WO1999045136A1 - Methods for assaying type iii secretion inhibitors - Google Patents

Abstract

A method of identifying compounds that specifically inhibit type III secretion systems that are used by several Gram-negative animal and plant pathogens to secrete virulence factors that are critical in causing disease. The compounds identified by this method are used as new antibacterial therapeutics. Specific inhibitors of the enteropathogenic Escherichia coli (EPEC) type III secretion system, that block EPEC signaling in host cells are identified by the use of specific molecular tools that have been developed with EPEC, including specific antibodies to secreted proteins and genetic fusions of epitope tags to genes encoding these secreted products. Promising compounds identified with the EPEC system are tested for their ability to inhibit type III secretion systems in other medically important pathogens.

Description

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METHODS FOR ASSAYING TYPE III SECRETION INHIBITORS FIELD OF THE INVENTION This invention relates to the development of a novel antibacterial therapeutics, and more particularly to inhibitors of the type III secretion system.

BACKGROUND OF THE INVENTION

Antibiotics have been used for years to successfully treat diverse bacterial infections, with spectacular results. As a result, many pharmaceutical companies have redeployed their resources to examine other pharmaceutical areas. However, bacterial resistance to antibiotics has been increasing exponentially over the past few years. Many pathogens are now resistant to several antibiotics. In some cases, the diseases they cause are not treatable with conventional antibiotics. Such resistance may continue to rapidly increase in the future. Despite the past successes of antibiotics, there have been no new classes of antibiotics developed in the past two decades. New variations on existing drugs have been introduced, but resistance to these compounds usually arises within a year. A new strategy to develop novel therapeutics to treat bacterial infections is desperately needed.

The knowledge of the molecular mechanisms of pathogenesis has increased rapidly over the past decade. The molecular mechanisms of toxins, adherence, invasion, intracellular parasitism, and regulation have been studied in increasing detail. Each virulence factor encompasses different mechanisms, making the development of a broad spectrum inhibitor impossible. To develop an ideal anti-infective, the bacterial virulence mechanism to be inhibited should be conserved among many pathogens, be specific for virulence mechanisms, and not be present in infected eukaryotic host cells.

Some conserved virulence mechanisms that could be a target for a therapeutics are the two-component regulatory systems. However, these systems are not specific for virulence factors. They are used in several bacterial housekeeping systems. Additionally, they have also been identified in eukaryotic systems, increasing the risk of infected eukaryotic host toxicity if an inhibitor was developed.

A different bacterial virulence mechanism is the type III secretion system. The type HI secretion pathway was only recently discovered. It is conserved in many diverse Gram- negative pathogens. The function of the pathway is to secrete virulence factors only. The type III secretion pathway is not involved in housekeeping functions nor has it been found in normal flora.

SUMMARY OF THE INVENTION

The invention provides a method for identifying compounds that specifically inhibit type III secretion systems, used by several pathogens to secrete virulence factors that are critical in causing disease. The method identifies inhibitors of bacterial type HI secretion systems, by contacting bacteria having a bacterial type HI secretion system with a compound suspected of inhibiting the bacterial type III secretion system, under conditions that allow secretion; and detecting the secretion of a type El-secreted polypeptide. In the practice of the method, a reduced level of secretion is indicative of inhibition of bacterial type III secretion systems. The invention also provides kits useful for the practice of the methods of the invention.

The compounds identified by the method of the invention are used as new antibacterial agents. First, specific inhibitors of the enteropathogenic Escherichia coli (EPEC) type III secretion system, that block EPEC signaling in infected eukaryotic host cells are identified by the use of specific molecular tools have been developed with EPEC, including specific antibodies to secreted proteins and genetic fusions of epitope tags to genes encoding these secreted products. Second, promising compounds identified with the EPEC system are tested for their ability to inhibit type HI secretion systems in other medically important pathogens. Third, these compounds are tested for their ability to inhibit disease in relevant animal disease model systems. This process thereby identifies potential novel therapeutics. - 3 -

DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a polyacrylamide gel. Various plant extracts were assayed for inhibitory compounds which might block type HI secretion system. EPEC was contacted with plant extracts and the resulting EPEC-secreted proteins were subjected to electrophoresis. EspC and EspB are EPEC-secreted proteins. EPEC in lanes 1 and 12 were not contacted with plant extract, cfm, an EPEC strain defective for secretion, in lanes 2 and 11 were also not contacted with plant extract. Several plant extract samples gave decreased secretion levels based on this primary assay and blockage of EspC. Although the extracts were not killing the bacteria, the compounds in the plant extracts decreased generalized secretion in the bacteria, both type III and other systems.

FIGURE 2 is a polyacrylamide gel. Various soil microbe extracts were assayed for inhibitory compounds which might block type IH secretion system. EPEC was contacted with soil microbe extracts and the resulting EPEC-secreted proteins were subjected to electrophoresis. EspC and EspB are EPEC-secreted proteins.EPEC in lane 1 was not contacted with plant extract cfm in lane 2 is an EPEC strain defective for secretion and was also not contacted with plant extract. Only type III mediated proteins were being inhibited. EspC was found in normal amounts in the supernatant, but type HI secreted proteinswere not.

DETAILED DESCRIPTION OF THE INVENTION The invention provides a method for identifying compounds that specifically inhibit type HI secretion systems. A polynucleotide encoding a selectable marker is present in bacteria having a bacterial type HI secretion system. A compound suspected of inhibiting the bacterial type HI secretion system is contacted with the bacteria under conditions that allow secretion of the polypeptide encoded by the polynucleotide. The secreted polypeptide is detected. A reduced level of secretion is indicative of the inhibition of bacterial type III secretion systems. - 4 - The invention also provides kits useful for the practice of the methods of the invention.

The details of one or more embodiments of the invention are set forth in the description infra. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Antibacterial Agents

The invention provides a method for the identification of an antibacterial agent. As used herein, an "antibacterial agent" is a substance that can kill, inhibit the growth of, or reduce the virulence of bacteria. Among the antibacterial agents identified by the method of the invention are those that inhibit virulence factors secreted by type HI secretion systems. In contrast to previous antibiotics, inhibitors of type HI secretion do not kill or inhibit growth of pathogens. Instead, they block the secretion of virulence factors that are critical to causing disease. The type III secretion system is the first virulence mechanism that shows a large degree of conservation between diverse pathogens, and thus specific inhibitors of type IE secretion are broad spectrum therapeutics.

An antibacterial agent identified by the method of the invention can inhibit type HI secretion systems of medically important bacterial pathogens. Examples of medically important bacterial pathogens are provided infra. Identification and testing of these specific inhibitors of type III secretion is a major step to develop a novel therapeutic that would block the virulence of several important human, animal, and plant pathogens. The compoimds identified by the method of the invention as specific inhibitors of type HI secretion are identified as new antibacterial therapeutics. First, specific inhibitors of the enteropathogenic Escherichia coli (EPEC) type HI secretion system, that block EPEC signaling in infected eukaryotic host cells, are identified by the use of specific molecular tools, including specific antibodies to secreted proteins and genetic fusions of epitope tags to genes encoding these secreted products. The screening method identifies inhibitors of type IH secretion that either physically (e.g., stearically) or functionally inhibit secretion. Second, specific inhibitors of type HI secretion identified with the - 5 -

EPEC system are tested for their ability to inhibit type in secretion systems in other medically or economically important pathogens. Third, these compounds are tested for their ability to inhibit disease in relevant animal disease model systems. This three-step process thereby identifies potential novel therapeutics. The effectiveness of such novel therapeutics is readily determined to develop more potent derivatives.

Because of the extensive conservation of the type III secretion machinery (that can often be interchanged between various pathogens), an inhibitor of one pathogen blocks the type III pathway in many other pathogens. The identified inhibitor is thus a broad spectrum compound that inhibits disease production in several virulent organisms. This conserved secretion pathway is critical for pathogenesis in several human pathogens, including Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Salmonella typhi, Salmonella typhimurium, Salmonella enteritica, all other Salmonella species, Campylobacter species, all Shigella species, including S. flexneri and S dysenteriae, Pseudomonas aeruginosa, EPEC, enterohemorrhagic E. coli (EHEC), Hafrtia alvei, Bordetella sp. and Chlamydia species. In addition, the conserved secretion pathway is needed for disease in animal pathogens such as rabbit E. coli (RDEC-1), and Citrobacter rodentiii. It is also critical for disease production in several important plant pathogens. Several Gram negative pathogens cause economically important damage to plant crops using a conserved type HI secretion system, as do human pathogens. Plant pathogens inclide Pseudomonas syringae, P. solanacearum, and Xanthamonas campestris.

The benefit of a method for identifying specific inhibitors of type IH secretion is that these novel therapeutics are useful in the treatment of several human, animal, and plant diseases. Such compounds are also of significant scientific use to dissect the working mechanisms of type III secretion systems. Many gene products (approximately twenty) are needed for type III secretion. Inhibitors could be used to uncouple the system and identify at what stage inhibition occurs. Such work would include fractionation of bacteria to determine where secreted products are blocked during their secretion out of - 6 - bacteria, and defining which gene product is being affected. This would yield valuable information about a generalized secretion system utilized by several bacterial pathogens to cause disease. Determining whether blockage of virulence mechanisms attenuates disease is important for the study of microbial virulence factors, in addition to the antibiotic development area.

The assay for type III secretion depends on the presence of viable bacteria. When compounds are bactericidal or bacteriostatic, lower secretion levels are found or secretion is non-existent. Therefore, this assay for type Ifl secretion is also a bactericidal assay in the primary screens.

Compounds suspected of inhibiting type III secretion can be extracted and purified from the culture media or a cell by using known protein purification techniques commonly employed, such as extraction, precipitation, ion exchange chromatography, affinity chromatography, gel filtration and the like. A first line of evidence was found when screening various random plant extracts. Of 200 samples, three plant extract samples gave decreased secretion levels based on this primary assay and blockage of EspC. Although the extracts were not killing the bacteria, the compounds in the extracts decreased generalized secretion in the bacteria, both type III and other systems, as shown in FIGURE 1.

A second line of evidence to indicate that this assay does detect the secreted proteins. Various soil microbe extracts were assayed for inhibitory compounds which might block type III ssecretion system. The secondary screen, observing EPEC-secreted proteins on polyacrylamide gels, indicated that only the type III mediated proteins were being inhibited, as EspC was found in normal amounts in the supernatant, but no type HI proteins. However, these extracts contained proteases which degraded the EPEC type HI secreted proteins. EspC was not degraded in this assay because it is protease resistant, as shown in FIGURE 2. - 7 -

In both^" these cases, this assay has worked well for measuring the level of secretion of these proteins.

Bacteria

The method of the invention identifies antibacterial agents by contacting bacteria with a compound. Among the bacteria that can be contacted are Gram-negative bacteria. The term "Gram-negative bacteria" refers to a diverse group of prokaryotes whose cell wall stains pink (negative) in Gram stain. Gram-negative bacteria include spirochetes such as Treponema and Borrelia, Gram-negative bacilli including the Pseudomonadaceae, Legionellaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellaceae, Gram-negative cocci such as Neisseriaceae, anaerobic Bacteroides, and other Gram-negative bacteria including Rickettsia, Chlamydia, and Mycoplasma.

Gram-negative bacilli are important in clinical medicine. They include (1) the Enterobacteriaceae, a family that comprises many important pathogenic genera, (2) Vibrio, Campylobacter and Helicobacter genera, (3) opportunistic organisms (e.g., Pseudomonas, Flavobacterium, and others) and (4) Haemophilus and Bordetella genera. The Gram-negative bacilli are the principal organisms found in infections of the abdominal viscera, peritoneum, and urinary tract, as well secondary invaders of the respiratory tracts, burned or traumatized skin, and sites of decreased infected eukaryotic host resistance. Currently, they are the most frequent cause of life-threatening bacteremia. Examples of pathogenic Gram-negative bacilli are E. coli (diarrhea, urinary tract infection, meningitis in the newborn), Shigella species (dysentery), Salmonella typhi (typhoid fever), Salmonella typhimurium (gastroenteritis), Yersinia enterocolitica (enterocolitis), Yersinia pestis (black plague), Vibrio cholerae (cholera), Campylobacter jejuni (enterocolitis), Helicobacter jejuni (gastritis, peptic ulcer), Pseudomonas aeruginosa (opportunistic infections including bums, urinary tract, respiratory tract, wound infections, and primary infections of the skin, eye and ear), Haemophilus influenzae (meningitis in children, epiglottitis, otitis media, sinusitis, and bronchitis), and Bordetella pertussis (whooping cough). Vibrio is a genus of motile, Gram-negative - 8 - rod-shaped bacteria (family Vibrionaceae). Vibrio cholerae causes cholera in humans; other species of Vibrio cause animal diseases. E. coli colonize the intestines of humans and warm blooded animals, where they are part of the commensal flora, but there are types of E. coli that cause human and animal intestinal diseases. They include the enteroaggregative E. coli (ΕaggΕC), enterohemorrhagic E. coli (ΕHΕC), enteroinvasive E. coli (ΕIΕC), enteropathogenic E. coli (ΕPΕC) and enterotoxigenic E. coli (ΕTΕC). Uropathogenic E. coli (UPΕC) cause urinary tract infections. There are also neonatal meningitis E. coli (NMΕC).

The pathogenic bacteria in the Gram-negative aerobic cocci group include Neisseria, Moraxella (Branhamella), and the Acinetobacter . The genus Neisseria includes two important human pathogens, Neisseria gonorrhoeae (urethritis, cervicitis, salpingitis, proctitis, pharyngitis, conjunctivitis, pharyngitis, pelvic inflammatory disease, arthritis, disseminated disease) and Neisseria meningitides(memngitis, septicemia, pneumonia, arthritis, urethritis). Other Gram-negative aerobic cocci that were previously considered harmless include Moraxella (Branhamella) catarrhalis (bronchitis and bronchopneumonia in patients with chronic pulmonary disease, sinusitis, otitis media) has recently been shown to be an common cause of human infections.

Type HI secretion systems are found in many pathogenic organisms. The role of the ΕPΕC type III secretion system in these pathogens is to secrete proteins necessary for formation of the attaching/effacing (A/Ε) lesion (or pedestal). As used herein, the term "Attaching/Effacing bacteria" refers to a group of pathogenic organisms that adhere to infected eukaryotic host cells and cause localized accumulation of infected eukaryotic host actin beneath adherent organisms. Pathogens in this group include EPEC; EHEC, the causative agent of hemorrhagic colitis (nicknamed "hamburger disease" because it is often associated with ground beef) and hemolytic uremic syndrome; several EPEC-like animal pathogens that cause disease in rabbits, dogs, pigs, etc. (including rabbit enteropathogenic E. coli; RDΕC-1); Citrobacter rodentii, that causes neoplasia in mice; and Hafnia alvei, a minor human pneumonia pathogen. In all cases tested thus far, mutations in the type HI secretion system attenuate virulence completely.

EPEC is a leading cause of infant diarrhea and was the first E. coli shown to cause gastroenteritis. EPEC continues to be a significant cause of infantile diarrhea in developing nations contributing to high morbidity and mortality. EPEC forms small microcolonies on the surface of infected epithelial cells followed by intimate contact and localized degeneration of the epithelial brush border microvilli, cumulating in an A/E lesion. The A/E lesion is associated with the assembly of highly organized cytoskeletal structures in epithelial cells immediately beneath the adherent bacteria that include the cytoskeletal components actin, α-actinin, myosin light chain, ezrin, and talin.

A three-stage model describes EPEC pathogenesis. An initial localized adherence to epithelial cells, mediated by a type IV fimbria, is followed by the activation of infected eukaryotic host epithelial cell signal transduction pathways and intimate attachment to infected eukaryotic host epithelial cells. These final two steps are collectively known as attaching and effacing. The signal transduction in the eukaryotic host epithelial cells involves activation of eukaryotic host cell tyrosine kinase activity leading to tyrosine phosphorylation of a 90 kilodalton (kDa) infected eukaryotic host membrane protein, Hp90, and fluxes of intracellular inositol phosphate (IP₃) and calcium. Following this signal transduction, the bacteria adhere intimately to the surface of the epithelial cell, accompanied by damage to infected eukaryotic host epithelial cell microvilli and accumulation of cytoskeletal proteins beneath the bacteria.

EHEC has been linked to many food-bome outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome worldwide. The most common epidemiologically-associated food is ground beef. EHEC causes no disease symptoms in cattle, but beef and dairy cattle carry EHEC in their intestinal tracts. Contamination of carcasses occurs during slaughter operations. - 10 -

As used herein, the term "eukaryotic host" refers to organisms that are infected by the pathogens. The eukaryotic host may be human, animal, or plant. For example, a eukaryotic host for EPEC and EHEC is human. A eukaryotic host for RDEC-1 is rabbit; a rabbit model is provided in EXAMPLE V. Plants are eukaryotic hosts for Pseudomonas syringae, P. solanacearum, and Xanthamonas campestris. As used herein, the term "infected eukaryotic host" is a eukaryotic host that has been infected by a pathogen, whether or not the eukaryotic host is currently suffering from a disease caused by the pathogen. As used herein, the terms "eukaryotic host cell" and "infected eukaryotic host cell" refer to cells of the eukaryotic host. The term "infected eukaryotic host cell" includes both eukaryotic cells that are infected by the pathogen and the uninfected cells of an infected eukaryotic host.

The invention provides a method for identifying inhibitors of a type IH secretion system wherein the inhibition blocks signaling in infected eukaryotic host cells. For example, the signal transduction in EPEC-infected eukaryotic host epithelial cells involves activation of eukaryotic host cell tyrosine kinase activity leading to tyrosine phosphorylation of a 90 kDa infected eukaryotic host membrane protein, Hp90, and fluxes of intracellular inositol phosphate (TP₃) and calcium. Following this signal transduction, EPEC adheres intimately to the surface of the epithelial cell, accompanied by damage to infected eukaryotic host epithelial cell microvilli and accumulation of cytoskeletal proteins beneath the bacteria.

Type III Secretion Pathway

The method of the invention identifies antibacterial agents by contacting bacteria with a compound suspected of inhibiting the bacterial type III secretion system. As used in this invention, the term "type III secretion" and "type IH secretion pathway" refer to a specialized machinery to export molecules across a cell membrane. - 11 -

Gram-negative bacteria need specialized machinery to export molecules across their two membranes and the periplasm, a process critical for moving virulence factors to the bacterial surface where they can interact with eukaryotic host components. Gram- negative secretion has been divided into four major pathways. Type I secretion is used by a small family of toxins, with E. coli hemolysin being the prototype. The type II secretion system is the major export pathway used by most gram negative bacteria to export many molecules, including some virulence factors. It shares homology to mammalian drug resistance mechanisms. The type IV secretion system is encoded within the secreted product, that cleaves itself as part of the secretion mechanism. The prototype of this system is the Neisseria IgA protease, and this family includes a handful of other virulence factors. The most recently discovered secretion pathway is the type HI pathway.

A type III secretion systems was originally described as a secretion system for Yersinia- secreted virulence proteins ("Yops"). Yop secretion is critical for Yersinia virulence. In the case of Yersinia species, at least two of the Yop proteins that utilize a type HI export pathway have defined functions. YopH is a tyrosine kinase that phosphorylates eukaryotic host proteins, while YopΕ is translocated into the eukaryotic host cell where it leads to the disruption of infected eukaryotic host actin filaments. A homologous secretion system was then identified in several plant pathogens, including Pseudomonas syringae, P. solanacearum, and Xanthomonas campestris. These plant pathogens use this secretion pathway to secrete virulence factors (hairpins and others) that are critical for causing disease in plants. Although the secretion system is similar, harpins and Yops (i.e., the secreted virulence factors) are not related. More recently, several other type πi secretion systems have been identified in other pathogens, and in all cases shown to be necessary for virulence. These systems include the highly conserved invasion systems of Salmonella and Shigella species used to enter non-phagocytic cells and cause disease. Both Shigella and Salmonella species use such a mechanism to secrete proteins that are needed for bacterial invasion into eukaryotic host cells. A second type IH secretion system has been identified in Salmonella, that is critical for disease, although the secreted - 12 - products of this pathway and the virulence mechanisms have not yet been established. Finally, Pseudomonas aeruginosa has a type IH secretion system necessary for secretion of Exoenzyme S, a potent virulence factor.

The molecular mechanisms of this secretion pathway use ATP as an energy source. Secretion of proteins by the type III export pathways requires dedicated chaperone molecules. Many gene products (approximately twenty) are needed for type HI secretion, yet a molecular function for all but the ATPase has yet to be identified. The type IH pathway is quite different from other secretion pathways found in Gram-negative bacteria, although it bears some homology to flagella and filamentous phage assembly genes. It does not resemble any mammalian pathway (unlike the type II pathway, that has homologies with multi-drug resistance (MDR) in mammalian cells). It is not found in non-pathogenic isolates, and is always associated with disease production. The virulence factors secreted vary between pathogens, although components of the secretion machinery are interchangeable, at least for Salmonella, Shigella, and Yersinia. For Salmonella and Shigella species, this pathway is critical for invasion of eukaryotic host cells. For Yersinia species, it is needed to inject anti-phagocytic factors into infected eukaryotic host cells to prevent phagocytosis. For EPEC and EHEC, it is needed to mediate intimate interactions with epithelial cells in the gut. This bacterial secretion system also pumps bacterial virulence factors into the mammalian cell, that then mediate virulence and pathogenesis. For plant pathogens, it is needed to secrete harpins that damage plant cells. Thus the type III secretion systems are the first virulence factor- specific conserved pathways identified in bacteria, and represent ideal targets for potential inhibitors.

Polynucleotide The method of the invention identifies antibacterial agents by contacting bacteria containing a polynucleotide with a compound. As used herein, the term "polynucleotide" refers to a nucleic acid, e.g., a DNA or RNA molecule, that is not immediately contiguous with the 5' and 3' flanking sequences with which it normally is immediately - 13 - contiguόus when present in the naturally occurring genome of the organism from which it is derived. The term thus describes, for example, a nucleic acid that is incorporated into a vector, such as a plasmid or viral vector; a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that at which it naturally occurs); and a nucleic acid that exists as a separate molecule, e.g., a DNA fragment produced by polymerase chain reaction (PCR) amplification or restriction enzyme digestion, or an RNA molecule produced by in vitro transcription. The term also describes a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion protein. EXAMPLE I provides an EspB fusion protein with an HSN tag. EXAMPLE IH provides an EspA fusion protein with an HSV tag. EXAMPLE VI provides T7-tz^'r and ttr-HSV gene products.

The methods of the invention may use polynucleotides as templates in standard methods for production of proteins that are secreted by a type HI secretion system. These isolated nucleic acids can be ligated into vectors and introduced into suitable host cells for expression. Methods of stable transfer, meaning that the foreign DΝA is continuously maintained in the host, are known in the art. Methods of ligation and expression of nucleic acids within cells are provided by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989), incorporated herein by reference.

As used herein, "vectors" includes plasmids, DNA and RNA viral vectors, baculoviral vectors, vectors for use in yeast, and other vectors well known to those of skill in the art. Several types of vectors are commercially available and can be used to practice this invention. Examples of vectors useful in the practice of this invention include those as widely varied as the low-copy vector pMW118, the positive-selection suicide vector pCVD442, and the commercially available pBluescript II SK(+) (Stragene, La Jolla, CA). - 14 -

When the vector is a plasmid, it generally contains a variety of components including promoters, signal sequences, phenotypic selection genes, origins of replication sites, and other necessary components as are known to those of skill in the art. Promoters most commonly used in prokaryotic vectors include the lacZ promoter system, the alkaline phosphatase phoA promoter, the bacteriophage λPL promoter (a temperature sensitive promotor), the tac promoter (a hybrid trp-lac promoter regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter. For example, the low-copy vector pMWl 18 under control of the lac∑ promoter may be used. A signal sequence is typically found immediately 5' to the nucleic acid encoding the peptide, and is thus transcribed at the amino terminus of the polypeptide. In EXAMPLE VI, constructions of Tl-tir and tzr-HSV were constructed in pET28a and pET28b set of vectors, then cloned into a pACYl 84-based vector for expression.

Typical phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell. For example, ampicillin resistance gene (amp) and the tetracycline resistance gene (tet) are readily employed for this purpose. Construction of suitable vectors containing polynucleotides encoding type El-secreted polypeptide are prepared using standard recombinant DNA procedures well known to those of skill in the art.

As used herein, in the context of genetic methods, the term "host cell" refers to cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

The methods of the invention may use a host cell containing a vector having a polynucleotide encoding the type El-secreted polypeptide. Methods of expressing polynucleotide sequences in bacteria are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known - 15 - in the art. Such vectors are used to incorporate DNA sequences of the invention. Among the pro kary otic organisms that may serve as host cells are E. coli strain JM101, E. coli K12 strain 294 (ATCC number 31,446), E. coli strain W3110 (ATCC number 27,325), E. coli XI 776 (ATCC number 31,537), E. coli XL-1 Blue (Stratagene), and E. coli B; however, many other strains of E. coli, such as HB101, NM522, NM538, NM539 and many other species and genera of prokaryotes can be used as well. Besides the E. coli strains listed supra, other enterobacteriaceae such as Salmonella typhimunium or Serratia marcesans and various Pseudomonas species can all be used as hosts. In one embodiment, the organism is an Attaching/Effacing pathogen. Among the Attaching/Effacing pathogens that may be transformed are EPEC, EHEC, and RDEC- 1. In EXAMPLE VII, a plasmid is grown in two EPEC host cells: in UMD864 and in cfm\4.2Λ as a negative control.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired. For another example, triparental conjugation may be used to genetically introduce vector into E. coli, especially EPEC, EHEC, or RDEC-1 The transformed cells are selected by growth on an antibiotic, commonly tetracycline (tet) or ampicillin (amp), to which they are rendered resistant due to the presence of tet or amp resistance genes on the vector. - 16 -

Polypeptide

The method of the invention identifies antibacterial agents by contacting bacteria containing a polynucleotide with a compound suspected of inhibiting the bacterial type III secretion system, where the polynucleotide encodes a polypeptide. As used herein, the term "polypeptide" encompasses type El-secreted proteins, any naturally occurring allelic variant thereof as well as manufactured recombinant forms. As used herein, polypeptides encompass both naturally occurring and recombinant forms, i.e., non- naturally occurring forms of the protein and the peptide that are sufficiently identical to naturally occurring peptide to have a similar function of being secreted by a type El secretion system. Examples of such polypeptides include, without limitation, the EspA, EspB, intimin, and Tir polypeptides from EPEC, EHEC, and RDEC-1. Also included in the invention are polypeptides having sequences that are substantially identical to the sequence of a type IE-secreted polypeptide, including EspA, EspB, intimin, and Tir polypeptide. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence only by conservative amino acid substitutions, for example, substitutions of one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine), or by one or more non-conservative substitutions, deletions, or insertions, provided that the polypeptide retains the ability to be secreted by a type IE secretion system and at least one epitope. For example, one or more amino acids can be deleted from an EspA, EspB, intimin, and Tir polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its ability to be secreted by a type HI secretion system. For example, amino- or carboxyl-terminal amino acids that are not required for biological activity, can be removed. Such modifications can result in the development of smaller active polypeptides. - 17 -

The terni "polypeptides" also includes recombinant polypeptides that can be secreted by the type El secretion system. Recombinant methods for producing fusion proteins are described infra.

Polypeptides Secreted by the Type I I Pathway The method of the invention identifies antibacterial agents by contacting bacteria containing a polynucleotide with a compound suspected of inhibiting the bacterial type III secretion system, where the polynucleotide encodes a polypeptide secreted by the bacterial type III secretion system. The release of proteins by many pathogenic microorganisms is integral to their pathogenicity. Many Gram-negative pathogenic organisms have genetic loci encoding many genes that collectively form a type El export pathway responsible for the export and assembly of surface appendages or secretion of proteins. Recently, some of the bacterial components involved in pedestal formation have been identified. EPEC possess a virulence plasmid that encodes the bundle forming pilus and a positive virulence factor regulator, Per. All of the genes encode products necessary for pedestal formation are found within a 35 kilobase pair (kb) pathogenicity island in the E. coli chromosome. Within the Locus of Enterocyte Effacement (LEE) region are several genes whose products have different functions, including a type IE secretion apparatus proteins, secreted effector molecules and their chaperones, and intimin.

EPEC secretes at least five proteins: 110 kDa (EspC); 40 kDa; 39 kDa; 37 kDa (EspB); and 25 kDa (EspA) into culture media under certain conditions. At least two of these bacterial proteins (EspA and EspB) are necessary for activating EPEC induced signals in epithelial cells. These signals include calcium and inositol phosphate fluxes, and tyrosine phosphorylation of Hp90. These proteins, except EspC, are secreted by a type IE secretion system that is encoded by the sep machinery. This sep locus is homologous to similar type IE secretion systems found in other enteric pathogens including Yersinia, Shigella, and Salmonella. Mutations in espA or espB, or those in the type IE secretion - 18 - system (sep and cfm) result in organisms that are unable to signal or induce binding of intimin to epithelial cell surfaces. A 35 kb LEE locus encodes the sep cluster, eαeA, e,spA and espB.

As used herein, the term "EspA" (for EPEC-secreted [or signaling] protein A) refers to a polypeptide that is a secreted protein from enteropathogenic or enterohemorrhagic E. coli and has a molecular weight of about 25 kDa as determined by SDS-PAGE. EspA is an enteropathogenic E. co/z-secreted protein necessary for activating epithelial cell signal transduction, intimate contact, and formation of attaching/effacing lesions, processes correlated with disease. Although EspA is required for EPEC invasion into epithelial cells, it bears no significant homology to the Shigella Ipa invasins.

As used herein, the term "espA" refers to polynucleotide encoding the EspA polypeptide. These polynucleotides include DNA, cDNA and RNA sequences that encode EspA. All polynucleotides encoding all or a portion of EspA are also included herein. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. All degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of EspA peptide encoded by the nucleotide sequence is functionally unchanged.

As used herein, the terms "espB" and "eαeA" refer to genes other than espA that encode enteropathogenic E. co//-secreted proteins. As used herein, the term "EspB" and "intimin" or "EaeA" refer to the proteins encoded by the espB and the eαeA genes, respectively. Intimin is the product of a bacterial chromosomal LEE locus, eaeA , and is a 94 kDa EPEC outer membrane protein that is needed for intimate adherence. Using human volunteers, it has been shown that intimin is needed for full EPEC virulence. Intimin (eαeA) mutants have been described by Kenny and Finlay (Infection & mmunity 65(7) 2528-2536, 1997). Mutants defective in eaeA form immature Attaching/Effacing (A/E) lesions and do not organize phosphotyrosine proteins and cytoskeletal components beneath adherent bacteria, although epithelial signal transduction is still activated. - 19 -

Intimin participates in reorganization of the underlying infected eukaryotic host cytoskeleton after other bacterial factors (EspA and EspB) stimulate epithelial signal transduction. Intimin binding to infected eukaryotic host cells also stimulates a second wave of signal transduction inside the mammalian cell, including tyrosine phosphorylation of phospholipase Cγ.

Another EPEC-secreted polypeptide has recently been identified as being secreted through the type El-secretion pathway. The protein (Hp90) associated with virulence in Attaching/Effacing bacteria, through its role as intimin receptor, is actually produced by the Attaching Effacing bacteria. Tir (for translocated intimin receptor; formerly Hp90) is secreted by pathogenic E. coli. Tir polypeptide is secreted by an Attaching/Effacing pathogen, such as EPEC or EHEC. Tir from EPEC is necessary for activating epithelial cell signal transduction, intimate contact, and formation of attaching/effacing lesions, processes correlated with disease. Tir has a molecular weight of about 78 kDa as determined by SDS-PAGE, but when obtained from epithelial cells (Hp90) has a molecular weight of about 90 kDa as determined by SDS-PAGE. Although the Tir protein secreted from EPEC is predicted to encode a 56.8 kDa protein, a molecular mass of about 78 kDa was observed for the secreted protein, which may reflect some additional bacterial modification or abnormal migration due to amino acid composition or structural features. Tir is predicted to have two transmembrane domains with the six tyrosine residues, potential kinase substrates, in the C-terminal half.

EspA, EspB, and intimin are secreted by EHEC as well as by EPEC. A homologous tir gene in EHEC has also been cloned. However, EHEC does not cause tyrosine phosphorylation of its receptor, indicating differences between these two pathogens.

Several pathogenic Gram-negative bacteria use type IE secretion systems to cause various effects in infected eukaryotic host cells. EPEC is the first pathogen that is recognized to use a type IE system to insert a bacterial receptor into infected eukaryotic host cell. Other pathogens may also use this strategy, especially for those in which the - 20 - mammaϊian receptor has not been identified. Tir also represents the first bacterial protein that is tyrosine phosphorylated in host cells. Other virulence factors may be inserted into host cells by type IE systems become modified inside the infected eukaryotic host cell.

A major benefit of this assay is that it has a built in control, a 110 kDa EPEC-secreted protein called EspC. EspC does not use the type El system. Instead it uses a different secretion system. An inhibitor that is type IE secretion specific does not block secretion of EspC.

Glyceraldehyde-3 -phosphate dehydrogenase is a glycolytic enzyme that is normally a cytoplasmic enzyme in most cells. However, EPEC uses a type El secretion system to secrete glyceraldehyde-3 -phosphate dehydrogenase. Supernatant from bacteria may be assayed colorimetrically, using a standard enzymatic assay, to detect the secretion of glyceraldehyde-3 -phosphate dehydrogenase or to identify specific inhibitors of type El secretion.

Conditions That Allow Secretion The methods of the invention identifies antibacterial agents by contacting bacteria with a compound suspected of inhibiting the bacterial type IH secretion system under conditions that allow secretion of type IE-secreted polypeptide. As used herein, the term "conditions that allow secretion" refers to suitable conditions such that the nucleic acid is transcribed and translated and isolating the polypeptide so produced. The polypeptide produced may be a protein secreted into the media. Media includes a fluid, substance or organism where microbial growth can occur or where microbes can exist. Such environments can be, for example, animal tissue or bodily fluids, water and other liquids, food, food products or food extracts, and certain inanimate objects. For example, microbes may grow in Luria-Bertani (LB) media. It is not necessary that the environment promote the growth of the microbe, only that it permits its subsistence. - 21 -

Detecting Secretion of Polypeptide

The method of the invention identifies antibacterial agents by contacting bacteria with a compound suspected of inhibiting the bacterial type HI secretion system, and detecting secretion of a type Hi-secreted polypeptide.

The secreted polypeptide may contain a selectable marker. As used herein, a "selectable marker" may be any polypeptide sequence or feature of the secreted polypeptide that can be detected. The methods of the invention may therefore use recombinant polynucleotides, produced by inserting a nucleic acid encoding a selectable marker into the polynucleotide encoding a type IE-secreted polypeptide. For example, a recombinant polynucleotide may include a first polynucleotide, which encodes a type IE-secreted polypeptide, operably linked to a second polynucleotide, which encodes a selectable marker. The resulting recombinant polypeptide contains the selectable marker, which is then secreted by a type IE secretion system. The term "operably linked" refers to functional linkage between a promoter sequence and the structural gene or genes in the case of a fusion protein, regulated by the promoter nucleic acid sequence. The operably linked promoter controls the expression of the polypeptide encoded by the structural gene (e.g., the fusion protein).

A selectable marker may be an expressed reporter gene that can be monitored by a functional assay or assay for a protein product. The reporter gene product is a polypeptide that provides an assayable or measurable expression product in order to allow detection of secretion of the reporter gene product. Such reporter genes include, but are not limited to, reporter genes such as chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, a β-galactosidase gene, a luciferase gene, a green fluorescent protein gene, guanine xanthine phosphoribosyltransferase genes, and antibiotic resistance genes (e.g., neomycin phosphotransferase). Expression and secretion of the reporter gene product is indicative of secretion by a type El secretion system. An advantage of using - 22 - alkaline phosphatase as a reporter is that the enzyme has activity only when it is secreted outside of the bacterial inner membrane, so bacteria need not be removed from the supernatant for assay.

The methods of the invention may also use antibodies that are immunoreactive or bind to epitopes of the type El-secreted polypeptides. The term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. As used herein, the term "epitope tag" refers to a selectable marker present on the type IE-secreted polypeptide to which antibodies can bind. The epitope tag may be present on a native type IE-secreted polypeptide, or may be encoded by a recombinant polynucleotide constructed by operably linking a polynucleotide encoding a type IE-secreted polypeptide to a polynucleotide encoding the epitope tag, to form a recombinant fusion protein.

The methods of the invention may use methods for producing fusion proteins. Because EspA, EspB, intimin, and Tir are type IE-secreted protein, they are useful as fusion partners for cloning and expressing other peptides and proteins. For example, EspA, EspB, intimin, or Tir fused to a protein of interest is recombinantly produced in a host cell, e.g., E. coli, and the fusion protein is secreted into the culture medium in which the transformed host is grown. The flision protein can be isolated by anti-EspA, EspB, intimin, or Tir antibodies followed by cleavage of Tir from the peptide or protein of interest. ELISA or other immunoaffinity methods can be used to identify the EspA, EspB, intimin, or Tir fusion protein. The method for producing a fusion protein includes growing a host cell containing a polynucleotide encoding EspA, EspB, intimin, or Tir operably linked to a polynucleotide encoding a polypeptide or peptide of interest under conditions that allow expression and secretion of the fusion polypeptides and isolating the fusion polypeptide. - 23 -

The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, Fab', F(ab')₂, Fv, and single chain antibody that can bind the epitope. These antibody fragments retain some ability to bind selectively with antigen or receptor. The antibody may consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (see, Kohler, et al (Nature 256: 495, 1975); Current Protocols in Molecular Biology (Ausubel et al, ed., 1989)). Methods of making these fragments are known in the art. See, for example, Harlow and Lane (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1997), incorporated herein by reference. If needed, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the peptide or a peptide to which the antibodies are raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies. See, e.g., Colligan et al. (Unit 9, Current Protocols in Immunology, Wiley Interscience, 1997) incorporated by reference). Antibodies, including polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies and the like, have with the ability to bind with high immunospecificity to the type IE-secreted proteins, peptides or nucleotide sequences of the invention, or fragments thereof, or to the epitope tags. These antibodies can be unlabeled or suitably labeled. Antibodies may be employed in known immunological procedures for qualitative or quantitative detection of these proteins or peptides in cells, tissue samples, sample preparations or fluids. Such probes or antibodies can be used to identify proteins or genes that may be involved in the virulence of other pathogens, including but not limited to polypeptides or polynucleotides from Gram- negative bacteria.

The methods of the invention may detect type El-secreted polypeptide, and thus the inhibition of type El secretion systems, in a sample, and detecting binding of the antibody to the polypeptide. For example, the method involves contacting a sample from - 24 - a subject with an antibody to EspA, EspB, intimin, or Tir polypeptide. Binding is indicative of the presence of type IE-secreted polypeptide (for example, EspA, EspB, intimin, or Tir polypeptide) in the sample. As used herein, the term "sample" includes material derived from a subject. Such samples include but are not limited to hair, skin samples, tissue sample, cultured cells, cultured cell media, and biological fluids. As used herein, the term "tissue" refers to a mass of connected cells (e.g., CNS tissue, neural tissue, or eye tissue) derived from a human or other animal and includes the connecting material and the liquid material in association with the cells. As used herein, the term "biological fluid" refers to liquid material derived from a human or other animal. Such biological fluids include but are not limited to blood, plasma, serum, serum derivatives, bile, phlegm, saliva, sweat, amniotic fluid, and cerebrospinal fluid (CSF), such as lumbar or ventricular CSF. As used herein, the term "sample" also includes solutions containing the isolated polypeptide, media into which the polypeptide has been secreted, and media containing host cells that produce the Tir polypeptide. For example, a sample may be a protein sample that is to be resolved by SDS-PAGE and transferred to nitrocellulose for Western immunoblot analysis. The quantity of sample required to obtain a reaction may be determined by one skilled in the art by standard laboratory techniques. The optimal quantity of sample may be determined by serial dilution.

As described supra, ELIS A or other immunoaffinity methods can be used to identify type El-secreted polypeptides, including EspA, EspB, intimin, or Tir fusion protein. As used herein, the term "ELIS A" refers to an enzyme-finked immunosorbant assay method for detecting antigens or antibodies using enzyme-substrate reactions. The enzymes are generally coupled to antibodies (either to antibodies specific for the antigen or to anti- immunoglobulin). The amount of enzyme conjugate determined from the turnover of an appropriate substrate.

An ELIS A assay useful for identifying inhibitors of type IE secretion is provided in EXAMPLE VE. A positive response in the provided ELIS A assay indicates that proteins were secreted by the type IE system, and hence, secretion was not inhibited. When no - 25 - color is detected in the ELISA plate, either type IH secretion is blocked, or the bacteria were kille or slowed down in their growth and so did not secrete these proteins. Typical ELISA readings of an absorbance of between 1-2 are negative (i.e., secretion occurred) and readings less than about 0.2 are positive, indicating that there is some inhibition of secretion. As a positive control, a polynucleotide encoding the fusion protein is introduced into a type HI secretion defective mutant. The tranformed mutant bacteria do not secrete this protein because they lack a type IE secretion system, indicating the maximun level of inhibition.

Derivative Antibacterial Agents Once antibacterial agents are identified, chemical synthesis of derivative molecules and testing for type IE secretion inhibition provides more potent and specific inhibitors. The polypeptide inhibitors of type El secretion systems identified by the method of the invention can be obtained using any of several standard methods. For example, polypeptides can be produced in a standard recombinant expression systems, chemically synthesized, or purified from tissues in which they are naturally expressed. See, e.g., Ausubel et al, supra). One skilled in the art can purify polypeptides using standard protein purification methods and the purity of the polypeptides can be determined using standard methods including, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis. Derivatives include protein and polypeptide analogs and peptidomimetics. Alternatively, peptides can be chemically synthesized using synthesis procedures known to one skilled in the art. Preferably, an automated peptide synthesizer is used with NTmoc amino acids on a polyethylene glycol-polystyrene (PEGPS) graft resin. Suitable linkers such as a peptide amide linker (PAL) can be used, for example, to create carboxamide end groups. - 26 -

Isolation and purification of specific inhibitors of type HI secretion that are polypeptides, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.

Compositions identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (see, Saiki et al. (Bio/Technology 3: 1008-1012, 1985)), allele-specific oligonucleotide probe analysis (see, Conner et al. (Proc. Natl. Acad. Sci. USA 80: 278, 1983)), oligonucleotide ligation assays (OLAs) (see, Landegren et al. (Science 241: 1077, 1988)), and the like. Molecular techniques for DNA analysis have been reviewed by Landegren et al. (Science 242: 229-237, 1988).

Additional inhibitors of a type IE secretion system are obtained by isolating polynucleotides homologous to the polynucleotides that encode specific inhibitors of type IE secretion pathways. Polynucleotides encoding the peptide can be isolated by methods well known in the art. These isolated nucleic acids can be ligated into vectors and introduced into suitable host cells for expression. Methods of ligation and expression of nucleic acids within cells are well known in the art. See, Sambrook et al, supra. Isolated polynucleotides encoding identified inhibitors of type IE secretion systems may be obtained by standard methods. The term "isolated polynucleotide" describes, for example, a nucleic acid that is incorporated into a vector, such as a plasmid or viral vector; a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that at which it naturally occurs); and a nucleic acid that exists as a separate molecule, e.g., a DNA fragment produced by PCR amplification or restriction enzyme digestion, or an RNA molecule produced by in vitro transcription. The term also describes a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion protein. - 27 -

Polynucleotides that encode inhibitors of type IE secretion systems identified by a method of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization techniques that are well known in the art. These include, but are not limited to (1) hybridization of libraries with probes to detect homologous nucleotide sequences, (2) polymerase chain reaction (PCR) on DNA using primers capable of annealing to the DNA sequence of interest, and (3) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features.

Screening procedures that rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes that correspond to a part of the sequence encoding the protein in question can be synthesized chemically or produced by fragmentation of the native sequence. Chemical synthesis requires that short, oligopeptide stretches of amino acid sequence be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency varies, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. - 28 -

An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42 °C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

Combinatorial chemistry methods may also be used to generate additional inhibitors of type IE secretion systems, using representative combinatorial chemistry methods described by Wong, et al. (Methods: A Companion of Methods in Enzymology 6: 404- 410, 1994). Using this approach, an amino acid at certain position can be substituted during a peptide synthesis process by a mixture of amino acids, yielding a mixture of several peptides. The peptides may then be screened using the method of the invention to identify inhibitors of type IE secretion systems.

Test Validation Procedures for Active Compounds

Compounds that are identified as inhibitors of type HI secretion systems are subjected to test validation procedures do determine whether these identified compounds are active compounds in related systems. First, verification of type IE secretion inhibition is performed using SDS-PAGE and Western blotting techniques. Then, through secondary screening of identified compounds, any cytotoxic effects of identified compounds are determined using mammalian cells to eliminate false positives. The inhibition of other pathogenic bacteria type IE secretion systems will be determined using a Salmonella invasion assay; a Salmonella macrophage survival assay; a Shigella invasion assay, a Yersinia anti-phagocytic assay, or a ti-Pseudomonas aeruginosa assay. Finally, animal models of disease caused by virulence factors secreted by a type El secretion system are used to determine the therapeutic effectiveness of the identified compounds. - 29 -

Secondary screening of identified compounds. A compound may appear to inhibit secretion in this ELISA assay for several reasons. The compound may inhibit general metabolic processes enough such that secretion is non-specifically inhibited, yet the bacteria still grow. Alternatively, the compound may specifically inhibit type El secretion. To directly identify specific type IE inhibitors, the protein profile of EPEC supematants are analyzed following growth in the compound. One of the five EPEC- secreted proteins, EspC (110 kDa), belongs to the IgA protease family that uses a different (type IN) secretion pathway, and mutants defective for type El secretion (such as cfm\4) still secrete EspC.

Supematants are prepared from wild-type EPEC grown under the conditions described above in the presence of various promising compounds. Supematants are purified and analyzed by polyacrylamide gel electrophoresis. Compounds are isolated that permit EPEC to still secrete the 110 kDa EspC, but not the other four secreted proteins. This profile is identical to that seen with type ET secretion mutants in EPEC (e.g., cfm\4). This assay provides an alternate yet sensitive method for determining the specificity of type IE secretion inhibition, and rule out non-specific effects.

Secondary screening of identified compounds may also be tested by in vitro toxicity testing. To rule out any other growth effects, standard growth curves in the presence of promising compounds are tested. In addition, to prepare for testing in cell cultures, the toxicity of promising compounds is then tested on cultured HeLa epithelial cells and J774 macrophages. This is done initially by trypan blue staining (live cells exclude trypan blue, dead ones are stained). Additionally, a MTT assay may be used to study the cyto toxicity of mammalian cells. The MTT assay may be performed according to the method of Patrick et al, (J. Infect. Dis. 165: 86572, 1992). This assay may be used to measure cytotoxicity of compounds on cultured HeLa and J774 cells. - 30 -

Secondary screening of identified compounds may also be tested by inhibition of EPEC- induced infected eukaryotic host cell signaling. EPEC strains with mutations in type IE secretion genes, or in at least two of the secreted proteins (EspA and EspB), are unable to activate several signals in infected eukaryotic host cells. These signals include HP90 tyrosine phosphorylation, calcium and inositol phosphate fluxes, cytoskeletal rearrangement, and intimate adherence). The identified compounds are tested for their ability to block these virulence mechanisms. Compounds are added to HeLa cells followed by infection with EPEC. Assays include rhodamine phalloidin staining to test for actin condensation, fluorescent staining with anti-phosphotyrosine antibodies, and determining if Hp90 becomes phosphorylated by using a Western blot probed with phosphotyrosine antibodies.

Inhibition of other type HI secretion systems. A compound which inhibits EPEC type El secretion systems can be tested for inhibition of other type IE secretion systems, as tested by inhibition of other Attaching/Effacing pathogens. There is a family of pathogens that use a type IE secretion system very similar to EPEC to form A/E lesions. These pathogens all secrete proteins highly homologous to EspA and EspB using PAGE and Western immunoblot analysis, and genetic probes. Such pathogens include the RDEC-1, EHEC, the causative agent of hamburger disease and hemolytic uremic syndrome, and Citrobacter rodentii, a mouse pathogen. These three organisms are grown in the presence of promising compounds to determine if they still secrete EspA and EspB. Compounds capable of blocking actin accumulation are detected using rhodamine phalloidin, and the other signals known to those of skill in the art.

A compound that inhibits EPEC type III secretion systems can be tested for inhibition of other type IE secretion systems, as tested by inhibition of Salmonella invasion. The gene products necessary for Salmonella invasion into non-phagocytic cells are all secreted by a type IE secretion system. Mutations in this secretion system result in non- invasive mutants that are avirulent when delivered orally. To determine if compounds that block EPEC secretion are also able to block Salmonella invasion, standard - 31 - gentamicin invasion assays are performed using S. typhimurium SL1344 and cultured HeLa cells. The assay of Salmonella invasion is performed according to the methods of Finlay (Curr. Top. Micrbiol. Immunol. 192: 163-85, 1994) and Tang et al. (J. Microbiological Methods. 18: 227-240, 1993). Briefly, gentamicin is added after bacterial infection of cultured cells to kill extracellular organisms while organisms that invaded the infected eukaryotic host cell and are inside are protected from gentamicin, and can be released by Triton X-100 detergent treatment followed by plating onto agar plates and counting colonies.

A compound which inhibits EPEC type El secretion systems can be tested for inhibition of other type IE secretion systems, as tested by inhibition of Shigella invasion. Shigella and enteroinvasive E. coli use a type IE secretion system to secrete molecules (Ipa's) that are critical for mediating invasion and disease. This system is homologous to the

Salmonella invasion system, and some genetic components are even interchangeable.

Compounds are tested for their ability to block Shigella flexneri invasion of HeLa cells using the assay described above for Salmonella. S. flexneri invasion is tested according to the methods of Finlay et al. (Biochimie. 70: 1089-99, 1988) and Mills et al. (Microbial

Path. 77.^* 409-23, 1994).

A compound which inhibits EPEC type IB secretion systems can be tested for inhibition of other type IE secretion systems, as tested by blockage of Yersinia enterocolitica antiphagocytic mechanisms. Yersinia species encode several virulence factors that are secreted by a type IE secretion system. Some of the secretion components are interchangeable with Salmonella and Shigella. However, the function of the Yersinia secreted proteins is completely opposite that of the invasive enterics; instead, Yersinia actually inserts secreted proteins into macrophages to block uptake (phagocytosis) into macrophages (1). By injecting a tyrosine phosphatase (YopH), an actin toxin (YopE), and a probable kinase, this pathogen inhibits phagocytosis. Again, this type El secretion- mediated process is needed for virulence. To determine if compounds that block EPEC secretion can also inhibit the Yersinia secretion system, standard phagocytic assays are - 32 - used that test Y. enterocolitica 808 lv (which contains the virulence plasmid) being internalized by the J774 mouse macrophage line. Controls include compounds that show no activity, and use of an isogenic Y. enterocolitica, 8081c, that is missing a virulence plasmid which encodes the type IE secretion machinery and the Yops.

A compound that inhibits EPEC type IE secretion systems can be tested for inhibition of other type III secretion systems, as tested by inhibition of Pseudomonas aeruginosa secretion of Exotoxin S. P. aeruginosa contains a homologue of a Yersinia type IE secretion chaperone. A virulence factor of P. aeruginosa, Exoenzyme S, uses a type HI secretion mechanism for its export. A simple protocol is used to examine whether P. aeruginosa Exoenzyme S is secreted is performed according to the method of Yahr et al. (J. Bacteriol. 177: 1169-78, 1995). This method involves collecting and concentrating P. aeruginosa strain PAK supernatant and using PAGE and Coomassie staining to look for the 53 and 49 kDa subunits. Controls include treatment with calcium, that inhibits ExoS secretion. Antibodies to ExoS verify the identity of the secreted proteins by Western analysis.

A compound that inhibits EPEC type El secretion systems can be tested for inhibition of other type IE secretion systems, as tested by inhibition of secretion of plant pathogens. Several plant pathogens use a type IE secretion system to secrete "harping" that are critical to cause disease in plants. A compound that is identified as having an ability to inhibit type IE mediated secretion of the above described human pathogens, it is of serious economic consideration to see if it could also be used to treat plant disease. Compounds are tested for their ability to inhibit harpin secretion and disease in plants.

Initial testing in relevant animal disease models. Initial testing in animal disease models may be performed by toxicity testing. The intimate interactions that occur between EPEC-secreted proteins and the eukaryotic host cell surface emphasize the complexity of eukaryotic host-pathogen interactions, and provide valuable tools to exploit and study cellular function and bacterial disease, in addition to potential uses in therapeutics. - 33 -

Compounds that block type El secretion systems can be tested in two relevant animal disease models: EPEC/EHEC in rabbits; and S. typhimurium in mice. Compounds typically are first tested for their toxicity to mice using standard methods known to those of skill in the art. Initially the pharmacokinetics of the drug are tested by measuring the levels of drug in the blood in the hours following oral drug ingestion. This information provides an indication of the bioavailability of the compound. Toxicity testing is also performed to determine the maximum tolerated dose. To do this, increasing concentrations of the drug are given to an animal up to a level of 1000 mg/kg.

Initial testing in animal disease models may be performed by inhibition of EPEC- mediated disease. Once initial toxicity and bioavailability studies have been completed, animal infection models are tested with the most promising compounds. These standard assays are used to determine the effect of promising compounds on RDEC-1 (the rabbit EPEC pathogen) and RDEC-1 containing the verotoxin (a well-known EHEC animal model) in rabbits. The amount of diarrhea is measured, and pathology is performed on infected animals to determine the extent of colonization with and without the drug. These studies indicate whether type El secretion compounds can affect the outcome of these infections.

Initial testing in animal disease models may be performed by inhibition of Salmonella typhimurium disease in the murine typhoid model. S. typhimurium infection in Balb/C mice leads to murine typhoid, ultimately resulting in animal death. Two routes of infection are used: oral (LD₅₀=10⁶) and intravenous ( L _Q=l& ). However, death as an endpoint depends on many factors, is difficult to interpret, and approval is difficult. Instead, animals can be sacrificed at various times, their livers and spleens homogenized, and the number of S. typhimurium in these organs counted according to the method of Leung et al. (Proc. Nat'l Acad. Sci. USA. 88: 11470-4, 1991). This technique is an excellent indicator of infectiveness. There are at least two type El secretion systems in S. typhimurium. The first is critical for invasion of epithelial cells, and is required for virulence. The second is needed for long term survival in the liver and spleen. To test the - 34 - ability of type III secretion inhibitors to block S. typhimurium virulence, the mice are given various doses of the compound at the same time as oral and IV infection. The dose used depends on the results of the toxicity and bioavailability tests. The ability of these compounds to alter organ colonization rates is an excellent indicator of the effectiveness of these compounds as potential therapeutics.

Kits

The invention provides kits for identifying compounds that specifically inhibit type El secretion systems. The kit is a carrier with two or more containers. As used herein, a "container" includes vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. The first container contains bacteria with a type IE secretion system. The bacteria contain a polynucleotide that encodes a polypeptide secreted by the type IE secretion system. The second container contains reagents for detecting secretion of the polypeptide. The function of the "reagents for detecting" is to detect polypeptides that are being or have been secreted from a bacteria by a type El secretion system. When the type IE-secreted polypeptide has an epitope that is bound by an antibody, the reagent for detecting may be that antibody. When the type El-secreted polypeptide contains a detectable marker that is an epitope tag (for example, HSV or T7), the detecting reagent may be an antibody that binds to that epitope (for example, anti-HSV and anti T7, respectively). When the type IE-secreted polypeptide contains a detectable marker that is an enzyme, the detecting reagent may be the substrate for the enzyme. When the enzyme is alkaline phosphatase, the detecting reagent is an alkaline phosphate substrate, for example, j?-nitrophenol. When the enzyme is β-galactosidase, the detecting reagent is a β-galactosidase substrate, for example, X- gal. When the detectable marker generates light (such as luciferase or green fluorescent protein), the detecting reagent may be a composition that amplifies or transforms the light signal. - 35 -

In one embodiment, the detecting reagent is detectably labeled. In a more specific embodiment, the label is selected from the group consisting of radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.

The following EXAMPLES are intended to illustrate but not limit the invention. While they are typical of those methods that might be used in the practice of the invention, other procedures known to those skilled in the art may alternatively be used.

EXAMPLE I SCREENING FOR INHIBITORS OF VIRULENCE FACTORS The purpose of this EXAMPLE was to develop a screening method for the identification of inhibitors of bacterial virulence factors. This screen involves the EPEC system.

Glyceraldehyde-3 -phosphate dehydrogenase is a glycolytic enzyme that is normally a cytoplasmic enzyme in most cells. However, EPEC uses a type IE secretion system to secrete glyceraldehyde-3 -phosphate dehydrogenase. Supernatant from bacteria was assayed colorimetrically, using a standard enzymatic assay, to detect the secretion of glyceraldehyde-3 -phosphate dehydrogenase or to identify specific inhibitors of type El secretion.

To develop a more sensitive assay, a gene encoding one of the EPEC-secreted proteins (espB) was fused to several well known molecules, including a HSV tag. The espB product is needed for signal transduction in mammalian cells. All of these gene fusions were still secreted out of EPEC. The supernatant of broth-grown organisms containing these fusions was collected, added to an ELISA plate, followed by standard ELISA technology with, for example, anti-HSV antibodies. A colorimetric readout indicated that the fusion protein was secreted. - 36 -

By using antibodies to EspB itself, such fusions were not needed. As a control, a fusion protein expressed in an EPEC mutant that contains a mutation in the secretion pathway was expressed, but not secreted. ELISA results with this mutant confirmed that it was not secreted, and there was no background due to bacterial lysis and release of the non- secreted protein.

EXAMPLE II

DEVELOPMENT OF AN ASSAY TO SCREEN FOR INHIBITORS

OF TYPE III SECRETION

One of the secreted proteins that is critical to activating signals in infected eukaryotic host cells is EspB. This protein is secreted into the supernatant via the type IE secretion system found in EPEC. Based on this knowledge, a simple and rapid ELISA-based screen was set up to test for EspB secretion out of EPEC. A plasmid (pMS21) was constructed that contains the region of espB that encodes the amino-terminal portion of EspB (needed to mediate type El secretion) fused to a Herpes Simplex Virus (HSV) sequence that encodes a sequence tag to which commercial antibodies are available. This plasmid was transformed into two EPEC strains: one that contains an espB mutation (UMD864) yet still secretes the other EPEC-secreted proteins that use the type IE secretion system; and cfm\4, a strain that is defective for type IE secretion (i.e., a negative control).

To assay for secretion, bacteria were grown standing overnight in tissue culture fluid in the presence of compounds to be tested. These conditions yielded significant EPEC- mediated secretion. The following day, bacteria were removed by centrifugation, and the supernatant placed into wells of a 96 well microtiter plate. A standard ELISA was performed on the supematants, with the primary antibody being anti-HSV tag. Under normal conditions, EPEC secretes the EspB-HSV fusion product, that is detected in the ELISA (A₄₈₀ -1-2). Using the strain defective for secretion (cfm\4), the A₄₈₀ was always less than 0.2. - 37 -

An additional benefit of this initial screen is that if the compound being tested is bactericidal (which is not the purpose of this screen), the bacteria do not grow overnight, which is easily noted.

This screen was used to assay approximately 200 plant extracts from medically important plants. See, FIGURE 1. Dilutions of 1/200-1/1000 (about 250 μg/ml) are appropriate. The assay was robust, reproducible, and sensitive. No specific type IE inhibitors were identified, although some extracts that slowed secretion in general (i.e., type II, El, and IV) were found.

EXAMPLE III DEVELOPMENT OF AN ASSAY TO SCREEN FOR INHIBITORS OF

BACTERIAL TYPE III SECRETION

The purpose of this EXAMPLE is to provide an assay to screen for inhibitors of bacterial type El secretion.

A polynucleotide encoding the EspA polypeptide is fused to several well known molecules, including a HSV tag. The gene fusion is still secreted out of enteropathogenic E. coli. A plasmid contains the genetic region of espA that encodes the amino-terminal portion of ΕspA (needed to mediate type ΕI secretion) fused to a Herpes Simplex Virus (HSV) sequence that encodes a sequence tag to which commercial antibodies are available. This plasmid is transformed into a strain that contains an espA mutation yet still secretes the other enteropathogenic E. co/z^'-secreted proteins that use the type ΕI secretion system. The supernatant of organisms containing these fusions is collected, added to an ΕLISA plate, followed by standard ΕLISA. A calorimetric readout indicates the fusion protein is secreted. - 38 -

This plasmid is also transformed into a strain that is defective for type El secretion (i.e. a negative control). When the fusion protein is expressed in this strain, the fusion protein is expressed but not secreted. ELISA results with this mutant confirm that it is not secreted.

EXAMPLE IV

THE TYPE III SECRETION APPARATUS AND ITS SECRETED PROTEINS, EspA AND EspB, FACILITATE DELIVERY OF TIR INTO EUKARYOTIC

HOST CELLS

The purpose of this EXAMPLE was to examine the role of the type El secretion apparatus and its secreted proteins on Tir delivery into HeLa cells. Strains containing mutations in genes encoding the type IE secretion apparatus (sep and cfm) or secreted proteins, EspA or EspB (espA or espB), do not produce a tyrosine-phosphorylated Tir in infected eukaryotic host cells. However, the non-tyrosine phosphorylated form of Tir might still be delivered to the eukaryotic host cell. Membrane extracts from EPEC or mutant infected HeLa cells were isolated and probed with anti-PY and anti-EPEC 78 kDa antibodies. (Anti-PY antibodies recognize phosphorylated proteins, including EPEC Tir.)

The tyrosine phosphorylated 90 kDa form of Tir was only detected with anti-PY antibodies in the EPEC and CVD206 infected membranes. The anti-EPEC 78 kDa antibodies recognized the same 90 kDa band in EPEC and CVD206 membrane extracts, but strains containing mutations in espA, espB, or cfml4 did not contain detectable levels of Tir in the membrane or cytoplasmic fractions. The 78 kDa form of Tir in EPEC or CVD206 infected cells was not detected, suggesting a rapid modification to 90 kDa. The bacterial form of the 78 kDa Tir protein was present in the insoluble fraction containing adherent bacteria of all these strains. Only the EPEC infected HeLa cell insoluble fraction contained the 90 kDa form of Tir that localizes to this fraction due to its interaction with intimin. - 39 -

Therefore, EspA and EspB, both secreted by the type El system, are needed for efficient delivery of Tir to the infected eukaryotic host membrane fraction.

EXAMPLE V RABBIT MODEL FOR EPEC VIRULENCE The purpose of this EXAMPLE was to provide a rabbit model for EPEC virulence. A natural rabbit EPEC infection model was used to demonstrate that EspA and EspB are critical for virulence.

RDEC-1 and its espA and espB mutant strains were inoculated by the orogastric route into young rabbits. Most RDEC-1 was found in the cecum and colon one week postinfection. However, the number of either mutant strain was greatly decreased in these tissues compared to the parent strain. RDEC-1 adhered specifically to the sacculus rotundas (follicle associated epithelium) and bacterial colonization was also observed in the cecum, indicating that the sacculus rotundas in the cecum is an important colonization site for this pathogen. The adherence levels of the EspA^" and EspB^" strains to the sacculus rotundas were 70 and 8000 times less than that of parent strain. These results show that the adherence ability and tissue tropism of RDEC-1 are dependent on EspA and ESPB. Furthermore, EspB appears to play a more critical role than EspA in bacterial colonization and pathogenesis. This is the first demonstration that the enteropathogenic E. coli secreted proteins, EspA and EspB, that are involved in triggering of infected eukaryotic host cell signal transduction pathways, are also needed for colonization and virulence.

Animal infections were performed as follows: Overnight bacterial cultures were collected by centrifugation and resuspended in one ml of phosphate-buffered saline. New Zealand white rabbits (weight 1.0 to 1.6 kg) were fasted overnight, then five ml of 2.5% sterile sodium bicarbonate and one ml of RDEC-1 or espA or espB strains (2.5x10¹⁰) were inoculated into the stomach using orogastric tubes. The same dosage of bacteria was inoculated into each rabbit the following day. - 40 -

Clinical assessments were performed as follows: Each rabbit was weighed daily and fecal shedding of bacteria were collected by rectal swabs and from stool pellets. Rectal swabs were rolled over one half of the surface of MacConkey plates containing nalidixic acid. Five stool pellets or same amount of liquid stool were collected from each rabbit and resuspended in three ml phosphate-buffered saline and 0.1 ml of each stool suspension was plated onto MacConkey plate containing nalidixic acid. The growth of nalidixic resistant colonies was scored as follows: O, no growth; 1, widely spaced colonies; 2, closely spaced colonies; 3, confluent growth of colonies.

Sampling and preparation of tissue were performed as follows: Tissues were excised immediately following sacrifice by intravenous injection of ketamine and overdosing with sodium phenobarbital.

The amount of bacterial colonization in intestinal tissues was assayed as follows: The intestinal segments (10 cm), except cecum, were doubly ligated at their proximal and distal ends, and dissected between the double ligated parts, then flushed with 10 ml of ice-cold phosphate-buffered saline. One gram of viscous contents from the cecum was added to 9 ml phosphate-buffered saline. The resulting phosphate-buffered saline suspensions were diluted and plated on MacConkey plates containing nalidixic acid.

The amount of bacterial adherence to intestinal tissues was assayed as follows: Tissue samples were excised using a 9 mm diameter cork punch, washed 3 times with phosphate-buffered saline, added to two ml of ice-cold phosphate-buffered saline, and homogenized with a homogenizer, then serial diluted samples were plated onto MacConkey plates. The numbers of bacteria adherent to each tissue per square centimeter were calculated as follows: CFU/cm² = the bacterial number/plate x dilution factor x 2 ml/0.452 - 41 -

These data indicate that these molecules and the functions they perform are critical for pathogenesis. The intimate interactions that occur between EPE- secreted proteins and the infected eukaryotic host cell surface emphasize the complexity of eukaryotic host- pathogen interactions, and provide valuable tools to exploit and study cellular function and bacterial disease.

EXAMPLE VI EPITOPE TAGGING OF Tir

The purpose of this EXAMPLE was provide Tir fusion proteins with epitope tags. Two genetic fusions were constructed linking the sequence encoding either the T7 or HSV epitopes to the 5' or 3' of tir, respectively.

Constmction of Tl -tir and t/r-HSV fusions were performed as follows: Briefly, the tir gene was amplified by PCR introducing unique restriction sites to enable in frame fusions with the T7 or HSV sequences in the pET28a and pET27b set of vectors (Novagen). The tagged tir gene, lacking the His tag, was then cloned into a pACYCl 84- based vector for expression. The resulting plasmids were transformed into the EPEC tir allelic deletion strain and used to infect HeLa cells. Fluorescence microscopy of infected cells revealed typical pedestal formation with co-localization of both T7 and HSV epitopes and actin in characteristic horseshoe patterns. This phenotypic complementation with the tir-HSV construct, that encodes tir but not the downstream orfU gene, indicates that the phenotype of the tir deletion mutation is not due to a polar effect on the orfU gene product.

The membrane fractions of cells infected with EPEC expressing T7 tagged Tir were also compared to those infected with the intimin mutant CVD206. T7-Tir was detectable in HeLa cell membrane extracts using antibodies against EPEC 78 kDa, T7, and PY. The addition of the T7 tag increased the apparent molecular mass of the Tir phosphorylated protein compared to that observed with CVD206. In contrast to T7-Tir, Tir itself did not cross-react with the T7 antibodies. - 42 -

T7 specific antibodies were used to immunoprecipitate membrane fractions of HeLa cells infected with CVD206 or the T7-Tir strain. As expected the T7 antibodies did not immunoprecipitate the 90 kDa Tir protein, while they precipitated the slightly larger T7-Tir fusion protein which was recognized by antibodies against either PY, T7, or EPEC 78.

Therefore, Tir fusion proteins containing epitope tags were produced and recognized by well-characterized antibodies that bind to the epitope tags.

EXAMPLE VII BACTERIAL TYPE III SECRETION SYSTEM INHIBITORS ASSAY: DETECTION OF SECRETED HSV-FUSION PROTEINS BY ELISA

The purpose of this EXAMPLE was to provide an easy ELISA assay has been developed to measure secretion. This assay is simple, and requires no special technology. It is also economical, because no expensive reagents are needed. It is reliable, reproducible, and could easily be automated for screening various collections of reagents to identify potential inhibitors of secretion. The detection assay is performed as follows:

1. Inoculate 1 ml DMEM with 10 μl of an overnight culture (or one colony) of MAS15 (pMS21 in UMD864; pMS21 in cfm\4.2.\ as negative control) and incubate at 37°C, 5% CO₂ overnight without shaking (Test compounds added for inhibition of secretion). 2. Spin down bacteria (2 min, 8,000 rpm) and transfer 100 μl of bacterial supernatant to 96-well ELISA plates.

3. Incubate at room temperature for 3 hours at room temperature (or 4°C overnight).

4. Remove supernatant and block for 2 hours at room temperature (or 4°C overnight) with 200 μl PBS, 4% BSA/well.

5. Remove blocking agent and incubate for 2 hours at room temperature with 150 μl anti-HSV antibody (M HSV; 1:2,500 in PBS, 1% BSA/well). - 43 -

6. Wash 3 times with PBS containing 0.05% Tween 20.

7. Add 150 μl anti-rabbit antibody (g M-HPO; 1 :5,000 in PBS 1% BSA/well) and incubate for 1 hour at room temperature.

8. Wash 5 times with PBS containing 0.05% Tween 20. 9. Develop with 100 μl OPD-solution (30 ml 0.1 M Citrate, 60 1 H₂O₂, one tablet OPD)/well. 10. Stop reaction with 100 μl 3M H₂SO well and read at 490 nm in ELISA-plate reader.

A number of embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

- 44 -CLAIMSWhat is claimed is:

1. A method for the identification of antibacterial agents, comprising: a) contacting bacteria containing a polynucleotide with a compound suspected of inhibiting the bacterial type IE secretion system, wherein the bacteria have a type El secretion system, wherein the polynucleotide encodes a polypeptide secreted by the type IE secretion system, and wherein the contacting is under conditions that allow secretion of the polypeptide; and b) detecting the secretion of the polypeptide, wherein a reduced level of secretion, compared to control bacteria not contacted with the compound, is indicative of the inhibition of the bacterial type El secretion system.

2. The method of claim 1 , wherein the polypeptide secreted by the bacterial type El secretion system contains a selectable marker.

3. The method of claim 2, wherein the selectable marker is an enzyme.

4. The method of claim 3, wherein the enzyme is glyceraldehyde-3 -phosphate dehydrogenase.

5. The method of claim 3, wherein the enzyme is chloramphenicol acetyl- transferase.

6. The method of claim 3, wherein the enzyme is alkaline phosphatase.

7. The method of claim 3, wherein the enzyme is ╬▓-galactosidase. - 45 -

8. The method of claim 3, wherein the enzyme is guanine xanthine phospho- ribosyltransferase.

9. The method of claim 3, wherein the enzyme is neomycin phosphotransferase.

10. The method of claim 2. wherein the selectable marker is luciferase.

11. The method of claim 2, wherein the selectable marker is green fluorescent protein.

12. The method of claim 2, wherein the selectable marker provides antibiotic resistance.

13. The method of claim 2, wherein the selectable marker is an epitope tag.

14. The method of claim 13, wherein the epitope tag is HSV.

15. The method of claim 13 , wherein the epitope tag is T7.

16. The method of claim 1 , wherein the polypeptide secreted by the bacterial type IE secretion system is a recombinant polynucleotide.

17. The method of claim 2, wherein the polypeptide secreted by the bacterial type IE secretion system is encoded by a Locus of Enterocyte Effacement

18. The method of claim 2, wherein the polypeptide is EspA.

19. The method of claim 2, wherein the polypeptide is EspB.

20. The method of claim 2, wherein the polypeptide is intimin. - 46 -

21. The method of claim 2, wherein the polypeptide is Tir.

22. The method of claim 1 , wherein the bacteria are Gram-negative bacteria.

23. The method of claim 22, wherein the Gram-negative bacteria are Attaching/Effacing bacteria.

24. The method of claim 23, wherein the Attaching/Effacing bacteria are EPEC.

25. The method of claim 23, wherein the Attaching/Effacing bacteria are EHEC.

26. The method of claim 23 , wherein the Attaching/Effacing bacteria are RDEC- 1.

27. The method of claim 1 , wherein the determining is by an ELISA assay.

28. The method of claim 1 , wherein the reduction in the the level of the bacterial type III secretion systems blocks signaling in infected eukaryotic host cells.

29. The method of claim 1 , further comprising: determining the secretion of a second polypeptide, wherein the second polypeptide is secreted by the bacteria other than by the type III scretetion system, and wherein a non-reduced level of secretion, compared to the control bacteria not contacted with the compound, is indicative of the inhibition of the bacterial type IE secretion system.

30. The method of claim 29, wherein the second polypeptide is EspC. - 47 -

31. A kit useful for the identification of specific inhibitors of a type El secretion system comprising: a) a first container containing bacteria containing a polynucleotide, wherein the bacteria have the type IE secretion system and wherein the polynucleotide encodes a polypeptide secreted by the type El secretion system; and b) a second container containing a reagent for detecting the secretion of the polypeptide.

32. The kit of claim 31 , wherein the reagent is detectably labeled.

33. The kit of claim 32, wherein the label is selected from the group consisting of radioisotope, a bioluminescent compound, a chemilummescent compound, a fluorescent compound, a metal chelate, and an enzyme.