxin is measured.
  • the method of screening of the invention may, in particular, allow the identification of compounds that affect specifically the growth of pathogenic strains of Salmonella.
  • the toxin-antitoxin systems of the invention are associated with pathogenicity in Salmonella.
  • the inventors have shown that the deletion of SehAl low the bacterial burden of mammals infected with Salmonella and therefore that an inhibitor of antitoxin SehAl impacts the Salmonella virulence.
  • Toxin-antitoxin (TA) modules are foreseen as targets to fight against antibiotic-resistant strains by using the toxin or disrupting the toxin-antitoxin system (for example see Engelberg-Kulka, et al. 2004 and Virginia S. Lioya, et al., plasmid 2010, William et al. 2012).
  • treatment or treating refers to both curative treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • the subject to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
  • the present invention also relates to a toxin of the invention for use in the treatment of Enterobacteriaceae infection.
  • the toxin of the invention acts as a novel antibiotic by affecting the growth of bacteria and may be used in the treatment of bacterial infection.
  • Enterobacteriaceae may be selected from the group consisting of Salmonella, Escherichia coli, Yersinia pestis, Klebsiella and Shigella.
  • Enterobacteriaceae infection is Salmonella infection.
  • bacterial infection is an infection with Salmonella enterica and more preferably Salmonella enterica of group I.
  • bacterial infection is an infection with Salmonella of enterica subspecies (group I) selected from the group consisting such as Typhi, Paratyphi, Enteritidis, Gallinarum, Heidelberg, Dublin, Agona and Newportin subspecies.
  • Salmonella infection may be entiritis, typhoid fever, blood stream infection, or focal infection such as meningitis, osteomyelitis, or abscess caused by Salmonella.
  • the present invention also relates to a method for the treatment of a bacterial infection in a subject in need thereof comprising administering to said subject an effective amount of the toxin of the invention.
  • the present invention also relates to a use of the toxin of the invention for the preparation of a medicament for the treatment of a bacterial infection.
  • the present invention also relates to an inhibitor of the antitoxin of the invention for use in the treatment of Salmonella infection.
  • Salmonella infection may be intestinal infection, typhoid fever, blood stream infection, or focal infection such as meningitis, osteomyelitis, or abscess.
  • the present invention also relates to a method for the treatment of a Salmonella infection in a subject in need thereof comprising administering to said subject an effective amount of an inhibitor of the antitoxin of the invention.
  • the inhibitor of the antitoxin by disrupting the toxin-antitoxin system acts as an antibiotic.
  • the antitoxin of the invention is specific of the Salmonella enterica species. Therefore, the inhibitor of the antitoxin of the invention would not provide side effects on the gut flora of the subject.
  • the present invention also relates to a use of an inhibitor of the antitoxin of the invention for the preparation of a medicament for the treatment of a Salmonella infection.
  • the present invention also relates to a method for detecting the presence of a pathogenic strain of Salmonella in a sample comprising the step of:
  • sehTl-sehAl and sehT2-sehA2 T/AT systems are present in the genus Salmonella enterica that affects mainly mammals and not Salmonella bongori that affect mainly cold-blood species.
  • sample may be any sample taken for the realization of in vitro assays in animals or humans or collected from food whatever the nature or from any liquid medium or solid gas may contain pathogens sought.
  • sample may be human or warm blood animal tissue homogenates, blood, feces and nasal and urethral mucous.
  • Preliminary enrichment of the bacteria present in the sample by cultivation may be possible before the identification test.
  • sehTl or sehAl are detected.
  • enterica subspecies such as Typhi, Paratyphi, Enteritidis, Gallinarum, Heidelberg, Dublin, Agona and Newportin but not the arizonae subspecies.
  • Enterica subspecies (group I) infect warm-blooded mammals, while Salmonella enterica subspecies arizonae (group Illb), also known to encode for SPI2, has been isolated from reptiles and is rarely associated with human infection.
  • the enterica subspecies group I are known to be responsible for 99% of mammalian infections (Baumler et al., 1997).
  • the present invention also relates to a kit for detecting the presence of a pathogenic strain of Salmonella in a sample comprising means for detecting the presence of the toxin of the invention or the antitoxin of the invention.
  • the presence of the toxin of the invention or the antitoxin of the invention may be detected at the nucleic acid level.
  • the means for detecting the presence of the toxin of the invention or the antitoxin of the invention are nucleic acid probes or primers that hybridize specifically to the nucleic acid molecule encoding the toxin or the antitoxin of the invention
  • primer and “probe” refer to the function of the oligonucleotide.
  • a primer is typically extended by polymerase or ligation following hybridization to the target but a probe typically is not.
  • a hybridized oligonucleotide may function as a probe if it is used to capture or detect a target sequence, and the same oligonucleotide may function as a primer when it is employed as a target binding sequence in an amplification primer.
  • any of the target binding sequences disclosed herein for amplification, detection or quantisation of the nucleic acid molecule encoding the toxin or the antitoxin of the invention may be used either as hybridization probes or as target binding sequences in primers for detection or amplification, optionally linked to a specialized sequence required by the selected amplification reaction or to facilitate detection.
  • Probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.
  • the method for detecting the presence of a pathogenic strain of Salmonella in a sample may comprise the step of:
  • nucleic acid probe or primer that selectively hybridizes to the nucleic acid molecule encoding the toxin or the antitoxin of the invention, -detecting the hybridization of the nucleic acid probe or primer with the nucleic acid molecule.
  • the nucleic acid molecule may be extracted using any methods known in the art, such as described in Sambrook et al., 1989.
  • nucleic acid molecule may be amplified by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • the means for detecting the presence of the toxin or the antitoxin comprise a binding partner able of selectively interacting with respectively the toxin or the antitoxin.
  • the method for detecting the presence of a pathogenic strain of Salmonella in a sample may comprise the step of:
  • the binding partner may be generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • Polyclonal antibodies directed against toxin of the invention, antitoxin of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against toxin of the invention, antitoxin of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler et al. Nature. 1975;256(5517):495-7; the human B-cell hybridoma technique (Cote et al Proc Natl Acad Sci U S A. 1983;80(7):2026-30); and the EBV-hybridoma technique (Cole et al., 1985, In Monoclonal Antibodies and Cancer Therapy (Alan Liss, Inc.) pp. 77-96).
  • Antibodies useful in practicing the present invention also include binding partners against toxin of the invention, antitoxin of the invention or a fragment thereof including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • the binding partners of the invention may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labeled", with regards to the binding partner is intended to encompass direct labeling of the binding partner by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the binding partner, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance.
  • a binding partner of the invention may be labeled with a radioactive molecule by any method known in the art.
  • radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88.
  • the toxin or the antitoxin of the invention are detected by Western blot.
  • cells from sample are lysed, for example with RIPA, and then sonicated. Then total proteins from sample are separated using gel electrophoresis, for example a SDS-PAGE. The proteins are transferred out of the gel and onto a membrane, typically polyvinyldiflroride or nitrocellulose, where they are probed using a labeled binding partner capable of selectively interacting with the toxin of the invention, the antitoxin of the invention or a fragment thereof.
  • a membrane typically polyvinyldiflroride or nitrocellulose
  • the toxin of the invention is detected by ELISA.
  • cells from sample are lysed, sonicated and microcentrifuged. After suitable dilution, the supernatant is added to wells of a microtiter plate that are coated with a set of primary antibodies. Then, after a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • Figure 1 shows that the atypical HigBA-type T/AT modules are present in pathogenic Salmonella.
  • Figure 2 shows that the SehAl antitoxin represses its own expression and controls the Salmonella growth.
  • FIG. 3 shows that the SehA2 antitoxin can act as a molecular backup of SehAl.
  • FIG 4 shows that sehl and seh2 are dispensable for replication in cultured cells.
  • Mouse RAW 264.7 macrophages (A) or human HeLa cells (B) were infected with wild-type or various sehl or seh2 mutant strains. AsifA and AssaV strains were used as controls. Cells were lysed at 2 and 16 hr post-infection for enumeration of intracellular bacteria. The values shown represent the fold increase calculated as a ratio of the intracellular bacteria between 16 and 2 hr and normalized to that of the wild- type strain. Values are means + SD of 3 independent experiments. One way ANOVA tests were used to determine whether the values were significantly different. P-values: ns, not significant; *, P ⁇ 0.05; P ⁇ 0.0005.
  • Figure 5 shows that sehl is transiently involved in Salmonella virulence upon peroral inoculation.
  • Mice were inoculated intraperitoneally (A) or perorally (B & C) with a 1: 1 mixture of two strains as indicated. Spleens were harvested two days (A), five days (C) or at various time points (B) post-inoculation and bacteria were enumerated. Each symbol represents a mouse, and horizontal bars correspond to the means +SD.
  • a one- sample t-test was used to determine whether a CI was significantly different of one, and unpaired t- tests to determine whether values were significantly different.
  • P- values ns, not significant; *, P ⁇ 0.05; **, P ⁇ 0.01; P ⁇ 0.0005.
  • Figure 6 shows that sehl and seh2 antitoxins bind to their promoter region and repress their own expression. Expressions of sehl-gfp and seh2-gfp are derepressed in minimum medium and inside RAW 264.7 mouse macrophages. Wildtype S. Typhimurium strains carrying a transcriptional fusion sehl-gfp, seh2-gfp or sifA-gfp (control) were grown in LB, minimum medium (MM) or inside RAW 264.7 mouse macrophages. Bacteria were collected at 4 (LB) or 16 (MM) hrs postinoculation or extracted from cells 16 hr post- infection and the relative fluorescence intensity of bacteria was determined by flow cytometry.
  • Strains were cultured in LB broth (Difco) or minimal medium (M9, glycerol 0.2%, MgS0 4 ImM, CaCl 2 200 mM, thiamine 1 mg/ml, casamino acids 1 mg/ml. Ampicillin (50 ⁇ g/ml), kanamycin (50 ⁇ g/ml), tetracycline (10 ⁇ g/ml) and chloramphenicol (50 ⁇ g/ml) were added when required. Bacterial suspensions were prepared from overnight LB cultures that were centrifuged and resuspended in fresh LB or minimal medium to an OD 6 oo of 1.
  • the inventors used the RASTA webtool and BLASTP of known bacterial toxins to detect putative TA modules in the S. Typhimurium genome. Finally, we selected genes organized into a putative operon of two genes.
  • Non-polar gene-deletion mutants were generated by the lambda Red recombinase system, using gene- specific primer pairs to amplify pKD4 kanamycin or pKD3 chloramphenicol resistance genes. All mutagenesis was performed in the 12023 wild- type strain. When required, Salmonella mutants were transformed with the pCP20 plasmid to excise the antibiotic cassette. Gene deletions were checked by PCR.
  • the sehl-gfp and seh2-gfp transcriptional fusions were generated by cloning PCR products corresponding to sehl and seh2 promoter regions, respectively, into pFPV25. These PCR products were digested with EcoRI and BamHI and ligated to pFPV25 digested with the same enzymes.
  • PCR products corresponding to Salmonella toxins were cloned in pDEST-17 using the Gateway system (Invitrogen, Ltd., Paisley, U.K.).
  • sehl and seh2 antitoxin genes were cloned in both pBAD- DEST49 and pMPM-K6.
  • primers contained the Ncol (5')/HindIII (3') restriction sites.
  • the resulting PCR products were digested with Ncol and Hindlll and ligated into pMPM-K6 digested with the same restriction enzymes, generating the plasmids pK6-sehAl and pK6-sehA2.
  • These plasmids contained the sehl and seh2 antitoxin genes under an arabinose inducible promoter. All constructs were confirmed by DNA sequencing.
  • Electrophoretic mobility shift assay (EMS A)
  • EMSA experiments were performed as described previously. PCR products corresponding to sehl and seh2 promoter regions were the same as used to generate sehl-gfp and seh2-gfp. These fragments (100 ng), were mixed with increasing concentrations of [His]6-tagged sehAl or sehA2 in PBS/50% glycerol. They were incubated 30 min at room temperature and then separated by electrophoresis in 6% polyacrylamide gels in IX Tris-borate-EDTA buffer. The DNA bands were visualized by staining with ethidium bromide.
  • RAW 264.7 and HeLa cell lines were grown in DMEM (GibcoBRL) supplemented with 10% foetal calf serum (FCS; GibcoBRL), 2 mM nonessential amino acids, and glutamine (GibcoBRL) at 37°C in 5% C02.
  • mice Eight- to ten-week-old C57BL/6 or 129S2 mice were inoculated intraperitoneally or perorally with equal amounts of two bacterial strains for a total of 105 bacteria per mouse.
  • the spleens were harvested 2 or 5 days after inoculation, as indicated, and homogenized. Bacteria were recovered and enumerated after plating a dilution series onto LB agar with the appropriate antibiotics.
  • Competitive indexes (CI) were determined for each mouse. The CI is defined as the ratio between the mutant and wild-type strains within the output (bacteria recovered from the mouse after infection) divided by their ratios within the input (initial inoculum). Statistical analyses were performed using Prism (GraphPad, San Diego, CA, USA).
  • the inventors have done a bioinformatics analysis several sequenced genomes of bongori and enterica species, subspecies and serotypes using the RASTA program (Sevin and Barloy-Hubler, 2007) and identified 2 toxin-antitoxin (T/AT) modules in the Salmonella chromosome.
  • T/AT modules presented opposite orientations and were separated by a gene, which encodes an esterase.
  • the modules showed an atypical organization rarely found in other bacteria so far: the toxin was upstream the antitoxin gene in the operon (Fig. 1A).
  • the inventors named these genes as sehT (S. enterica HigB toxin) for the toxins and sehA (S. enterica HigA antitoxin) for the antitoxins.
  • the modules were named the sehTl-sehAl and sehT2-sehA2 T/AT modules, respectively.
  • the inventors detected the presence of the sehTl-sehAl and sehT2-sehA2 T/AT modules in all sequenced serotypes belonging to the enterica species and specifically in the enterica subspecies (group I) such as Typhi, Paratyphi, Enteritidis, Gallinarum, Heidelberg, Dublin, Agona and Newport (Fig. IB).
  • group I enterica subspecies
  • the inventors did not find these T/AT modules in bongori species.
  • Enterica subspecies (group I) infect warm-blooded mammals, while Salmonella enterica subspecies arizonae (group Illb), also known to encode SPI2, has been isolated from reptiles and is rarely associated with human infection.
  • Salmonella enterica subspecies arizonae has been suggested to be an intermediate in evolution between the bongori and enterica species (Baumler et al., 1997). Strikingly, the inventors detected, in the arizonae subspecies the presence of the sehT2-sehA2 module but not the sehTl-sehAl module (Fig. IB). These observations suggest that the sehT2-sehA2 module was initially acquired, followed by the sehTl- sehAl . The SehTl -SehAl module is present in enterica subspecies (group I) known to be responsible for 99% of mammalian infections (Baumler et al., 1997).
  • the AsehAl antitoxin mutant grew slower than the wt, mainly at the entrance of the stationary phase (Fig. 2A). This mutant growth could be complemented by the pBAD-SehAl plasmid. The attenuated phenotype of the AsehAl mutant could not be observed when the inventors deleted both the SehAl antitoxin gene and sehTl toxin gene (Fig. 2A). This result demonstrates the importance of the SehAl antitoxin in the control of the toxicity of SehTl. One explanation would be that in the absence of the antitoxin, the toxin inhibits the bacterial growth.
  • the HigBA antitoxin is a transcriptional repressor of its own expression.
  • the inventors evaluated the expression of the sehTl -sehAl operon by using a transcriptional fusion (sehTl -gfp) that contained the promoter region of the sehTl -sehAl operon.
  • Different strains were transformed with the sehTl -gfp plasmid and grown in LB medium. Then the transcriptional expression was measured by flow cytometry. The expression of the sehTl -gfp fusion was 50-fold de-repressed in the absence of SehAl antitoxin (Fig.
  • SehAl negatively regulated its own expression.
  • the inventors purified a [His] 6 tagged version of SehAl protein and performed an electrophoretic mobility shift assay experiments.
  • the SehAl antitoxin specifically bound to the sehTl promoter region (Fig. 2C), but not to the STM3559-STM3558 (another toxin- antitoxin found in Salmonella) promoter region used as negative control. SehAl bound with high affinity to its promoter region, while Mycobacterium tuberculosis HigA binding affinity is above 50 ⁇ (Fivian-Hughes and Davis, 2010).
  • sehT2-sehA2 a transcriptional fusion (sehT2-gfp) containing the promoter region of the sehT2-sehA2 operon.
  • sehT2-gfp a transcriptional fusion
  • SehA2 antitoxin specifically bound to its promoter region indicating that sehA2 encodes an antitoxin, which directly repressed the expression of the sehT2-sehA2 operon.
  • the inventors found two palindromic sequences in the promoter region of the sehT2-sehA2 operon.
  • the palindromic sequences and the nucleotide sequences between the palindromes were both different from those found in the sehTl-sehAl promoter region.
  • the inventors sought to determine if the transcription of seh loci is altered in response to conditions encountered during interactions with animal cells. Stressful conditions increase the proteolytic cleavage of antitoxins that have a higher susceptibility to degradation than toxins. This results in both the freeing of toxins and the increased transcription-rate of TA operons. Wild- type S. Typhimurium carrying transcriptional reporters were grown in minimal medium known to induce the expression of genes necessary for intracellular survival/replication or were used to infect RAW 264.7 mouse macrophages. As a control, the inventors used a transcriptional reporter for sifA, which is required for intracellular replication and up regulated in these growth conditions (Fig. 6).
  • Sehl and Seh2 are dispensable for replication inside HeLa and RAW 264.7 cells; ii) SehTl but not SehT2 restricts weakly the growth of S. Typhimurium in cultured cells; iii) that over-expression of either antitoxin confers to Salmonella an intracellular hyper-replicative phenotype.
  • the inventors found that after mixed peroral inoculation a Asehl strain was significantly attenuated (0 0.52+0.18) (Fig. 5B).
  • mice The attenuation of a Asehl mutant upon peroral inoculation of mice suggested that Salmonella encounters conditions favouring the degradation of the antitoxin and consequently an enhanced transcription of the locus.
  • the inventors inoculated mice with wild-type S. Typhimurium carrying a transcriptional reporter for sehl. Bacteria extracted from organs were examined for the expression sehl-gfp by flow cytometry and compared to reference strains grown in LB. Most Salmonella extracted from inoculated mice showed GFP fluorescence levels that were comparable to that of wild- type S. Typhimurium grown in LB. This indicates that the majority of bacteria presented a basal or slightly increased level of sehl expression.
  • GFP hlgh bacteria corresponded to Salmonella in which expression of sehl is de-repressed.
  • MN mesenteric lymph nodes
  • GFP hlgh bacteria were hardly detected the spleen of mice inoculated intraperitoneally, while this population was significantly more prevalent in the spleen and MLNs of mice inoculated perorally. Yet, in these conditions, the percentage of GFP hlgh Salmonella did not exceed 5%.
  • the mean fluorescence of total bacteria was significantly increased in populations extracted from perorally but not from intraperitoneally inoculated mice.
  • the mean fluorescence of GFP hlgh bacteria was significantly higher in bacteria extracted form MLNs in comparison to spleens. Considering that after having crossed the intestinal barrier the bacteria reach MLNs before the spleen, these results confirm that activation of the sehl module is an early event in the process of infection through oral ingestion.
  • SPI2 is the main virulence determinant of S. Typhimurium
  • the inventors analyzed whether attenuation mediated by SehAl was SPI-2-dependent. The inventors first studied the fate of PipB2 and SseJ and showed that the translocation of these two SPI2 effector proteins into macrophages was not altered in the absence of SehAl. Moreover, an AsehAl AssaV double mutant was even more attenuated than the AsehAl mutant. The inventors concluded that the attenuation of the AsehAl mutant was not dependent on SPI-2. DISCUSSION
  • Type II TA modules are inhibitors of translation that induce bacterial dormancy. Being in a dormant state help bacteria to survive harmful environments.
  • This study presents evidence that virulent strains of Salmonella possess specific TA modules and shows that one of these is beneficial in the early phase of the infection by the natural route.
  • the inventors identified eleven toxin/antitoxin systems in S. Typhimurium and examined the presence of homologous genes across Salmonellae.
  • the heatmap generated from clustering by both strains and TA modules genes highlighted a set of four toxin/antitoxin systems that are found across serovars of S. enterica infecting warm-blooded animals, while the nonpathogenic species and strains of S. enterica, which are associated with coldblooded animals, have no or a low number of TA modules, respectively.
  • Sehl plays a role in an early step of the infection that is temporally localized between the arrival in the stomach and the triggering of a systemic infection.
  • the CI in perorally inoculated mice was the same at 5 and 18 days post- infection, thus confirming that Sehl plays a transient role and is not involved in the systemic phase of the infection.
  • Sehl helps to survive the biochemical stresses (NO, antibacterial peptide%) and/or the innate immune cellular defences present in the gut and the subepithelial environment. Indeed, this possibility is supported by the presence of Salmonella with a high activation profile of the sehl promoter in MLNs, into which Peyer's patches are drained.
  • the HigB toxin of E. coli K12 cleaves mRNAs positioned at the ribosomal A-site and stops the translation.
  • the mechanism of action of SehTl is unknown but, considering that SehTl presents 40% identity with HigB, these toxins are likely to have identical targets.
  • SehTl activity is dependent on an additional mechanism. Indeed, a AsehAl strain, which expresses a high level of free toxin, presents only a partial growth defect in LB or in cultured cells. In contrast, the virulence of a AsehAl mutant is severely impaired in the mouse model as its defect is comparable to the strongly attenuated AssaV mutant.
  • toxins and antitoxins form hetero- oligomers that are toxin-inhibiting complexes and that bind their promoter region.
  • the studies of the inventors have shown that SehAl forms dimers whereas SehTl forms tetramers and aggregates. The aggregation of SehTl might result from a non-native folding and explain its lack of interaction with the antitoxin. However, is it also possible that SehAl and SehTl do not interact.
  • the dimer of SehAl binds a palindromic stretch of its promoter and could well be the functional unit that represses sehl transcription.
  • SehAl inhibits effectively the operon expression even in absence of its cognate toxin.
  • the activity of the toxin in mice seems to be regulated by the aforementioned additional factor rather than by SehAl.
  • SehTl Stress conditions leading to afully active SehTl are poorly experienced in cultured cells. This is shown by the limited activation of the sehl promoter and the weak growth defect of a Asehl mutant. This suggests that Sehl does not support the survival/replication of intracellular bacteria. Another possibility is that HeLa and RAW264.7 cells are not representative of cell types Salmonella encounters during the early phase after oral inoculation. Sehl might help to survive the bactericidal activity of immune cells that are recruited to the infection sites. Supporting this observation is the fact that Salmonella are not killed and survive in dendritic cells and that this survival is independent of virulence factors important in macrophages.
  • PemK toxin of Bacillus anthracis is a ribonuclease: an insight into its active site, structure, and function. J Biol Chem. 2010 Mar 5;285(10):7254-70. Epub 2009 Dec 17. Baumler, A. J. 1997. The record of horizontal gene transfer in Salmonella. Trends Microbiol. 5: 318-322.
  • Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence- specific and frame- dependent mRNA cleavage. Mol. Microbiol. 71: 1071-1087.
  • RASTA-Bacteria a web-based tool for identifying toxin- antitoxin loci in prokaryotes. Genome Biol. 8:R155. Shea, J. E., Beuzon, C. R., Gleeson, C, Mundy, R., and Holden, D. W. 1999. Influence of the Salmonella typhimurium pathogenicity island 2 type III secretion system on bacterial growth in the mouse. Infect Immun. 67: 213-219. Winther, K. S., and Gerdes, K. 2009. Ectopic production of VapCs from Enterobacteria inhibits translation and trans-activates YoeB mRNA interferase. Mol. Microbiol. 72: 918-930.

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Abstract

L'invention concerne un/des marqueur(s) du pouvoir pathogène de Salmonella et leurs utilisations.
PCT/EP2013/063900 2012-07-02 2013-07-02 Marqueur du pouvoir pathogène de salmonella WO2014006025A2 (fr)

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