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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0258—Escherichia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Gram-negative bacteria
  • C07K16/1228—Enterobacterales (O), e.g. Citrobacter (G), Serratia (G), Proteus (G), Providencia (G), Morganella (G) or Yersinia (G)
  • C07K16/1232—Escherichia (G)
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • G01N2333/245—Escherichia (G)

Definitions

• the present invention relates generally to the virulence of pathogenic organisms and more specifically to virulence factors associated with enteropathogenic bacteria.
• Antibiotics have been used for years to successfully treat diverse bacterial infections.
• bacterial resistance to antibiotics has been an increasing problem over the past few years.
• Many pathogens are now resistant to several antibiotics, and m some cases, the diseases they cause are no longer treatable with conventional antibiotics.
• 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 short period of time.
• Gram-negative bacteria utilize specialized machinery to export molecules across their two membranes and the piroplasm, a process critical for moving virulence factors to the bacterial surface where they can interact with host components.
• Gram- negative secretion has been divided into four major pathways. First, the Type I secretion is used by a small family of toxins, with E. coli hemolysin being the prototype. Second, the type II secretion system is the major export pathway used by most Gram-negative bacteria to export many molecules, mcluding some virulence factors; it shares homology to mammalian drug resistance mechanisms.
• the type IV secretion system is encoded within the secreted product, which cleaves itself as part of the secretion mechanism; the prototype of this system is the Neisseria IgA protease.
• the most recently discovered secretion pathway is the type HI pathway.
• Type L ⁇ secretion systems were originally described as a secretion system for Yersinia secreted virulence proteins, YOPs, which are critical for Yersinia virulence. A homologous secretion system was then identified in several plant pathogens, mcluding
• SUBSOTUTE SHEET (RULE 26) Pseudomonas syringae, P. solanacearurn, w ⁇ Xantharnonas carnpestris. These plant pathogens use this secretion pathway to secrete virulence factors (ha ⁇ ins and others) tiiat are required for causing disease in plants. Although the secretion system is similar, harpins and YOPs (ie. the secreted virulence factors) are not homologous polypeptides. Several other type HI secretion systems necessary for virulence have more recently been identified in other pathogens. These systems include the invasion systems Salmonella and Shigella use to enter cells and cause disease.
• Exoenzyme S a potent virulence factor.
• Enteropathogenic Escherichia coli is a leading cause of infant diarrhea and was the first E coli shown to cause gastroenteritis. Enteropathogenic E. coli activates the host epithelial cells' signal transduction pathways and causes cytoskeletal rearrangement, along with pedestal and attaching/effacing lesion fo ⁇ nation.
• a three-stage model has describes enteropathogenic E coli pathogenesis.
• the signal transduction in the host epithelial cells involves activation of host cell tyrosine kinase activity leading to tyrosine phosphorylation of a 90 kilodalton host membrane protein, Hp9.0, and fluxes of intracellular inositol phosphate (LP,) and calcium. Following this signal transduction, the bacteria adheres intimately to the surface of the epithelial cell, accompanied by damage to host epithelial cell microvilli and accumulation of cytoskeletal proteins beneath the bacteria.
• the present invention is based on the discovery a protein associated with virulence in pathogenic bacteria, for example enteropathogenic E. coli.
• EspA is a secreted protein, it is ideally suited for use in a fusion protein linked to a polypeptide of interest.
• the type HI secretion pathway is an ideal target for potential inhibitors, because it is a virulence factor-specific conserved pathway identified in bacteria.
• the present invention provides a method for identifying inhibitors of type HI secretion systems. The relevance of this invention is directed toward the development of new antibacterial therapeutics. In contrast to other antibiotics, the compounds identified by the method of this invention will not kill or inhibit growth of pathogens; instead, the compounds will block the secretion of virulence factors that are critical to causing disease.
• FIGURE 1 shows the nucleotide and deduced amino acid sequence of espA (herein referred to as SEQ ID NO: 1 and SEQ ID NO:2, respectively). Potential ribosome binding sites are underlined. Nucleotides included in primers Donne-99 and Donne- 100, flanking the deletion engineered in pLCL 121 , are shaded.
• FIGURE 2 shows the construction of a non-polar mutation in espA.
• Primers Donne-90 and the reverse primer were used to amplify a fragment containing a 5 1 portion of the gene, which was cloned into pCRscript to create pLCLl 19.
• Primers Donne-100 and the universal primer were used to amplify a fragment containing a 3' portion of the gene, which was cloned into pCRscript to create pLCL120.
• New Nrul sites were incorporated into both Donne-99 and Donne-100 so that the Nrul-Sall fragment of ⁇ LCL120 could be cloned into pLCLl 19 to create pLCL121, which has a 150bp deletion within the espA gene.
• a Smal fragment from pUCl 8K containing the aphA-3 kanamycin resistance gene was cloned into the Nrul site of pLCLl 21 to create pLCLl 22. This insertion results in a transcriptional fusion of the aphA-3 gene and a translational fusion of the 3' end of the espA gene, with preservation of the espA reading frame.
• the ribosome binding sites are underlined.
• FIGURE 3 shows a genetic map of the plasmids containing RDEC-1 (A) and enteropathogenic Escherichia coli (EPEC) (B) espA, espD, and espB genes. Arrows indicate positions that stop codon insertions were made in espA and espB (A), and the frame shift mutation engineered into the BgHT site in espD (B). The 250 base pair deletion in espB is marked by / /. Solid and clear boxes represent open reading frames and predicted open reading frames. Restriction enzymes are indicated as follows: Bam*
• FIGURE 4 shows the nucleotide sequence of RDEC-1 espA (A) (herein referred to as SEQ ID NO: 3 and SEQ ID NO:4, respectively) and espB (B) (herein referred to as SEQ ID NO: 5 and SEQ LD NO:6, respectively).
• Asterisks indicate stop codons.
• Potential ribosome binding sites are underlined.
• Predicted amino acid sequences of EspA and EspB are aligned in C) (SEQ ID NO:7-14). Shaded areas represent identity.
• RDEC-1 espA U80908
• RDEC-1 espB U80796
• enteropathogenic Escherichia coli strain E2348/69 espA Z54352
• enteropathogenic Escherichia coli strain E2348/69 espB Z21555
• enteropathogenic Escherichia coli strain E2348/69 espD Y092278
• enterohemorrhagic Escherichia coli strain EDL933 serotype 0157 espB X96953
• enterohemorrhagic Escherichia coli strain 413/89-1 serotype 026 espB X99670
• the present invention provides a polypeptide, called EspA, which is secreted by pathogenic E. coli, such as the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli.
• pathogenic E. coli such as the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli.
• Diagnosis of disease caused by such pathogenic E. coli can be performed by standard techniques, such as those based upon the use of antibodies which bind to EspA to detect the protein, as well as those based on the use of nucleic acid probes for detection of nucleic acids encoding EspA polypeptide.
• the invention also provides isolated nucleic acid sequences encoding EspA polypeptide, EspA peptides, a recombinant method for producing recombinant EspA, antibodies which bind to EspA, and a kit for the detection of EspA-producing E. coli.
• the invention also provides a method of immunizing a host with EspA to induce a protective immune response to EspA.
• EspA for EPEC secreted [or signaling] protein ⁇ refers to a polypeptide which is a secreted protein from enteropathogenic or enterohemorrhagic E. coli and has a molecular weight of about 25 kilodaltons as determined by SDS-PAGE. EspA is an enteropathogenic E. c ⁇ //-secreted protein s necessary for activating epithelial cell signal transduction, intimate contact, and formation of attaching and effacing lesions, processes correlated with disease.
• An example of epithelial cells are cells.
• polypeptide encompasses any naturally occurring allelic variant thereof as well as manufactured recombinant forms.
• EspA o polypeptides encompass both naturally occurring and recombinant forms, / ' . e., non- naturally occurring forms of the protein and the peptide that are sufficiently identical to naturally occurring EspA peptide to have a similar function of causing pathogenicity. Examples of such polypeptides include the EspA polypeptides from enteropathogenic and enterohemorrhagic E coli, but are not limited to them.
• Protein and polypeptides s include derivatives, analogs and peptidomimetics.
• EspA peptides can be chemically synthesized using synthesis procedures known to one skilled in the art.
• an automated peptide synthesizer is used with N'Fmoc amino acids on a polyethylene glycol-polystyrene (PEGPS) graft resin.
• PGPS polyethylene glycol-polystyrene
• Suitable linkers such as a peptide amide linker (PAL) can be used, for example, to create carboxamide end groups.
• substantially pure is used herein to describe a molecule, such as a polypeptide (e.g., an EspA polypeptide, or a fragment thereof) that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated.
• a substantially pure molecule such as a polypeptide
• a substantially pure molecule can be at least 60%, by dry weight, the molecule of interest.
• One skilled in s the art can purify EspA 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.
• polyacrylamide gel electrophoresis e.g., SDS-PAGE
• column chromatography e.g., high performance liquid chromatography (HPLC)
• amino-terminal amino acid sequence analysis e.g., amino-terminal amino acid sequence analysis.
• EspA polypeptides included in the invention can have one of the amino acid sequences of EspAs from human or rabbit enteropathogenic E. coli, for example, the amino acid sequence of Figure 1 or Figure 4.
• Figures 1 and 4 can be characterized by being approximately 25 kD as determined by SDS-PAGE.
• 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 at least one EspA-specific activity or an EspA-specific epitope.
• one or more amino acids can be deleted from an EspA polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.
• amino- or carboxyl-terminal amino acids that are not required for EspA biological activity can be removed. Such modifications can result in the development of smaller active EspA polypeptides.
• EspA polypeptides included in the invention are polypeptides having amino acid sequences that are at least 50% identical to the amino acid sequence of an EspA polypeptide, such as any of EspAs in Figures 1 and 4.
• the length of comparison in determining amino acid sequence homology can be, for example, at least 15 amino acids, for example, at least 20, 25, or 35 amino acids.
• Homology can be measured using standard sequence analysis software (e.g., Sequence Analysis Software Package of the
• the invention also includes fragments of EspA polypeptides that retain at least one EspA-specific activity or epitope.
• an EspA polypeptide fragment containing, e.g., at least 8-10 amino acids can be used as an immunogen in the production of EspA-specific antibodies.
• the fragment can contain, for example, an amino acid sequence that is conserved in EspAs.
• the above-described EspA fragments can be used in immunoassays, such as ELISAs, to detect the presence of EspA-specific antibodies in samples.
• the EspA polypeptides of the invention can be obtained using any of several standard methods.
• EspA polypeptides can be produced in a standard recombinant expression systems (see below), chemically synthesized (this approach may be limited to small EspA peptide fragments), or purified from tissues in which they are naturally expressed (see, e.g., Ausubel, et al, supra).
• the invention also provides isolated nucleic acid molecules that encode the
• nucleic acids that encode EspAs as in Figures 1 and 4 are included in the invention. These nucleic acids can contain naturally occurring nucleotide sequences (see Figures 1 and 4), or sequences that differ from those of the naturally occurring nucleic acids that encode EspAs, but encode the same amino acids, due to the degeneracy of the genetic code.
• the nucleic acids of the invention can contain DNA or RNA nucleotides, or combinations or modifications thereof
• isolated nucleic acid is meant 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 contiguous 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
• SUt*STmJTE SHEET (RULE 26) 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.
• the nucleic acid molecules of the invention can be used as templates in standard methods for production of EspA gene products (e.g., EspA RNAs and EspA polypeptides; see below).
• EspA polypeptides (and fragments thereof) and related nucleic acids such as (1 ) nucleic acids containing sequences that are complementary to, or that hybridize to, nucleic acids encoding EspA polypeptides, or fragments thereof (e.g., fragments containing at least 12, 15, 20, or 25 nucleotides); and (2) nucleic acids containing sequences that hybridize to sequences that are complementary to nucleic acids encoding EspA polypeptides, or fragments thereof (e.g., fragments containing at least 12, 15, 20, or 25 nucleotides); can be used in methods focused on their hybridization properties.
• nucleic acid molecules can be used in the following methods: PCR methods for synthesizing EspA nucleic acids, methods for detecting the presence of an EspA nucleic acid in a sample, screening methods for identifying nucleic acids encoding new EspA family members, and therapeutic methods.
• the invention also includes methods for identifying nucleic acid molecules that encode members of the EspA polypeptide family in addition to EspAs shown in Figures 1 and 4.
• a sample e.g., a nucleic acid library, such as a cDNA library, that contains a nucleic acid encoding an EspA polypeptide is screened with an EspA-specific probe, e.g., an EspA-sp. -dfic nucleic acid probe.
• EspA-specific nucleic acid probes are nucleic acid molecules (e.g., molecules containing DNA or RNA nucleotides, or combinations or modifications thereof) that specifically hybridize to nucleic acids encoding EspA polypeptides, or to complementary sequences thereof.
• EspA-specific probe in the context of this method of invention, refers to probes that bind to nucleic acids encoding EspA polypeptides, or to complementary sequences thereof, to a detectably greater extent than to nucleic acids encoding other polypeptides, or to complementary sequences thereof.
• the term "EspA-specific probe” thus includes s probes that can bind to nucleic acids encoding EspA polypeptides (or to complementary sequences thereof).
• the invention facilitates production of EspA-specific nucleic acid probes
• Methods for obtaining such probes can be designed based on the amino acid sequence alignments shown in Figures 1-3.
• the probes which can contain at least 12, e.g.,ai least o 15, 25, 35, 50, 100, or 150 nucleotides, can be produced using any of several standard methods (see, e.g., Ausubel, et al., supra). For example, preferably, the probes are generated using PCR amplification methods .
• primers are designed that correspond to EspA-conserved sequences, which can include EspA-specific amino acids, and the resulting PCR product is used as a probe to screen a nucleic acid library, s such as a cDNA library.
• a nucleotide sequence encoding EspA was identified generally following this process based upon the analysis of the sequences of EspA in Figures 1 and 4.
• PCR primers are typically designed to contain at least 15 nucleotides, for example 15-30 nucleotides.
• EspA-specific primers containing 21 nucleotides, which encode EspA peptides containing 7 amino acids am described as follows.
• most or all of the nucleotides in such a probe encode EspA-conserved amino acids, including EspA-specific amino acids.
• primers containing sequences encoding peptides containing at least 40% EspA-conserved amino acids can be used.
• Such a primer, containing 21 nucleotides can include sequences encoding at least 3 EspA-conserved amino acids.
• the primer can contain sequences encoding at least one EspA-specific amino acid, for example, up to 7 EspA-specific amino acids. Once EspA-specific amino acid sequences are selected as templates against which primer sequences are to be designed, the primers can be
• SUBSTTTUTE SHEET (RULE 2r3) synthesized using, e.g., standard chemical methods.
• primers should be designed to include appropriate degenerate sequences, as can readily be determined by one skilled in the art.
• the term "espA” refers to polynucleotide encoding the EspA polypeptide. These polynucleotides include DNA, cDNA and RNA sequences which 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. For example, a espA polynucleotide can be subjected to site-directed mutagenesis. The espA polynucleotide sequence also includes antisense sequences. 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.
• nucleic acid encompasses RNA as well as single and double-stranded DNA and cDNA.
• the polynucleotide encoding EspA includes the nucleotide sequence in FIGURE 1 and 4, as well as nucleic acid sequences complementary to that sequence. A complementary sequence may include an antisense nucleotide.
• the sequence is RNA, the deoxynucleotides A, G, C, and T of FIGURE I and 4 are replaced by ribonucleotides A, G, C, and U, respectively.
• fragments of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the protein of FIGURE 1 or 4 under physiological conditions.
• nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, 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
• 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.
• DNA sequences of the invention can be obtained by several methods.
• the DNA can be isolated using hybridization techniques which 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.
• Oligonucleotide probes which 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
• SLBSTTTUTE SHEET (RULE 26) single-stranded DNA or denatured double-stranded DNA. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.
• the invention provides nucleic acid sequences encoding the EspA polypeptides, vectors and host cells containing them and methods of expression. After a peptide of EspA is isolated, nucleic acids 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, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, incorporated herein by reference).
• the terms "e.$pB” and “e ⁇ eA” refer to genes other than espA that encode enteropathogenic E. co/i-secreted proteins.
• the term “EspB” and “EaeA” refer to the proteins encoded by the espB and the e ⁇ eA genes, respectively.
• the invention provides vectors containing polynucleotides encoding the EspA polypeptide.
• the plasmid (pMSD2) with an intact espA can restore secretion of the EspA protein in an espA deficient strain.
• vectors includes plasmids, DNA and RNA viral vectors, baculovirai vectors, vectors for use in yeast, and other vectors well known to those of skill in the art.
• 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 pMWl 18, the positive-selection suicide vector pCVD442, and the commercially available pBluescript H SK(+) (Stragene, La Jolla, CA).
• the vector When the vector is a plasmid, it generally contains a variety of components including promoters, signal sequences, phenotypic selection genes, origins of replication sites, nd 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 pho A promoter, the bacteriophage ⁇ PL promoter (a temperature sensitive promotor), the tac promoter (a hybrid trp-lac promoter regulated by the lac
• a signal sequence is typically found immediately 5' to the nucleic acid encoding the peptide, and will thus be transcribed at the amino terminus of the fusion protein.
• Typical phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell.
• ampicillin resistance gene (amp) and the tetracycline resistance gene (/er) are readily employed for this purpose.
• the aphA-3 cassette, encoding a gene for resistance to kanamycin (kan) may be cloned into the region of vector containing polynucleotides encoding the
• EspA polypeptide for selection of the vector on kanamycin plates.
• Plasmid DNA procedures well known to those of skill in the art. Isolated polynucleotides encoding the EspA polypeptide to be combined to form the vector are cleaved and ligated together in a specific order and orientation to generate the desired vector.
• the invention provides a host cell containing a vector having a polynucleotide encoding the EspA polypeptide.
• the polynucleotides of the present invention can be used to produce transformed or transfected cells for enhanced production of the expressed EspA.
• EspA can be isolated from transformed cells by standard methods well known to those of skill in the art.
• the protein could be isolated, for example, using immunoafFinity purification.
• DNA sequences encoding EspA can be expressed in vitro by DNA transfer into a suitable host cell.
• "Host cells” are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell.
• progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included
• SUBSTfTUTE SHEET (RULE 26) when the term "host cell" is used.
• Methods of stable transfer meaning that the foreign DNA is continuously maintained in the host, are known in the art.
• the EspA polynucleotide sequences may be inserted into a recombinant expression vector.
• recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the EspA genetic sequences.
• Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
• the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
• Polynucleotide sequences encoding EspA can be expressed in either prokaryotes or eukaryotes.
• Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
• Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
• 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.
• the host is prokaryotic, such as E. coli
• competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art.
• MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
• triparental conjugation may be used to genetically introduce vector into E. coli, especially enteropathogenic E. coli or rabbit enteropathogenic E. coli.
• the transformed cells are selected by growth on an antibiotic, commonly tetracycline (te/) or ampicillin (amp), to which they are rendered resistant due to the presence of tet or amp resistance genes on the vector.
• te/ tetracycline
• amp ampicillin
• cells are selected on the basis of resistance to kanamycin and sucrose.
• Eukaryotic cells can also be cotransformed with DNA sequences encoding the EspA of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
• Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic ceils and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
• a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
• Isolation and purification of microbial expressed polypeptide, 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.
• prokaryotic organisms which may serve as host cells are £ coli strain JM101, E coli K.12 strain 294 (ATCC number 31 ,446), £ coli strain W3110 (ATCC number 27,325), E coli XI 776 (ATCC number 31 , 537), E. coli XL-lBroe (Stratagene), and E coli B; however, many other strains of £.
• prokaryotic host cell is enteropathogenic E coli. In another specific embodiment, the prokaryotic host cell is rabbit enteropathogenic £ coli.
• yeast strains such as PS23-6A, W301-18A, LUO, D234-3, INVSC1, INVSC2, YJJ337.
• Promoter and enhancer sequences such as gal 1 and pEFT- 1 are useful.
• Vra-4 also refers to yeast strains such as PS23-6A, W301-18A, LUO, D234-3, INVSC1, INVSC2, YJJ337.
• Promoter and enhancer sequences such as gal 1 and pEFT- 1 are useful.
• Vra-4 also provides yeast strains such as PS23-6A, W301-18A, LUO, D234-3, INVSC1, INVSC2, YJJ337.
• Promoter and enhancer sequences such as gal 1 and pEFT- 1 are useful.
• Vra-4 also refers to yeast strains such as PS23-6A, W301-18A, LUO, D234-3, INVSC1, INVSC2, YJJ337.
• Sequences useful as functional origins of replication include arsl and 2 ⁇ circular plasmid.
• the Gram-negative bacteria are a diverse group of organisms and include Spirochaetes 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 which comprises many important pathogenic genera, (2) Vibrio, Campylobacter and Helicobacter genera, (3) opportunistic organisms (e.g.,
• 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 host resistance. Currently, they are the most frequent cause of life-threatening bacteremia. Examples of pathogenic Gram-negative bacilli are £.
• 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 rod-shaped bacteria (family Vibrionaceae). Vibrio cholerae causes cholera in humans; other species of Vibrio cause animal diseases. £. coli colonize the intestines of humans and warm blooded animals, where they are part of the commensal flora, but there are types of £ coli that cause human and animal intestinal diseases. They include the enteroaggregative £ coli
• the Neisseria species include N. cinerea, N. gonorrhoeae, N. gonorrhoeae subsp. kochii, N. lactamica, N. meningitidis, N. polysaccharea, N. mucosa, N. sicca, N. sub ⁇ ava, the asaccharolytic species N.flavescens, N. caviae, N. cuniculi and N. ovis.
• the strains of Moraxella (Branhamella) catarrhalis are also considered by some taxonomists to be Neisseria.
• Other related species include Kingella, Eikenella, Simonsietta, Alysietta, CDC group EF-4, and CDC group M-5.
• Veillonella are Gram-negative cocci that are the anaerobic counterpart oi Neisseria. These non-motile diplococci are part of the normal flora of the mouth.
• Neisseria 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(mewngi ⁇ s, septicemia, pneumonia, arthritis, urethritis).
• 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.
• the EspA polypeptides of the invention can also be used to produce antibodies which are immunoreactive or bind to epitopes of the EspA polypeptides.
• Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the
• SUBSiTTUTE SHEET (RULE 26) protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).
• antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, Fab', F(ab')j, and Fv that can bind the epitope. These antibody fragments retain some ability selectively to bind with its antigen or receptor and are defined as follows:
• Fab the fragment that contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and part of one heavy chain;
• Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and part of the heavy chain; two Fab' fragments are obtained per antibody molecule;
• Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
• Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable peptide linker as a genetically fused single chain molecule.
• An epitope is 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, 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.
• the invention also provides peptide epitopes for use in designing espA specific nucleotide probes or anti-EspA antibodies.
• 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 antibodies of the invention including polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies and the like, have with the ability to bind with high immunospecificity to the EspA proteins, peptides or nucleotide sequences of the invention, or fragments thereof. These antibodies can be unlabeled or suitably labeled. Antibodies of the invention can be used for affinity purification of EspA for example. Antibodies of the invention 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. Antibodies of the invention may be employed in known immunological procedures for qualitative or quantitative detection of the nucleotide sequences or portions thereof.
• the invention provides a method for detecting EspA polypeptide in a sample, including contacting a sample from a subject with an antibody to EspA polypeptide;
• sample includes material derived from a mammalian or human subject or other animal. Such samples include but are not limited to hair, skin samples, tissue sample, cultured cells, cultured cell media, and biological fluids.
• EspA polypeptide can be detected in
• HeLa cell e.g., human
• culture e.g., human
• 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.
• CNS tissue e.g. , CNS tissue, neural tissue, or eye tissue
• 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.
• CSF cerebrospinal fluid
• sample also includes solutions containing the isolated polypeptide, media into which the polypeptide has been secreted, and media containing host cells which produce the EspA polypeptide.
• a sample may be a protein samples which 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.
• the presence of EspA polypeptide in the sample is indicative of infection by enteropathogenic £ coli.
• the presence of EspA polypeptide in the sample is indicative of infection by enterohemorrhagic £ coli.
• Proteins, protein fragments, and synthetic peptides of the invention are projected to have numerous uses including prognostic, therapeutic, diagnostic or drug design applications. Proteins, protein fragments, and synthetic peptides of the invention will provide the basis for preparation of monoclonal and polyclonal antibodies specifically immunoreactive with the proteins of the invention.
• the invention provides a method of immunizing a host susceptible to disease caused by EspA-producing £ coli, by administering to a host with the polypeptide of claim 1; and inducing a protective immune response in the host to EspA polypeptide. The infection of the host by EspA-producing organism is thereby prevented.
• the EspA-producing organism is an £ coli strain.
• the £ coli strain is either enteropathogenic or enterohemorrhagic JE, atli.
• the invention provides a method of ameliorating disease caused by EspA-producing organism, by immunizing a host with EspA polypeptide and inducing an immune response in the host to the EspA polypeptide.
• the EspA-producing organism is an £. coli strain.
• the £ coli strain is either enteropathogenic or enterohemorrhagic E. coli.
• the invention provides a method for detecting espA polynucleotide in a sample, by contacting a sample suspected of containing espA polynucleotide with a nucleic acid probe that hybridizes to espA polynucleotide; and detecting hybridization of the probe with espA polynucleotide.
• the detection of hybridization is indicative oiespA polynucleotide in the sample.
• the invention provides an organism with a mutated espA gene.
• Preferred organisms in which an espA gene may be mutated include but are not limited to bacteria.
• the bacteria in which an espA gene may be mutated are £ coli.
• the E. coli in which an espA gene may be mutated are enteropathogenic and enterohemorrhagic £. coli.
• the invention provides a recombinant method for producing espA polynucleotide, including inserting a nucleic acid encoding a selectable marker into the polynucleotide encoding EspA polypeptide.
• the resulting polynucleotide encodes a recombinant EspA polypeptide containing the selectable marker.
• a selectable marker may be a herpes simplex virus (HSV) tag, for which there are commercially available antibodies.
• HSV herpes simplex virus
• the invention provides a recombinant method for producing EspA polypeptide, by growing a host cell containing a polynucleotide encoding EspA polypeptide under conditions which allow expression and secretion of EspA polypeptide; and isolating the polypeptide.
• Methods of producing polypeptides and peptides recombinantly are within the scope of this invention.
• the term "conditions which allow expression and 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.
• 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.
• LB Luria-Bertani
• the invention provides a method to identify a compound which inhibits bacterial type LH secretion systems, by introducing the polynucleotide encoding a selectable marker into bacteria having a bacterial type HI secretion system; growing the bacteria under conditions which allow growth of bacteria and secretion of the polypeptide encoded by the polynucleotide; contacting a compound suspected of inhibiting the bacterial type LH secretion system with the bacteria; inducing the expression of the polypeptide; and detecting the secretion of the polypeptide.
• a lack of secretion is indicative of the inhibition of bacterial type HI secretion systems.
• the term "type HI secretion" and "type III secretion” pathway refer to a specialized machinery to export molecules across a cell membrane.
• the type HI secretion pathway uses adenosine triphosphate (ATP) as an energy source.
• ATP adenosine triphosphate
• the type ⁇ l secretion pathway is different than other secretion pathways found in Gram-negative bacteria, although it is homologous to flagella and filamentous phage assembly genes. It does not resemble any mammalian pathway. It is always associated with disease production.
• the virulence factors secreted by the type HI secretion pathway vary between pathogens, although components of the type HI secretion machinery are interchangeable, at least for Salmonella, Shigella, and Yersinia
• polypeptide or nucleotide sequences of the invention can be used to identify compounds or compositions which interact (e.g., bind) with them and affect their biological activity. Such effects include inhibition or stimulation of EspA activity or secretion.
• the invention provides a method for producing a nonpathogenic organism, by generating a mutation in a polynucleotide encoding EspA polypeptide; inserting a nucleic acid sequence encoding a selectable marker into the site of the mutation; introducing the mutated espA polynucleotide into a chromosomal espA gene of an organism to produce a mutation in the chromosomal espA gene; and selecting organisms having the mutation.
• mutation refers to a change in the nucleotide sequence of a gene, in particular, the polynucleotide encoding EspA polypeptide. Mutations include mutations producing EspA polypeptide with a different amino acid sequence, missense mutations (including frame shift mutations), nonsense mutations (including knockout mutations), and recombinant genetic techniques which produce fusion proteins containing part of the EspA polypeptide.
• the nucleic acid sequence encoding a selectable marker encodes resistance to kanamycin.
• the aphA-3 cassette encoding a gene for resistance to kanamycin (kan) may be cloned into the polynucleotides encoding the EspA polypeptide for selection of the mutated espA polynucleotide on kanamycin plates to produce a knockout mutation.
• kan kanamycin
• Preferred organisms in which to practice the invention include but are not limited to bacteria.
• the organism which is used to generate a mutation in a polynucleotide encoding EspA polypeptide is £ coli.
• the E. coli that may be transformed are enteropathogenic and enterohemorrhagic £ coli.
• the invention provides a method of activating tyrosine kinase activity in a host cell by adding both mutant espA-deficient organisms that express Eae polypeptide and mutant e ⁇ eA-deficient organisms that express EspA polypeptide to a host cell and binding the bacteria to the host cell, thereby activating host cell tyrosine kinase activity in the cell.
• the activation of host cell tyrosine kinase activity in the cell causes the tyrosine phosphorylation of a 90 kilodalton host membrane protein, Hp90, and fluxes of intracellular inositol phosphate (LPj) and calcium.
• LPj intracellular inositol phosphate
• an eae A mutant can be used to complement an espA mutant for invasion when these two mutant strains were used to co-infect HeLa ceils.
• the invention thus provides a useful scientific method to investigate pathogenesis by cell biology.
• kits containing one or more antibodies of the invention as well as a nucleotide based kit.
• the kit is useful for the detection of EspA polypeptide and is a carrier means compartmentalized to receive in close confinement a container containing an antibody which binds to EspA polypeptide.
• a “container means” includes vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
• the antibody which binds to EspA polypeptide is detectably labeled.
• 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 kit is useful for the detection of an espA polynucleotide and is a carrier means compartmentalized to receive in close confinement a container containing the nucleic acid probe that hybridizes to espA polynucleotide.
• nucleic acid probe that hybridizes to espA polynucleotide is detectably labeled.
• 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.
• EspA is a secreted protein, it is useful as a fusion partner for cloning and expressing other peptides and proteins.
• EspA fused to a protein of interest is recombinantly produced in a host cell, e.g. , £ coli, and the fusion protein is secreted into the culture media in which the transformed host is grown.
• the fusion protein can be isolated by anti-EspA antibodies followed by cleavage of EspA from the peptide or protein of interest. ELISA or other immunoaffinity methods can be used to identify the EspA fusion protein.
• the invention provides a method of producing an EspA fusion protein including growing a host cell containing a polynucleotide encoding EspA operably linked to a polynucleotide encoding a polypeptide or peptide of interest under conditions which allow expression and secretion of the fusion polypeptides and isolating the fusion polypeptide.
• operably linked or associated 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. t the fusion protein).
• the purpose of this Example is to characterize espA, a gene responsible for the attaching and effacing activity of enteropathogenic £ coli.
• the espA gene encodes the 25 kilodalton secreted protein and is located on the enteropathogenic £ coli genome
• the DNA sequence of the enteropathogenic E. coli Locus of Enterocyte Effacement between e ⁇ eA and espB was determined. DNA sequencing was performed as follows: The Sall-BglU DNA fragment of the Locus of Enterocyte Effacement spanning from within e ⁇ eA to upstream (5 1 ) of espB was cloned into the commercially available plasmid pBluescript to create the plasmid pLCL109. A series of nested DNA deletions was made from the end of the plasmid pLCL109 closer to the e ⁇ eB gene.
• DNA deletions of plasmid pLCL109 were used as templates to determine the nucleotide sequence of both strands of DNA using oligonucleotide primers synthesized as needed, [a- 3S S]dATP, and the Sequenase enzyme. DNA sequence data were analyzed with the package developed by the Genetics Computer Group of the University of Wisconsin.
• the purpose of this Example was to construct a mutation in an espA gene on the chromosome of an organism.
• the purpose of this Example was to construct a mutation in an espA gene on a plasmid.
• the plasmid can then be used to create mutations in the espA gene of other organisms. Plasmids with a nonpolar mutation in espA gene were constructed. Furthermore, a mutant bacterial strain with a nonpolar mutation in its chromosomal espA gene was generated.
• FIGURE 2 An espA gene with a nonpolar mutation is described in FIGURE 2.
• the use of the polymerase chain reaction (PCR) generated a deletion with restriction sites that allow an aphA-3 cassette, encoding resistance to kanamycin, to be cloned into the deleted region.
• This aphA-3 cassette is preceded (5') by translation stop codons in all three reading frames and immediately followed (3") by a consensus ribosome binding site and a start codon.
• the insertion into the espA gene was engineered to retain the reading frame of the 3' end of espA and to therefore allow unaffected transcription and translation of downstream (3 ⁇ genes. DNA sequencing confirmed the reading frame of the mutation.
• the construction of a nonpolar mutation in the espA gene on a plasmid was performed by the polymerase chain reaction as follows:
• the PCR template was plasmid pLCLl 14, containing the CM- Bg ⁇ fragment of pLCL109 cloned into pBluescript.
• Two pairs of primers were used, the universal primer with Donne-99 and the reverse primer with Donne-100.
• Oligonucleotides Donne-99 and Donne-100 are nucleotides 4157 through 4140 and 4297 through 4324 of the SaU-BgUI fragment, respectively.
• an Nrul restriction site was engineered into the 5' end of both Donne-99 and Donne-100.
• Oligonucleotides were constructed at the Biopolymer Laboratory ofthe University of Maryland at Baltimore. PCR was performed on 50 ⁇ L samples in a minicyler. The PCR reaction was thirty cycles of D ⁇ A denaturation at 94 * C for one minute, annealing to the primers at 55°C for two minutes, and polynucleotide extension at 72°C for three minutes. The two resulting amplified fragments amplified
• SUBST ⁇ L ⁇ SHEET (RULE 26) were each cloned into the commercially available plasmid pCRscript to create pLCll 19 • and pLCLl 20, respectively.
• the insert of pLCLl 20 was then cloned into pLCLl 19 using Sail and Nrul to create pLCL121 containing the desired deletion.
• the 850 base pair aphA-3 kanamycin resistance cassette flanked by Smal sites was then inserted into the Nrul site of pLCL121.
• the mutated espA allele was cloned in the positive-selection suicide vector pCVD442 and introduced into wild-type enteropathogenic £ coli strain E2348/69 by allelic exchange.
• a espA mutant bacterial strain was constructed as follows: The Sail - Sad fragment from the plasmid with the aphA-3 kanamycin resistance which contained the interrupted espA gene was cloned into positive-selection suicide vector pCVD442 in
• DH5 ⁇ ir for introduction into E2348/69 by triparental conjugation or by electroporation in 0.1 cm cuvettes with an £ coli pulser set at 1.8 kV.
• An espA mutant was selected on modified LB kanamycin plates.
• the resulting enteropathogenic £ coli mutant strain, UMD872 was resistant to sucrose and kanamycin, and sensitive to ampicillin.
• Bacteria were stored at -70°C in 50% LB broth/50% glycerol (vol/vol) and grown on LB agar plates or LB broth with chloramphenicol (20 ⁇ g/ml), ampicillin (200 ⁇ g/ml), nalidixic acid (50 ⁇ g/ml), or kanamycin (50 ⁇ g/ml) added as needed.
• SUBST ⁇ TUTE SHEET confirmed by immunoprecipitation using an anti-EPEC antisera which reacts with tiie secreted EspA protein.
• Western analysis using anti-EPEC antisera showed no 25 kilodalton secreted protein.
• Secretion of the EspA protein was restored when the espA deficient strain was transformed a plasmid (pMSD2) with an intact espA.
• pMSD2 plasmid
• the increased production by the bacteria of EspA protein encoded by the plasmid reduced the secretion of the other proteins by the type LH secretion pathway.
• Example 4 Enteropathogenic £ coli ESPA Is Required for Invasion of Cultured Epithelial Cells The purpose of this Example was to examine whether the enteropathogenic £ coli EspA protein is involved in epithelial cell invasion. EspA protein is needed for triggering the host signal transduction pathway and invasion of host cells.
• Monolayers of an epithelial cancer cell were infected for three hours with either parental wild-type or espA mutant enteropathogenic £ coli strains.
• the number of adherent and intracellular (i.e., invasive) enteropathogenic £ coli was determined.
• the absolute number of bacteria adherent to HeLa cells varied between strains, according to the different growth rates between the mutant and parental enteropathogenic £ coli strains.
• the espA mutant strain UMD872 adheres efficiently to epithelial cells, it is deficient for invasion. However, UMD872 invaded HeLa monolayers at near wild-type levels when the espA gene was genetically complemented by the plasmid pMSD2, which encodes an intact espA gene.
• the purpose of this Example was to determine whether EspA is essential to induce signal transduction events in epithelial cells.
• Enteropathogenic £ coli induce tyrosine phosphorylation of a host cell 90 kilodalton membrane protein and subsequent accumulation of phosphorylated proteins, actin, and other cytoskeletal components beneath adherent bacteria.
• the ability of an espA mutant defective for invasion to induce these two signaling events in mammalian cells was examined.
• the espA mutant strain UMD872 was unable to induce phosphorylation of host Hp90.
• the ability to induce this phosphorylation event was restored by a plasmid (pMSD2) that encodes the EspA protein.
• the adherence and invasion assays were performed as follows: 10 J HeLa cells grown in DMEM were infected with the various enteropathogenic £ coli strains
• HeLa cells were grown at 37°C with 5% CO 2 in Dulbecco's Minimal Eagles Medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum. Monolayers were washed thrice in phosphate-buffered saline before lysing in DMEM (DMEM) supplemented with 10% (vol/vol) fetal calf serum. Monolayers were washed thrice in phosphate-buffered saline before lysing in DMEM (DMEM) supplemented with 10% (vol/vol) fetal calf serum. Monolayers were washed thrice in phosphate-buffered saline before lysing in DMEM (DMEM) supplemented with 10% (vol/vol) fetal calf serum. Monolayers were washed thrice in phosphate-buffered saline before lysing in DMEM (DMEM) supplemented with 10% (vol/vol)
• enteropathogenic £ co/i-secreted proteins and HeLa cellular proteins was accomplished as follows: Tissue culture plates were seeded overnight with
• HeLa cells Before infection, the media was replaced with DMEM minus mediionine/cysteine containing cycloheximide (100 ⁇ g/ml). HeLa cells were grown at 37°C with 5% COj in DMEM supplemented with 10% (vol/vol) fetal calf serum. Enteropathogenic £ coli was added (m.o.i. 100: 1) and incubated for 2.5 hours at 37°C in 5% COj incubator before adding 200 ⁇ g/ml 35 S cysteine/methionine for 30 minutes. The culture supernatant was removed and the bacteria pelleted by centrifugation (18,000x g, 10 minutes).
• the supernatant secreted proteins were precipitated by the addition of ice cold trichloroacetic acid (10% vol/vol) and incubated on ice for 60 minutes. Proteins were pelleted by centrifugation as above and resuspended in Laemelli sample buffer. Samples were resolved by 12% SDS-PAGE and protein profiles examined by autoradiography or transferred to nitrocellulose prior to probing with anti-EPEC antibodies.
• HeLa phosphotyrosine proteins were analyzed as follows: Infected HeLa monolayers were washed thrice with ice cold phosphate-buffered saline prior to lysis ia 1% Triton X-100 in the presence of protease inhibitors. The Triton insoluble and soluble
• SUBSiTTUTE SHEET (RULE 26) fraction were isolated, resuspended in Laemelli sample buffer, and analyzed for the presence of phosphotyrosine proteins by Western immunoblot analysis with anti-phosphotyrosine antibodies.
• the monolayers were then washed and fixed in 2.5% paraformaldehyde prior to staining for filamentous actin (using phallodin-rhodamine) or with anti-phosphotyrosine antibodies with an appropriate secondary fluorescein conjugated antibody.
• EspB in rabbit enteropathogenic £ coli RDEC-1
• the espA and espA genes were cloned and their sequences were compared to those of enteropathogenic £ coli (EPEC).
• the EspA protein showed some similarity (88.5% identity).
• the EspB protein was heterogeneous in internal regions (69.8% id ⁇ ⁇ ty), but was identical to one strain of enterohemorrhagic £ coli (EHEC).
• SUBS ⁇ rUTE SHEET enteropathogenic £ coli.
• Vent DNA polymerase was used for PCR to amplify chromosomal DNA from RDEC-1 and enteropathogenic £ coli strains.
• the PCR reaction was carried out for thirty cycles of denaturation at 94°C for one minute, annealing at 55°C for one minute, and elongation at 72°C for two minutes.
• the resulting 4.3 kilobase pair product was ligated into the commercially available plasmid pBluescript and both strands were sequenced.
• DNA sequencing was done as follows: The 4 J kilobase pair DNA fragment encoding the espA and espB genes was amplified by PCR using the primers AA01(+) and MSI 1(-), and RDEC-1 chromosomal DNA as the DNA template. The resulting blunt end fragment was digested with Sail and cloned into the Sall-Smal site of the commercially available plasmid pBluescript-H SK (+). The DNA sequence of RDEC-1 espA and both strands using the commercially available Taq DyeDeoxy kit ( Figure 3)
• EspB (314 amino acids) was 33,219 dalton.
• RDEC-1 EspA was somewhat similar to that of enteropadiogenic £ coli with 88.5% identity ( Figures 4A and 4B).
• RDEC-1 EspB protein was identical to the recently reported EspB from enterohemorrhagic £ coli strain 413/89-1 serotype 026, which was originally isolated from a calf and also isolated from patients with hemolytic uraemic syndrome, although two nucleotide differences occurred at positions 12 (T to C) and 7J9 base pair (G to T). Furthermore, RDEC-1 EspB showed 70.3% enc. 69.8% identity respectively to that of enterohemorrhagic £ coli serotype 0157 and enteropathogenic £ coli strains. Small sequence deletions were found in RDEC-1 and enterohemorrhagic £ coli (serotype 026 and 0157) EspB at the same positions when compared to the enteropathogenic £ coli sequences ( Figures 4A-C).
• Example 7 Characterization of RDEC-1 ESPA and EsnB The purpose of this Example was to investigate the function of EspA and
• inverse PCR was carried out using the ⁇ espA(+) and ⁇ espA(-) primers which contain a BglH restriction site and a stop codon using circular pORF23 as a DNA template.
• the PCR product was then blunt end ligated to obtain pORF23 ⁇ .
• the resulting plasmid contained a stop codon and a BglD site 235 base pair downstream (3 * ) from the esp A start codon, which was confirmed by DNA sequencing.
• the 1.1 kilobase pair Sall-Sacl DNA fragment containing the esp A mutation from pORF23 A was inserted into the same sites of the suicide vector pCVD442, which contains the sacB gene for positive selection and an ampicillin resistance gene, to obtain pAA23 ⁇ .
• the resulting plasmid was introduced into E. coli SMIO ⁇ pir and back conjugated into RDEC-1 harboring pACYCl 84.
• inverse PCR was carried out using the ⁇ espB(+) and ⁇ cspB(-) primers, and pBxb as a DNA template.
• pBxb contains the 1.4 kilobase pair Xbal fragment from pORF23B encoding espB cloned into the pBluescript vector.
• the resulting PCR product was self-ligated to obtain pBxb ⁇ that contained a stop codon and a BglH site introduced by the ⁇ espB(-) and ⁇ espB(+) primers.
• the resulting esp gene in pBXb ⁇ was deleted by 250 base pair, starting 154 base pair downstream (3*) of the espB start codon.
• the 1.1 kilobase pair Sall-Sacl site DNA fragment containing the ejpB mutation from pBxbA was inserted into the same site of the pCVD442 to obtain pAABxb ⁇ .
• the resulting plasmid was transformed into £ coli SMIO ⁇ pir and back conjugated into RDEC-1 harboring pACYCl 84.
• pAABxb ⁇ was introduced into AAF001 ⁇ A (EspA ) strain.
• Three RDEC-1 non-polar mutant strains were confirmed by their phenotypes which maintain resistance to sucrose and chloramphenicol, and sensitivity to ampicillin. To confirm the stop codon
• back mutation Mutations back to wild type (“back mutation") were made to confirm that suicide vectors do not affect respective flanking region or other loci.
• Two back mutant strains were obtained by bans conjugation of the suicide vectors pAA23 and pAABxb into AAFOO 1 ⁇ A (EspA) and AAF001 ⁇ B (EspB) strains.
• the resulting back mutant strains, AAF003 and AAF004 were confirmed by PCR and BglH digestion.
• the construction of back mutations in EspA and EspB strains was done as follows: The 1.1 kilobase pair Sall-Sacl DNA fragment from pORF23 containing esp A was inserted into the Sall-Sacl sites of ⁇ CVD442 to obtain pAA23.
• the 1.4 kilobase pair Sall-Sacl fragment of pBxb was inserted into the Sall-Sacl site of pCDD442 to obtain pAAFBxb.
• pAA23 and pAABxb were introduced into SM ⁇ pir and bans conjugated into AAFOO IAA and AAFOO 1 AB.
• the resulting back mutant strains were confirmed as described above and designated as AAF002 (EspA+) and AAF003 (EspB+).
• pMWespAB was digested with BglH which has a restriction site in espD open reading frame, blunt ended with Klenow fragment, and then self-ligated to obtain pMW6espD.
• pMWespAB was also digested with BglH and BamHI, and then self ligated to obtain pMWespB.
• the PMWespAB was digested with BglH-Sall, and filled with Klenow Fragment, then self-ligated to obtain pMWespA.
• RDEC-1 secreted two proteins with similar mobility to enteropathogenic £ coli EspA and EspB.
• RDEC-1 secreted proteins were prepared as follows: Bacterial overnight > cultures were diluted 1:100 into DMEM and incubated to an optical density of 1.0 at 600 nm (OD600). For RDEC-1 mutant strains containing enteropathogenic £ coli esp A ami espB recombinant plasmids, isopropylthiogalactoside (LPTG) was added in DMEM to induce transcription. Bacteria were removed by centrifugation (18,000 x g, 10 mir»it.»y) and the supernatant precipitated by addition of 10% ice-cold trichloroacetic acid, and incubated on ice for one hour. After centrifugation, the pellets were resuspended in
• EspA, EspB, and EspA/EspB proteins were tested for their secretion profile and Western blot analysis.
• EspB whose gene is located downstream (3 1 ) from ejpA, was still secreted in the mutant strain AAF00 ⁇ A (EspA ), indicating that the stop codon insertion mutation does not affect downstream gene expression.
• EspA mutant strain AAF00 ⁇ A
• the amount of the other secreted proteins were decreased in the EspA “ , EspB', EspA/EspB " strains when compared to wild type RDEC-1 strain. Furthermore, the decrease of detectable secretion of the 40 kilodalton and 39 kilodalton proteins in the
• EspA/EspB " strain is greater than that found in EspA " and EspB ' strains.
• Secretion of enteropathogenic £ coli proteins, except EspC, arc mediated by a type HI secretion system encoded by the sep cluster. It is possible that truncation of EspA or EspB by inserting a stop codon may interfere with this secretion pathway or feedback regulation of this system, thereby affecting secretion of the other type HI dependent secreted proteins.
• Esp proteins are needed for triggering of the host signal transduction pathway. Enteropathogenic £ coli EspA and EspB proteins induce host signal transduction pathways resulting in accumulation of tyrosine phosphorylated proteins, cytoskeletal actin, and other components beneath the adherent bacteria. To determine whether
• RDEC-1 EspA and EspB trigger these events in HeLa cells
• cytoskeletal actin add tyrosine-phosphorylated proteins were stained with rhodamine-phallodin and fluorescently labelled anti-phosphotyrosine antibody.
• the level of accumulation of cytoskeletal actin and tyrosine-phosphorylated protein beneath the attached RDEC-1 is lower than that of enteropathogenic £ coli, these behaviors were indistinguishable to that of enteropathogenic £ coli.
• RDEC-1 EspA”, EspB " , and EspA/EspB" strains did not accumulate cytoskeletal act in or tyros ine- phosphorylated proteins beneath the attached bacteria.
• the back mutant strains AAF003 and AAF004 accumulated these proteins similar to the parental strains.
• Enteropathogenic £ coli EspB also did not complement RDEC-1 EspA in co- infection experiment Therefore, functionally EspA and EspB are similar in RDEC-1 and enteropathogenic £ coli with respect to activating host signal transduction pathways, although both proteins need to be secreted by the same strain.
• Tyrosine-phosphorylated Hp90 could be detected by immunoblotting when HeLa cells were infected with enteropathogenic £ coli. Tyrosine-phosphorylated Hp90 could not be detected with RDEC-1 infected cells, even though tyrosine phosphorylated proteins could be observed under adherent RDEC-1 bacterial cells by immunofluorescence.
• Enterohemorrhagic £ coli does not induce tyrosine phosphorylation in HEp-2 and T84 cells as judged by immunofluorescence microscopy.
• the sequencing results showed that RDEC-1 EspB is more similar to that of enterohemorrhagic £ coli than enteropathogenic £ coli.
• These results in this Example show that the lower accumulation of tyrosine-phosphorylated proteins during RDEC-1 infection is due to lower adherence efficiency of RDEC-1 because of differences in adhesion levels or heterogeneity of Esp proteins or both.
• SUBSTTTUTE SHEET (RULE 26) wild type RDEC-1 strain, indicating that adherence is independent of EspA and EspB expression.
• the invasive ability of wild type RDEC-1 was about ninety times lower than that of enteropathogenic £ coli, this ability was further decreased in the mutant strains EspA, EspB ' , and EspAVEspB " .
• invasion was restored by back mutation strains AAF002 and AAF003.
• RDEC-1 EspB was more similar to that of enterohemorrhagic £ coli radier than enteropathogenic £ coli, perhaps emphasizing the role of EspB in invasion. These findings strongly support that Esp heterogeneity affects the invasive ability of enteropathogenic £ coli, RDEC-1 , and enterohemorrhagic £ coli.
• EspD mutant affects EspA and EspB secretion.
• Enteropathogenic £ coli contains an open reading frame, espD, located between esp A and espB.
• espD open reading frame
• the plasmid pMWespD encoding enteropathogenic £ coli esp A, ⁇ espD (frame shift mutation at the BglH site), arid espO was constructed and introduced into the RDEC-1 double mutant strain, AAF001 AAB.
• truncated enteropathogenic £ coli EspD may affect the secretion of EspA and EspB in AAFOO 1 AAB [pMW ⁇ espD] due to interference in type HI secretion system. Whether or not EspD is directly involved in this secretion system is still unclear.
• Enteropathogenic £ coli and RDEC-1 secreted proteins are tightly controlled by temperatures, which correspond to their relevant host body temperatures. Temperature regulates the expression of enteropathogenic £ coli and enterohemorrhagic £ coli 413/89-1 secreted proteins. EspB expression was greatly increased when the temperature was shifted from 20°C to 37°C. Because EspA and EspB proteins are regulated by appropriate host body temperatures, wild type enteropathogenic £ coli and RDEC-I strains were inoculated into DMEM and the secreted proteins were prepared following incubation at various temperatures, then analyzed by SDS-PAGE. Expression of enteropathogenic £ coli secreted proteins were visible at 33°C and reached maximal secretion level at 36°C.
• RDEC-1 and enteropathogenic £ coli secreted proteins correlated with their natural host's body temperature. This explains their strict host specificity and the lack of enteropathogenic £ coli infection in rabbits or other animals. Animal infection studies using RDEC-1 esp A and espB strains will provide information about the role of these secreted proteins in virulence, and may possibly be useful for vaccine studies.
• RDEC-1 and its esp A 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.
• 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 esp A or espB strains (2.5x10 10 ) were inoculated into the stomach using orogastric tubes. The same dosage of bacteria was inoculated into each rabbit the following day.
• 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.
• 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 purpose of this Example is to provide an assay to screen for inhibitors of bacterial type HI 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 £ coli.
• a plasmid contains the genetic region of esp A that encodes the amino-terminal portion of EspA (needed to mediate type HI secretion) fused to a Herpes Simplex Virus (HSV) sequence that encodes a sequence tag to which commercial antibodies are available.
• HSV Herpes Simplex Virus
• This plasmid is transformed into a strain that contains an ejrpA mutation yet still secretes the other enteropathogenic £ co/i-secreted proteins that use the type HI secretion system. The supernatant of organisms containing these fusions is collected, added to an ELISA plate, followed by standard ELISA. A calorimetric readout indicates the fusion protein is secreted.
• This plasmid is also transformed into a strain which is defective for type HI secretion (i.e. a negative control).
• a strain which is defective for type HI secretion i.e. a negative control.
• 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.
• SUBST ⁇ TUTE SHEET (RULE 26) reagents are needed. It is automated and used to screen reagents to identify inhibitors of bacterial type HI secretion.
• bacteria are grown standing overnight in tissue culture fluid in the presence of compounds to be tested. These conditions yield enteropathogenic £ co/i-mediated secretion. The following day, bacteria are removed by centrifugation, and the supernatant placed into wells of a 96 well microtiter plate. A standard ELISA is performed on the supernatants. If the compound being tested is bactericidal, the bacteria do not grow overnight
• a polynucleotide encoding another polypeptide secreted by enteropathogenic £ coli is fused to several well known molecules.
• a polynucleotide encoding EspB polypeptide was fused a HSV tag, to which commercial antibodies are available.. The gene fusion was still secreted out of enteropathogenic £ coli.
• This plasmid was transformed into a strain that contained an espA mutation yet still secretes the other enteropathogenic £ co/j-secreted proteins that use the type HI secretion svstem. The supernatant of organisms containing these fusions is collected, added to an ELISA plate, followed by standard ELISA technology with, for example, anti-HSV antibodies. This screen was used to assay plant extracts from medically important plants. Dilutions of 1/200-1/1000 (about 250 ⁇ g/ml) are appropriate. Promising compounds are rescreened in the EOS A secretion assay to check for reproducibility.

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