WO1992017785A1 - Detection of salmonella - Google Patents

Detection of salmonella

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Publication number
WO1992017785A1
WO1992017785A1 PCT/US1992/002591 US9202591W WO1992017785A1 WO 1992017785 A1 WO1992017785 A1 WO 1992017785A1 US 9202591 W US9202591 W US 9202591W WO 1992017785 A1 WO1992017785 A1 WO 1992017785A1
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phop
gene
pagc
salmonella
strain
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PCT/US1992/002591
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French (fr)
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Samuel I. Miller, Iii
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The General Hospital Corporation
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255Salmonella (G)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1235Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Salmonella (G)

Abstract

A method of detecting Salmonella in a sample including contacting the sample with an antibody specific for a PhoP regulated gene product, allowing the antibody to form immune complexes with Salmonella, and detecting the immune complexes as an indication of the presence of Salmonella in the sample.

Description

DETECTION OF SALMONELLA The invention relates to the immunological detection of Salmonella . Background of the Invention

This invention was made in the course of work supported by the United States Government, which has certain rights in the invention.

Enteric fevers and diarrheal diseases, e.g., typhoid fever and cholera, are major causes of morbidity and mortality throughout the developing world. Hook et al., 1980, In Harrison's Principles of Internal Medicine, 9th Ed., 641-848, McGraw Hill, New York. Salmonella species cause a spectrum of clinical disease that includes enteric fevers and acute gastroenteritis, Hook et al., 1980, supra .

S . typhi , the bacterium that causes typhoid fever, can only infect man. The narrow host specificity of S . typhi has resulted in the extensive use of S. enter iditis typhimurium infection of mice as a laboratory model of typhoid fever, Carter et al., 1984 J. Exp. Med. 139:1189. S. typhimurium infects a wider range of hosts, causing acute gastroenteritis in man and a disease similar to typhoid fever in the mouse and cow. Recent studies have begun to define the molecular basis of Salmonella typhimurium virulence, Miller et al., 1989, Proc. Natl. Acad. Sci. USA 86:5054, hereby incorporated by reference. Salmonella typhimurium strains with mutations in the positive regulatory regulon phoP are markedly attenuated in virulence for BALB/c mice. The phoP regulon is composed of two genes present in an operon, termed phoP and phoQ . The phoP and phoQ gene products are highly similar to other members of bacterial two-component transcriptional regulators that respond to environmental stimuli and control the expression of a large number of other genes. A mutation at one of these phoP regulatory region regulated genes, page, confers a virulence defect. Strains with page, phoP, or phoQ mutations afford partial protection to subsequent challenge by wild-type S. typhimurium .

Summary of the Invention In general the invention features a method of detecting Salmonella in a sample. The method includes contacting the sample with an antibody specific for a phoP regulated gene product, e.g., the product of a pag gene, e.g, page, or a prg gene, e.g., prgA, prgB, prgC, prgD, prgE, prgF, prgG, or prgH; allowing the antibody to form immune complexes with Salmonella , and detecting the immune complexes as an indication of the presence of Salmonella in the sample.

The invention also features a purified antibody, e.g., a monoclonal antibody, against a phoP regulated gene product, e.g., against a pag gene, e.g., page, or a prg gene, e.g. , prg A, prgB, prgC, prgD, prgE, prgF, prgG, or prgH.

Specific for a phoP regulated gene product, as used herein, refers to an antibody which binds to a Salmonella phoP regulated gene product but not to lysates of another gram negative bacterium, e. g. , E. coli . A purified preperation, as used herein, refers to an antibody preperation in which antibodies of the desired specificity constitute at least 50% (wt) , and preferrably at least 80% (wt) , of the antibodies in the preperation. The phoP regulatory region, as used herein, is a two-component regulatory system that controls the expression of pag and prg genes. It includes the phoP locus and the phoQ locus. phoP regulatory region regulated genes, or phoP regulated genes, as used herein, refer to genes such as pag and prg genes. pag, as used herein, refers to a gene which is positively regulated by the phoP regulon. prg, as used herein, refers to a gene which is negatively regulated by the phoP regulon. phoP regulated genes are involved in virulence. Virulent bacteria generally contain genes which encode virulence proteins specific to the organism. Therefore, the use of virulence gene products allow the detection of disease causing organisms without interference by or detection of comensal organisms. Because they interact with host organism structures, virulence proteins are generally secreted or located on the membrane. Thus phoP regulated gene products are likely to posses two properties important to proteins used as targets for the immunological identification of a microorganism.

The invention provides a rapid and economic assay for Salmonella in a variety of samples, including food, water, agricultural products, e.g., poultry, products, blood, urine, and feces.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.

Description of the Preferred Embodiments The drawings will first be described. Drawings

Fig. 1 is a map of the restriction endonuclease sites of the pagC locus.

Fig. 2 is a map of the DNA sequence of the pag C region (Sequence ID No. 1) . Strain Deposit

PhoPc strain CS022 (described below) has been deposited with the American Type Culture Collection (Rockville, MD) and has received ATCC designation 10428. The phoP system

The phoP regulatory region includes two regulatory genes, phoP and phoQ, and is essential for full virulence, survival within macrophages, and defensin resistance of Salmonella typhimurium. The phoP and phoQ proteins have amino acid similarity to the gene products of other bacterial two-component which control the synthesis of many proteins in response to environmental signals. The PhoP and PhoQ gene products are essential for the transcriptional activation of a number of unlinked phoP-activated genes, the pag loci. Another set of genes, the prg loci, are repressed by the wild type function of PhoP. As discussed below, a phoPc (constitutive) mutation results in the constitutive induction of pag genes and the constitutive repression of prg genes.

The phoP constitutive allele (PhoPc) . pho-24 . results in derepression of multiple paσ loci.

Using diethyl sulfate mutagenesis of S. typhimurium LT-2, Ames and co-workers isolated strain TA2367 pho-24 (all strains, materials, and methods referred to in this section are described below) , which contained a phoP locus mutation that resulted in constitutive production of acid phosphatase in rich media, Kier et al., 1979, J. Bacteriol. 138:155, hereby incorporated by reference. This phoP-regulated acid phosphatase is encoded by the phoN gene, a pag locus, Kier et al., 1979, supra. Miller et al., 1989, supra. To analyze whether the pho-24 allele increased the expression of other pag loci the effect of the pho-24 allele on the expression of other pag loci recently identified as transcriptional (e.g., pagA and pagB) and translational (e.g., pagC) fusion proteins that required phoP and phoQ for expression, Miller et al., 1989, supra, was determined, pag gene fusion strains, isogenic except for the pho-24 allele, were constructed and assayed for fusion protein activity. PhoPc derivatives of the pagA: :Mu dJ and pagB::Mu dJ strains produced 480 and 980 U, respectively, of .-galactosidase in rich medium, an increase of 9- to 10-fold over values for the fusion strains with a wild-type phoP locus, see Table 1.

TABLE 1. Bacterial strains and properties

A. Acid phosphatase; B, ϊ-galactosidase; C, alkaline phosphatase.

Gift of Ning Zhu and John Roth. The page : : TnphoA gene fusion produced 350 U of alkaline phosphatase, an increase of three- to fourfold over that produced in strain CS119, which is isogenic except for the pho-24 mutation, Miller et al., 1989, supra. These results compare with a ninefold increase in the acid phosphatase activity in strain CS022 on introduction of the pho-24 allele. Therefore, these available assays for pag gene expression document that the pho-24 mutation causes constitutive expression of pag loci other than phoN.

The phoP constitutive allele results in repression of multiple prσ loci. Whole-cell proteins of strain CS022 were analyzed to estimate the number of protein species that could be potentially regulated by the PhoP regulon. Remarkably, analysis by one-dimensional polyacrylamide gel electrophoresis of the proteins produced by strains with the PhoP phenotype indicated that some protein species were decreased in expression when many presumptive pag gene products were fully induced by the pho-24 mutation. The proteins decreased in the PhoPc strain might represent products of genes that are repressed by the PhoP regulator. Genes encoding proteins decreased by the pho-24 allele are designated prg loci, for phoP-repressed genes. Comparison of wild- type, PhoP~ , and PhoPc mutant strain proteins shows that growth in LB medium at 37°C represents repressing conditions for pag gene products and derepressing conditions for prg gene products.

To estimate the total number of potentially PhoP- regulated gene products, the total cell proteins of wild- type and PhoPc mutant strains grown in LB were analyzed by two-dimensional gel electrophoresis. Approximately 40 species underwent major fluctuation in expression in response to the pho-24 mutation. Virulence defects of the PhoPc strain Remarkably, strains with the single pho-24 mutation were markedly attenuated for virulence in mice. The number of PhoPc organisms (2 x 105) that killed 50% of BALB/c mice challenged (LD50) by the intraperitoneal (i.p.) route was near that (6 x 105) of PhoP~ bacteria. Miller et al., 1989, supra. The PhoPc strains had growth comparable to wild-type organisms in rich and minimal media. The PhoPc mutants were also tested for alterations in lipopolysaccharide, which could explain the virulence defect observed. Strain CS022 had normal sensitivity to phage P22, normal group B reactivity to antibody to O antigen, and a lipopolysaccharide profile identical to that of the parent strain, as determined by polyacrylamide gel electrophoresis and staining.

Strains, materials and methods The strains, materials, and methods used in the PhoP regulon work described above are as follows.

American Type Culture Collection (ATCC) strain 14028, a smooth virulent strain of S . typhimurium, was the parent strain for all virulence studies. Strain TT13208 was a gift from Nang Zhu and John Roth. Strain TA2367 was a generous gift of Gigi Stortz and Bruce Ames, Kier et al. , 1979, supra. Bacteriophage P22HT int was used in transductional crosses to construct strains isogenic except for phoP locus mutations , Davis et al. , 1980, Advanced Bacterial Genetics, p. 78, 87. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, hereby incorporated by reference. Luria broth was used as rich medium, and minimal medium was M9, Davis et al., 1980, supra. The chromogenic phosphatase substrate 5-bromo™4- chloro-3indolyl phosphate (XP) was used to qualitatively access acid and alkaline phosphatase production in solid media. Derivatives of S . typhimurium ATCC 10428 with the pho-24 mutation were constructed by use of strain TA2367 as a donor of the purB gene in a P22 transductional cross with strain CS003 AphoP ApurB , Miller et al., 1989, supra. Colonies were then selected for the ability to grow on minimal medium. A transductant designated CS022 (phenotype PhoPc) that synthesized 1,750 U of acid phosphatase in rich medium (a ninefold increase over the wild-type level in rich medium) was used in further studies.

Derivatives of strains CS022 and CS023 pho-24 phoN2 zxx: :6251Tnl0d-Cam, and acid phosphatase-negative derivative of CS022, containing pag gene fusions were constructed by bacteriophage P22 transductional crosses, using selection of TnphoA- or Mu dJ-encoded kanamycin resistance. Strains were checked for the intact pag gene fusion by demonstration of appropriate loss of fusion protein activity on introduction of a phoP105 : :TnI0d or phoP102: :Tn20d-Cam allele. Assays of acid phosphatase, alkaline phosphatase, and ?-galactosidase were performed as previously described, Miller et al., 1989, supra and are reported in units as defined in Miller, 1972, Experiments in molecular genetics, p. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, hereby incorporated by reference.

In the mouse virulence and vaccination studies bacteria grown overnight in Luria broth were washed and diluted in normal saline. The wild-type parent strain of CS022 (ATCC 10428) was used for all live vaccine challenge studies. This strain has a 50% lethal dose (LD50) for naive adult BALB/c mice of less than 20 organisms when administered by intraperitoneal (i.p.) injection and 5xl04 when administered orally in NaHCOj. Mice were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, Mass.) and were 5 to 6 weeks of age at initial challenge. All i.p. inoculations were performed as previously described. Miller et al., 1989, supra. Oral challenge experiments were performed with bacteria grown in LB broth and concentrated by centrifugation. The bacteria were resuspended in 0.1 M NaHC03 to neutralize stomach acid, and administered as a 0.5-ml bolus to animals under ether anesthesia. Colony counts were performed to accurately access the number of organisms administered. All challenge experiments were performed 1 month after i.p. inoculation and 6 weeks after oral challenge. Challenge inoσula were administered by the same route as vaccinations. The care of all animals was under institutional guidelines as set by the animal are committees at the Massachusetts General Hospital and Harvard Medical School.

Protein electrophoresis was performed as follows. One-dimensional protein gel electrophoresis was performed by the method of Laemmli, 1970, Nature 227:680, hereby incorporated by reference, on whole-cell protein extracts of stationary-phase cells grown overnight in Luria broth. The gels were fixed and stained with Coomassie brilliant blue R250 in 10% acetic acid-10% methanol. Two- dimensional protein gel electrophoresis was performed by method of O'Farrell, 1975, J. Biol. Chem. 250:4007, hereby incorporated by reference, on the same whole-cell extracts. Isoelectric focusing using 1.5% pH 3.5 to 10 ampholines (LKB Instruments, Baltimore, Md.) was carried out for 9,600 V h (700 V for 13 h 45 in) . The final tube gel pH gradient extended from pH 4.1 to pH 8.1 as measured by a surface pH electrode (BioRad Laboratories, Richmond, Calif.) and colored acetylated cytochrome pi markers (Calbiochem-Behring, La Jolla, Calif.) run in an adjacent tube. The slab gels were silver stained, Merril et al., 1984, Methods Enzy ol. 104:441, hereby incorporated by reference.

The isolation of phoP regulated genes

PhoP regulated genes (i.e., pag and prg genes) can be identified and cloned by mutagenizing a Salmonella strain with a transposon which carries a marker gene, e.g., the lacZ gene, or a gene encoding alkaline phosphatase, and screening for phoP regulated expression of the transposon-borne marker. phoP regulated expression can be identified by comparing the expression of the inserted marker gene in wild type and in phoP regulatory region mutant backgrounds.

The expression of pag genes is positively regulated by PhoP, thus an insertion into a pag will be characterized by relatively high expression in a PhoP+ or PhoPc background, and relatively low expression in a PhoP~ background, on rich medium. The expression of prg genes is negatively regulated by PhoP. Thus, an insertion of a transposon borne marker into a prg gene will be characterized by relatively low expression in a PhoP+ or PhoPc background, and by relatively high expression in a PhoP~ background, on rich medium.

Once insertions at putative prg and pag loci are identified the insertionally disrupted genes can be cloned and used to identify and clone wild type genomic DNA corresponding to the site of the insertional mutation.

This method of isolating pag and prg genes is described in Miller et al., 1989, supra , and herein. The paσC gene and pagC gene product Strains, materials, and methods The following strains, materials, and methods were used in the cloning of pagC and in the analysis of the gene and its gene product.

Rich media was Luria broth (LB) and minimal media was M9, Davis et al., 1980, supra. The construction of S . typhimurium strain CS119 pagCl: : nphoA phoN2 zxx::6251 TnlOd-Cam was previously described. Miller et al., 1989, supra. American Type Culture Collection (ATCC) S . typhimurium strain 10428 included CS018 which is isogenic to CS119 except for phoP105: : Tnl0d, Miller et al., 1989, supra, CS022 pho-24 , Miller et al., 1990, J. Bacteriol. 172:2485-2490, hereby incorporated by reference, and CS015 phoP102 : :Tnl0d-cam, Miller et al., 1989, supra. Other wild type strains used for preparation of chromosomal DNA included S. typhimurium LT2 (ATCC 15277) , S. typhimurium Ql and S . dry pool (Dr. J. Peterson U. Texas Medical Branch, Galveston) , and Salmonella typhi T 2 (Dr. Caroline Hardegree, Food and Drug

Administration) . pLAFR cosmids were mobilized from E. coli to S. typhimurium using the E. coli strain MM294 containing pRK2013, Friedman et al., 1982, Gene 18:289- 296, hereby incorporated by reference. Alkaline phosphatase (AP) activity was screened on solid media using the chromogenic phosphatase substrate 5-bromo-4- chloro-3-indolyl phosphate (XP) . AP assays were performed as previously described, Brickman et al., 1975, J. Mol. Biol. £6:307-316, hereby incorporated by reference, and are reported in units as defined by Miller, Miller, 1972, supra, pp. 352-355.

One dimensional protein gel electrophoresis was performed by the method of Laemmli, 1970, Nature, 227:680-685. hereby incorporated by reference, and blot hybridization using antibody to AP was performed as previously described, Peterson et al., 1988, Infect. Immun. .56:2822-2829, hereby incorporated by reference. Whole cell protein extracts were prepared, from saturated cultures grown in LB at 37°C with aeration, by boiling the cells in SDS-pagE sample buffer, Laemmli, 1970, supra. Two dimensional gel electrophoresis was performed by the method of O'Farrell, 1975, J. Biol. Chem. 250:4007. hereby incorporated by reference. Proteins in the 10% polyacryla ide slab gels were visualized by silver staining, Merril et al., 1984, Methods in

Enzymology, 104:441, hereby incorporated by reference.

Chromosomal DNA was prepared by the method of Mekalanos, 1983, Cell, 3_5:253-263, hereby incorporated by reference. DNA, size fractionated in agarose gels, was transferred to nitrocellulose (for blot hybridization) by the method of Southern, 1975, J. Mol. Biol. £8:503-517, hereby incorporated by reference. DNA probes for Southern hybridization analysis were radiolabeled by the random primer method, Frinberg et al. , 1984, supra. Plasmid DNA was transformed into E. coli and Salmonella by calcium chloride and heart shock, Mekalanos, 1983, supra, or by electroporation using a Genepulser apparatus (Biorad, Richmond, Ca.) as recommended by the manufacturer, Dower et al., 1988, Nucl. Acids Res. 1.6:6127-6145, hereby incorporated by reference. DNA sequencing was performed by the dideoxy chain termination method of Sanger et al., 1977, Proc. Natl. Acad. Sci. USA, 4.:5463-5467, hereby incorporated by reference, as modified for use with Sequenase (U.S. Biochemical, Cleveland, Ohio) . Oligonucleotides were synthesized on an Applied Biosystems Machine and used as primers for sequencing reactions and primer extension of RNA. Specific primers unique to the two ends of TnphoA, one of which corresponds to the alkaline phosphatase coding sequence and the other to the right IS50 sequence, were used to sequence the junctions of the transposon insertion.

Construction of a S . typhimurium cosmid gene bank in pLAFR3 and screening for clones containing the wild type pagC DNA was performed as follows. DNA from S. typhimurium strain ATCC 10428 was partially digested using the restriction endonuclease Sau3A and then size selected on 10-40% sucrose density gradient. T4 DNA ligase was used to ligate chromosomal DNA of size 20-30 kilobases into the cosmid vector pLAFR3, a derivative of pLAFRl, Friedman et al., 1982, Gene 1^:289-296, hereby incorporated by reference, that was digested with the restriction endonuclease BamEI . Cosmid DNA was packaged and transfected into E. coli strain DH5-α using extracts purchased from Stratagene, La Jolla, Ca. Colonies were screened by blot hybridization analysis.

The analysis of proteins produced from cloned DNA by in vitro transcription/translation assays was analyzed as follows. These assays were performed with cell free extracts, (Amersham, Arlington Heights, Illinois) , and were performed using conditions as described by the manufacturer. The resultant radiolabeled proteins were analyzed by SDS-pagE.

RNA was purified from early log and stationary phase Salmonella cultures by the hot phenol method, Case et al., 1988, Gene .72:219-236, hereby incorporated by reference, and run in agarose-formaldehyde gels for blot hybridization analysis, Thomas, 1980, Proc. Natl. Acad. Sci. USA 2Z:5201, hereby incorporated by reference. Primer extension analysis of RNA was performed as previously described, Miller et al., 1986, Nuc. Acids. Res. 14:7341-7360, hereby incorporated by reference, using AMV reverse transcriptase (Promega, Madison, Wisconsin) and synthesized oligonucleotide primers complementary to nucleotides 335-350 and 550-565 of the pagC locus.

Construction of a pacrC insertional mutation pagC was identified by the insertion of TnphoA. TnphoA is a transposon which carries the marker alkaline phosphatase. A pagC: :TnphoA insertion in a phoP+ background had a 20 fold increase in expression of the marker as compared to a pagC: :TnphoA insertion in a phoP deletion background. See Miller et al., 1989, supra . Random transposon mutagenesis of S . typhimurium was performed as described in Miller et al., 1989, supra , by using MudJ, described in Hughes et al., 1988, Mol. Gen. Genet. 119:9. hereby incorporated by reference, TnlOd-Cam, described in Eliot et al., 1988 Mol. Gen. Genet. 213:332, hereby incorporated by reference, and Bender et al., 1986, Cell 4J5:801, and TnphoA, described in Taylor et al., 1989, J. Bact 121:1870, hereby incorporated by reference. Escherichia coli strain CS118 was used as a phoA-negative recipient for TnphoA mutagenesis of plasmid DNA with TnphoA as described in Gutierrez et al., 1987, J. Mol . Biol . 195:289. hereby incorporated by reference.

Identification of an 18 kDa protein missing in a pacrC mutant of S . typhimurium pagC mutant strain CS119 was analyzed by two dimensional protein electrophoresis to detect protein species that might be absent as a result of the TnphoA insertion. Only a single missing protein species, of approximately 18 kD and pI-8.0, was observed when strains, isogenic except for their transposon insertions, were subjected to this analysis. This 18 kDa species was also missing in similar analysis of Salmonella strains with mutations phoP and phoQ . Though two-dimensional protein gel analysis might not detect subtle changes of protein expression in strain CS119, this suggested that a single major protein species was absent as a result of the pagC: : nphoA insertion.

Additional examination of the 2-dimensional gel analysis revealed a new protein species of about 45 kDa that is likely the pagC-Ap fusion protein. The pagC-AP fusion protein was also analyzed by Western blot analysis using antisera to AP and found to be similar in size to native AP (45 kDa) and not expressed in PhoP-S. typhimurium. Cloning of the paσC: :TnphoA insertion Chromosomal

DNA was prepared from S. typhimurium strain CS119 and a rough physical map of the restriction endonuclease sites in the region of the pagC: : nphoA fusion was determined by using a DNA fragment of TnphoA as a probe in blot hybridization analysis. This work indicated that digestion with the restriction endonuclease eσoRV yielded a single DNA fragment that included the pagC: :TnphoA insertion in addition to several kilobases of flanking DNA. Chromosomal DNA from strain CS119 was digested with JϊσoRV (blunt end) and ligated into the bacterial plasmid vector pUC19 (New England Biolabs) that had been digested with the restriction endonuclease Smal (blunt end) . This DNA was electroporated into the E. coli strain DH5-α (BRL) and colonies were plated onto LB agar containing the antibiotics kanamycin (TnphoA encoded and ampicillin (pUC19 encoded) . A single ampicillin and kanamycin resistant clone containing a plasmid designated pSMlOO was selected for further study.

A radiolabeled DNA probe from pSMlOO was constructed and used in Southern hybridization analysis of strain CS119 and its wild type parent ATCC 10428 to prove that the pagC: :TnphoA fusion had been cloned. The probe contained sequences immediately adjacent to the transposon at the opposite end of the alkaline phosphatase gene [iϊpal endonuclease generated DNA fragment that included 186 bases of the right IS50 of the transposon and 1278 bases of Salmonella DNA (Fig. 1) . As expected, the pSMlOO derived probe hybridized to an 11- 12 kb AσcJ endonuclease digested DNA fragment from the strain containing the transposon insertion, CS119. This was approximately 7.7kb (size of TnphoA) larger than the 3.9 kB AccJ fragment present in the wild type strain that hybridizes to the probe. In addition, a derivative of plasmid pSMlOO, pSMlOl (which did not allow expression of the pagC-PhoA gene fusion off the lac promoter) , was transformed into phoP- (strain Cs015) and phoN- (strain CS019) Salmonella strains and the cloned AP activity was found to be dependent on phoP for expression. Therefore we concluded that the cloned DNA contained the pagC: :TnphoA fusion.

Cloning of the wild type paσC locus DNA and its complementation of the virulence defect of a S . typhimurium paαC mutant The same restriction endonuclease fragment described above was used to screen a cosmid gene bank of wild type strain ATCC 10428. A single clone, designated pWP061, contained 18 kilobases of S . typhimurium DNA and hybridized strongly to the pagC DNA probe. pWP061 was found to contain Salmonella DNA identical to that of pSMlOO when analyzed by restriction endonuclease analysis and DNA blot hybridization studies. Probes derived from pWP061 were also used in blot hybridization analysis with DNA from wild type and CS119 S . typhimurium. Identical hybridization patterns were observed to those seen with pSMlOO. pWP061 was also mobilized into strain CS119, a pagC mutant strain. The resulting strain had wild type virulence for BALB/c mice (a D50 less than 20 organisms when administered by IP injection) . Therefore the cloned DNA complements the virulence defect of a pagC mutant strain. Since, a wild type cosmid containing pagC locus DNA was found to complement the virulence defect of a pagC mutant S. typhimurium strain, it was concluded that the pagC protein is an 188 amino acid (18 kDa) membrane (see below) protein essential for survival within icrophages and virulence of S. typhimurium .

Physical mapping of restriction endonuclease sites. DNA sequencing, and determination of the paσC gene product Restriction endonuclease analysis of plasmid pSMlOO and pWP061 was performed to obtain a physical map of the pagC locus, and, in the case of PSMlOO, to determine the direction of transcription (Fig. 1) . DNA subclones were generated and the TnphoA fusion junctions were sequenced, as well as the Salmonella DNA extending from the ifpal site, 828 nucleotides 5' to the phoA fusion junction, to the J_*σoRI site 1032 nucleotides 3' to the TnphoA insertion (Fig. 1 and 2) . The correct reading frame of the DNA sequence was deduced from that required to synthesize an active AP gene fusion. The deduced amino acid sequence of this open reading frame was predicted to encode a 188 amino acid protein with a predicted pI+8.2. This data were consistent with the 2- D polyacrylamide gel analysis of strain CS119 in which an 18 kDa protein of approximate pI+8.0 was absent. No other open reading frames, predicted to encode peptides larger than 30 amino acids, were found.

The deduced amino acid sequence of the 188 amino acid open reading frame contains a methionine start codon 33 amino acids from the fusion of pagC and AP (Fig. 2) . This 33 amino acid page contribution to the fusion protein was consistent with the size observed in Western blot analysis and contains a hydrophobic N-terminal region, identified by the method of Kyle et al., 1982, J. Mol. Biol. 157:105-132. hereby incorporated by reference, that is a typical bacterial signal sequence. Von Heinje, 1985, J. Mol. Biol. 184.:99-105, hereby incorporated by reference. Specifically, amino acid 2 is a positively charged lysine, followed by a hydrophobic domain and amino acid 24 is a negatively charged aspartate residue. A consensus cleavage site for this leader peptide is predicted to be at an alanine residue at amino acid 23, Von Heinje, 1984, J. Mol. Biol. 121:243-251, hereby incorporated by reference. The DNA sequence also revealed a typical ribosomal binding site. Shine et al., 1974, Proc. Natl. Acad. Sci. USA 21:1342-1346, hereby incorporated by reference, at 6-2 nucleotides 5' to the predicted start of translation (Fig. 2) nucleotides 717- 723) . This suggested that the open reading frame was, in fact, translated and further supported the assumption that this was the deduced amino acid sequence of the pagC protein interrupted by the TnphoA insertion (Fig. 2) .

In vitro synthesis of proteins by the cloned paσC locus To detect if other proteins were encoded by pagC and to determine the approximate size of the pagC gene product, an in vitro coupled transcription/translation analysis was performed. A 5.3 kilobase J?coRI fragment of pWP061 was inserted into pUC19 so that the pagC gene would not be expressed off the lac promotor. This plasmid was used in an in vitro coupled transcription- translation assay. A single protein of approximately 22 kilodaltons was synthesized by the cell free system. The size was compatible with this being the precursor of the pagC protein containing its leader peptide. These data further support the conclusion the single and the single pagC gene product had been identified.

Identification of the pacrC encoded RNA An approximately 1100 nucleotide RNA is encoded by pagC. The pagC gene is highly expressed by cells with a phoP constitutive phenotype of pag activation, as compared to wild type and phoP constitutive phenotype of pag activation, as compared to wild type and phoP- bacteria. In these blot hybridization experiments page is only detected in wild type cells grown in rich media during stationary growth. This result, coupled with previous work. Miller et al., 1989, supra, Miller et al., 1990, supra, demonstrates that pagC is transcriptionally regulated by the phoP gene products and is only expressed during early logarithmic phase growth in rich media by cells with a phoP constitutive phenotype. The size of the pagC transcript is approximately

500 nucleotides greater than that necessary to encode the 188 amino acid protein. Primer extension analysis of Salmonella RNA using oligonucleotide primers specific for pagC sequence was performed to determine the approximate start site of transcription and to determine whether these nucleotides might be transcribed 5• or 3' to the 188 amino acid pagC gene product. Primer extension analysis with an oligonucleotide predicted to be complementary to nucleotides 550-565 of page, 150 nucleotides 51 to the predicted start codon, resulted in an approximately 300 nucleotide primer extension product. Therefore a primer further upstream was constructed complementary to nucleotides 335-350 of pagC and used in a similar analysis. A primer extension product of 180 nucleotides was observed to be primer specific. This is consistent with transcription starting at nucleotide 170 (Fig. 2) . Upstream of the predicted transcriptional start, at nucleotides 153-160, a classic RNA polymerase binding site was observed with the sequence TATAAT at - 12 nucleotides as well as the sequence TAATAT at -10 nucleotides. No complete matches were observed for the consensus RNA polymerase recognition site (TTGACA) 15-21 nucleotides upstream from the -10 region. AT -39 (126- 131) nucleotides (TTGGAA) , -38 (127-132) nucleotides (TTGTGG) , and -25 (135-140) nucleotides (TTGATT) are sequences that have matches with the most frequently conserved nucleotides of this sequence.

Based on the above results transcription was predicted to terminate near the translational stop codon of the 188 amino acid protein (nucleotide 1295, Fig. 2) . Indeed, a stem loop configuration was found at nucleotides 1309-1330 that may function as a transcription terminator. This was consistent with the lack of evidence of open reading frames downstream of the 188 amino acid protein and the lack of synthesis of other transcription/translation using the cloned pagC DNA. This further suggests that the pagC: :TnphoA insertion inactivated the synthesis of only a single protein. pagC Mutant Strains Are Attenuated For Virulence Salmonella typhimurium strains with a page mutation are most likely inactivated for the phoP- regulated gene product, as these strains are attenuated for virulence by at least 1,000-fold. pagC is common to many Salmonella strains but lacking in many other bacteria. The presence of the page gene was also demonstrated in other strains of S . typhimurium, as well as in S . typhi , and S . dry pool . All Salmonella strains examined demonstrated similar strong hybridization to an 8.0 kb JSσoRV and a 3.9 kb Aσcil restriction endonuclease fragment suggesting that pagC is a virulence gene common to Salmonella species.

The pagC gene probe from nucleotides -46 (with 1 as the first base of the methionine to 802 (PstI site to the BglII site) failed to cross hybridize to DNA from Citrobacter freundii, Shigella flexneri, Shigella sonnei, Shigella dysenterial, Escherichia coli, Vibrio cholerae, Vibrio vulnificus, Yersenia entero colitica, and Klibsiella pneumonia.

Lipopolysaccharide patterns and p22 bacteriophage sensitivity appear to be the same in phoP' , phoPc , and phoP+, bacgrounds, suggesting that phoP regulated genes do not encode flagellar or lipopolysaccharide flagellar or conventional lipopolysaccharide antigen products. Additionally, phoP mutants are motile, suggesting that phoP regulated genes do not encode flagellar antigens. Production of antibodies to the products of phoP regulatory region regulated genes

Antibodies to the products of the phoP regulatory region-regulated genes can be made by methods known to those in the art. Polyclonal or monoclonal antibodies can be used in the methods of the invention.

Antibodies can be generated by challenge with any antigen which results in the production of antibodies which bind to a pag or prg gene product and which are specific for Salmonella . Antibodies to a given pag or prg gene product can be produced, e.g., by challenge with Salmonella lysates; a naturally occurring example of pag or prg gene product, or fragments thereof; recombinant examples of pag or prg gene product, or fragments thereof; synthetic pag or prg gene product, or fragments or homologs thereof; and in general, peptides which, regardless of their origin, length, or degree of exact or partial homology with a naturally occurring pag or prg gene product, produce an antibody which binds specifically to the pag or prg gene product of interest. Antibodies thus produced, whether polyclonal or monoclonal, are screened for suitability by the ability to bind to an epitope present on the naturally occurring pag or prg gene product in question. Antibodies can be tested for specificity to

Salmonella by methods known to those skilled in the art. For example an antibody preparation which, under a given set of conditions, binds to Western blots of Salmonella lysates but which fails to bind to Western blots of lysates of other gram negative bacteria expected to be present in the sample to be tested, e.g., E. coll . , are specific for Salmonella .

Anti-pacrC antibodies

The peptide Arg-Gly-Val-Asn-Val-Lys-Tyr-Arg-Tyr- Glu-Asp-Asp-Ser-Phe (Sequence No. 2) (the "pagC fragment") , which corresponds to amino acid residues 47- 60 of the predicted page gene product of S . typhimurium, was produced by solid state synthesis, by methods known to those skilled in the art. The pagC fragment was conjugated to keyhole limpet hematocyanin (KLH) , by methods known to those skilled in the art, and injected into rabbits. Polyclonal antibodies were recovered and absorbed against a crude lysate of E. coli . This step was necessary because essentially all sera from rabbits contain antibodies reactive with E. coli . The serum was tested prior to absorption and found to exhibit a high titre against the page fragment. The post-absorbed antiserum bound to Western blots of Salmonella lysates but not to Western blots of E. coli lysates. The difference in signal strength between the E. coli blot and the Salmonella blot appeared to be at least 100 fold.

The anti-pagC antiserum was used to demonstrate that pagC is an outer membrane protein. Fractions of Salmonella outer membranes, and of total Salmonella protein minus outer membranes, were prepared by methods known to those skilled in the art. Western blots of each fraction were probed with the anti-pagC fragment antibody. All signal was confined to the outer membrane blot. Anti-pro* gene product antibodies

TnphoA transposon insertions were used (as described above) to identify and recover several prg genes. TnphoA insertions identify envelop or membrane proteins because the marker encoded by TnphoA, alkaline phosphatase, must dimerize in order to be active. Dimerization requires passage through the periplasmic space, and thus the presence of active alkaline phosphatase indicates insertion into a gene which encodes a secreted, periplasmic, or membrane protein.

A prg gene product, or a fragment or homolog thereof, can be used to stimulate the production of anti- prg antibodies. Antibodies are selected as described above. Monoclonal antibodies to the products of phoP regulated genes

Monoclonal antibody are prepared by fusing spleen cells from a mammal which has been immunized against a pag or prg encoded antigen, with an appropriate myeloma cell line. The resultant product is cultured in a standard HAT (hypoxanthine, aminopterin and thymidine) medium to yield hybridomas.

The immunized spleen cells may be derived from any mammal, e.g., mice. The animal is first immunized by injection of the chosen antigen. When the animal shows sufficient antibody production against the antigen, as determined by conventional assay methods, it is given a booster injection of the antigen, and then killed so that the immunized spleen may be removed. The fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.

Hybridomas are selected by screening for those which produce antibodies which bind to a molecule carrying the epitope against which they were generated, e.g., in the case of a the pagC fragment, hybridomas could be screened with the pagC fragment peptide or with all or an appropriate portion of naturally occurring or recombinant page protein. Detection of Salmonella The formation of an immune complex between antibodies against the products of phoP regulated genes and their target antigens can be used to detect the presence of Salmonella in a sample. A wide variety of suitable methods for detecting the formation of immune complexes, including liquid phase and solid phase assays, are known to those skilled in the art and any of them can be used in conjunction with the antibodies of the invention for the detection of Salmonella . Immunoassays are commonly carried out, at least in part, on solid supports, e.g., glass fiber membranes. Two of the most common forms of immunoassay which employ solid supports are competitive and sandwich formats. Typical competitive formats are described e.g., in Litt an et al., U.S. Patent 4,540,659, hereby incorporated by reference; and a typical sandwich assay by David et al., U.S. Patent 4,376,110, hereby incorporated by reference.

In a sandwich assay the anti-phoP gene product antibody can be bound to a solid support, contacted with the sample to be tested, the bound antibody and sample allowed to incubate, excess sample removed, and the antigen-antibody complexes contacted with a second antibody which binds to an epitope on the antigen (or on the Salmonella cell carrying the antigen) other than that recognized by the anti-phoP gene product antibody. The epitope recognized by the second antibody need not be on the pagC gene product but may be any epitope on the surface of the bound Salmonella cell. Other embodiments are with the following claims.

What is claimed is: COMPUTER SUBMISSION OF DNA AND AMINO ACID SEQUENCES (1) GENERAL INFORMATION:

(i) APPLICANT: Miller, Samuel I.

(ii) TITLE OF INVENTION: Detection of Salmonella (iϋ) NUMBER OF SEQUENCES:2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb storage

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM:IBM P.C. DOS (Version 3.30)

(D) SOFTWARE: WordPerfect (Version 5.0)

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:07/678,409

(B) FILING DATE: 29 March 1991

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Clark, Paul T.

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 00786/084001

(XX) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2320

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 1:

GTTAACCACT CTTAATAATA ATGGGTTTTA TAGCGAAATA CACTTTTTTA TCGCGTGTTC 60 AATATTTGCG TTAGTTATTA TTTTTTTGGA ATGTAAATTC TCTCTAAACA CAGGTGATAT 120 TTATGTTGGA ATTGTGGTGT TGATTCTATT CTTATAATAT AACAAGAAAT GTTGTAACTG 180 ATAGATATAT TAAAAGATTA AATCGGAGGG GGAATAAAGC GTGCTAAGCA TCATCGTGAA 240 TATGATTACA GCGCCTGCGA TGGCATATAA CCGTATTGCG GATGGAGCGT CACGTGAGGA 300 CTGTGAAGCA CAATGCGATA TGTTCTGATT ATATGGCGAG TTTGCTTAAT GACATGTTTT 360

TAGCCGAACG GTGTCAAGTT TCTTAATGTG GTTGTGAGAT TTTCTCTTTA AATATCAAAA 420 TGTTGCATGG GTGATTTGTT GTTCTATAGT GGCTAAAGAC TTTATGGTTT CTGTTAAATA 480 TATATGCGTG AGAAAAATTA GCATTCAAAT CTATAAAAGT TAGATGACAT TGTAGAACCG 540 GTTACCTAAA TGAGCGATAG AGTGCTTCGG TAGTAAAAAT ATCTTTCAGG AAGTAAACAC 600 ATCAGGAGCG ATAGCGGTGA ATTATTCGTG GTTTTGTCGA TTCGGCATAG TGGCGATAAC 660 TGAATGCCGG ATCGGTACTG CAGGTGTTTA AACACACCGT AAATAATAAG TAGTATTAAG 720 GAGTTGTT 728

ATG AAA AAT ATT ATT TTA TCC ACT TTA GTT ATT ACT ACA AGC GTT TTG 776 Met Lys Asn lie lie Leu Ser Thr Leu Val lie Thr Thr Ser Val Leu

5 10 15

GTT GTA AAT GTT GCA CAG GCC GAT ACT AAC GCC TTT TCC GTG GGG TAT 824 Val Val Asn Val Ala Gin Ala Asp Thr Asn Ala Phe Ser Val Gly Tyr 20 25 30

GCA CGG TAT GCA CAA AGT AAA GTT CAG GAT TTC AAA AAT ATC CGA GGG 872 Ala Arg Tyr Ala Gin Ser Lys Val Gin Asp Phe Lys Asn lie Arg Gly 35 40 45 GTA AAT GTG AAA TAG CGT TAT GAG GAT GAG TCT CCG GTA AGT TTT ATT 920 Val Asn Val Lys Tyr Arg Tyr Glu Asp Asp Ser Pro Val Ser Phe He 50 55 60

TCC TCG CTA AGT TAC TTA TAT GGA GAC AGA CAG GCT TCC GGG TCT GTT 968 Ser Ser Leu Ser Tyr Leu Tyr Gly Asp Arg Gin Ala Ser Gly Ser Val 65 70 75 80

GAG CCT GAA GGT ATT CAT TAC CAT GAC AAG TTT GAG GTG AAG TAC GGT 1016 Glu Pro Glu Gly He His Tyr His Asp Lys Phe Glu Val Lys Try Gly 85 90 95

TCT TTA ATG GTT GGG CCA GCC TAT CGA TTG TCT GAC AAT TTT TCG TTA 1064 Ser Leu Met Val Gly Pro Ala Tyr Arg Leu Ser Asp Asn Phe Ser Leu 100 105 110

TAC GCG CTG GCG GGT GTC GGC ACG GTA AAG GCG ACA TTT AAA GAA CAT 1112 Tyr Ala Leu Ala Gly Val Gly Thr Val Lys Ala Thr Phe Lys Glu His 115 120 125

TCC ACT CAG GAT GGC GAT TCT TTT TCT AAC AAA ATT TCC TCA AGG AAA 1160 Ser Thr Gin Asp Gly Asp Ser Phe Ser Asn Lys He Ser Ser Arg Lys 130 135 140

ACG GGA TTT GCC TGG GGC GCG GGT GTA CAG ATG AAT CCG CTG GAG AAT 1208 Thr Gly Phe Ala Trp Gly Ala Gly Val Gin Met Asn Pro Leu Glu Asn 145 150 155 160

ATC GTC GTC GAT GTT GGG TAT GAA GGA AGC AAC ATC TCC TCT ACA AAA 1256 He Val Val Asp Val Gly Tyr Glu Gly Ser Asn He Ser Ser Thr Lys 165 170 175

ATA AAC GGC TTC AAC GTC GGG GTT GGA TAC CGT TTC TGA AAAGC 1300

He Asn Gly Phe Asn Val Gly Val Gly Tyr Arg Phe 180 185

ATAAGCTATG CGGAAGGTTC GCCTTCCGCA CCGCCAGTCA ATAAAACAGG GCTTCTTTAC 1360

CAGTGACACG TACCTGCCTG TCTTTTCTCT CTTCGTCATA CTCTCTTCGT CATAGTGACG 1420

CTGTACATAA CATCTCACTA GCATAAGCAC AGATAAAGGA TTGTGGTAAG CAATCAAGGT 1480

TGCTCAGGTA GGTGATAAGC AGGAAGGAAA ATCTGGTGTA AATAACGCCA GATCTCACAA 1540

GATTCACTCT GAAAAATTTT CCTGGAATTA ATCACAATGT CATCAAGATT TTGTGACCGC 1600

CTTCGCATAT TGTACCTGCC GCTGAACGAC TACTGAAAAG TAGCAAGGTA TGTATTTTAT 1660

CCAGGAGAGC ACCTTTTTTG CGCCTGGCAG AAGTCCCCAG CCGCCACTAG CTCAGCTGGA 1720

TAGAGCATCA ACCTCCTAAG TTGATGGTGC GAGGTTCGAG GCCTCGGTGG CGGTCCAATG 1780 TGGTTATCGT ATAATGTTAT TACCTCAGTG TCAGGCTGAT GATGTGGGTT CGACTCCCAC 1840

TGACCACTTC AGTTTTGAAT AAGTATTGTC TCGCAACCCT GTTACAGAAT AATTTCATTT 1900

ATTACGTGAC AAGATAGTCA TTTATAAAAA ATGCACAAAA ATGTTATTGT CTTTTATTAC 1960

TTGTGAGTTG TAGATTTTTC TTATGCGGTG AATCCCCCTT TGCGGCGGGG CGTCCAGTCA 2020

AATAGTTAAT GTTCCTCGCG AACCATATTG ACTGTGGTAT GGTTCACCGG GAGGCACCCG 2080

GCACCGCAAT TTTTTATAAA ATGAAATTCA CACCCTATGG TTCAGAGCGG TGTCTTTTTA 2140

CATCAGGTGG GCAAGCATAA TGCAGGTTAA CTTGAAAGAT ACGATCAATA GCAGAAACCA 2200

GTGATTTCGT TTATGGCCTG GGGATTTAAC CGCGCCAGAG CGTATGCAAG ACCCTGGCGC 2260

GGTTGGCCGG TGATCGTTCA ATAGTGCGAA TATGAATGGT TACCAGCCGC CTGCGAATTC 2320

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (i) SEQUENCE CHARACTERISTICS:

(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 2:

Arg-Gly-Val-Asn-Val-Lys-Tyr-Arg-Tyr-Glu-Asp-Asp-Ser-Phe

Claims

Claims
1. A method of detecting Salmonella in a sample comprising contacting said sample with an antibody specific for a PhoP regulated gene product, allowing said antibody to form immune complexes with said Salmonella , and detecting said immune complexes as an indication of the presence of Salmonella in said sample.
2. The method of claim 1, wherein said PhoP regulated gene is a pag.
3. The method of claim 2, wherein said pag is page .
4. The method of claim 1, wherein said PhoP regulated gene is a prg.
5. A purified antibody against a phoP regulated gene product.
6. The purified antibody of claim 5, where said antibody is a monoclonal antibody.
7. The purified antibody of claim 5, wherein said phoP regulated gene is a pag.
8. The purified antibody of claim 7, where said antibody is a monoclonal antibody.
9. The purified antibody of claim 7, wherein said pag is page.
10. The purified antibody of claim 9, where said antibody is a monoclonal antibody.
11. The purified antibody of claim 5, wherein said phoP regulated gene is a prg gene.
12. The purified antibody of claim 11, where said antibody is a monoclonal antibody.
PCT/US1992/002591 1991-03-29 1992-03-30 Detection of salmonella WO1992017785A1 (en)

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EP0563311A1 (en) * 1990-12-18 1993-10-06 The General Hospital Corporation Improved vaccines
EP0663404A1 (en) * 1994-01-14 1995-07-19 MERCK PATENT GmbH Antibodies for the detection of Salmonella
US5599537A (en) * 1990-12-18 1997-02-04 The General Hospital Corporation Salmonella virulence genes
WO1997023622A1 (en) * 1995-12-22 1997-07-03 Smithkline Beecham Plc Novel tcts response regulator of staphylococcus aureus
WO1997023620A1 (en) * 1995-12-22 1997-07-03 Smithkline Beecham P.L.C. Novel tcts response regulator of staphylococcus aureus
US5695983A (en) * 1990-12-18 1997-12-09 The General Hospital Corporation Salmonella vaccines
US5843426A (en) * 1990-12-18 1998-12-01 The General Hospital Corporation Salmonella vaccines
WO2004092408A2 (en) * 2003-04-18 2004-10-28 Warnex Research Inc. Oligonucleotides for the detection of salmonella species
US6861247B1 (en) 1995-11-14 2005-03-01 The General Hospital Corporation Salmonella secreted proteins and uses thereof
WO2007027936A2 (en) * 2005-09-01 2007-03-08 The General Hospital Corporation Methods and compositions for the diagnosis of a salmonella spp. infection
WO2010057983A1 (en) * 2008-11-20 2010-05-27 Creatogen Laboratories Gmbh Salmonella marker vaccine

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INFECTION AND IMMUNITY, Volume 58, No. 11, issued November 1990, MILLER et al., "Characterization of Defensin Resistance Phenotypes Associated with Mutations in the phoP Virulence Regulon of Salmonella typhimurium", pages 3706-3710. *
JOURNAL OF BACTERIOLOGY, Volume 172, No. 5, issued May 1990, MILLER et al., "Constitutive Expression of the phoP Regulon Attenuates Salmonella Virulence and Survival within Macrophages", pages 2485-2490. *
JOURNAL OF BACTERIOLOGY, Volume 173, No. 1, issued January 1991, PULKKINEN et al., "A Salmonella typhimurium Virulence Protein is Similar to a Yersinia enterocolitica Invasion Protein and a Bacteriophage Lambda Outer Membrane Protein", pages 86-93. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA, Volume 86, No. 13, issued July 1989, MILLER et al., "A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence", pages 5054-5058. *
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US5674724A (en) * 1990-12-18 1997-10-07 The General Hospital Corporation Salmonella virulence genes
EP0563311A4 (en) * 1990-12-18 1995-08-09 Gen Hospital Corp Improved vaccines
US5599537A (en) * 1990-12-18 1997-02-04 The General Hospital Corporation Salmonella virulence genes
US6010901A (en) * 1990-12-18 2000-01-04 The General Hospital Corporation Salmonella virulence genes
US5843426A (en) * 1990-12-18 1998-12-01 The General Hospital Corporation Salmonella vaccines
US5674736A (en) * 1990-12-18 1997-10-07 The General Hospital Corporation Salmonella virulence genes
US5695983A (en) * 1990-12-18 1997-12-09 The General Hospital Corporation Salmonella vaccines
US5731196A (en) * 1990-12-18 1998-03-24 The General Hospital Corporation Salmonella virulence genes
EP0663404A1 (en) * 1994-01-14 1995-07-19 MERCK PATENT GmbH Antibodies for the detection of Salmonella
US5840513A (en) * 1994-01-14 1998-11-24 Merck Patent Gesellschaft Mit Beschrankter Haftung Antibodies directed to pagC peptides for the detection of salmonellae
US6861247B1 (en) 1995-11-14 2005-03-01 The General Hospital Corporation Salmonella secreted proteins and uses thereof
WO1997023620A1 (en) * 1995-12-22 1997-07-03 Smithkline Beecham P.L.C. Novel tcts response regulator of staphylococcus aureus
US5962259A (en) * 1995-12-22 1999-10-05 Smithkline Beecham Plc Response regulator polynucleotides
WO1997023622A1 (en) * 1995-12-22 1997-07-03 Smithkline Beecham Plc Novel tcts response regulator of staphylococcus aureus
WO2004092408A2 (en) * 2003-04-18 2004-10-28 Warnex Research Inc. Oligonucleotides for the detection of salmonella species
WO2004092408A3 (en) * 2003-04-18 2005-03-03 Daniel Plante Oligonucleotides for the detection of salmonella species
WO2007027936A2 (en) * 2005-09-01 2007-03-08 The General Hospital Corporation Methods and compositions for the diagnosis of a salmonella spp. infection
WO2007027936A3 (en) * 2005-09-01 2007-11-01 Gen Hospital Corp Methods and compositions for the diagnosis of a salmonella spp. infection
WO2010057983A1 (en) * 2008-11-20 2010-05-27 Creatogen Laboratories Gmbh Salmonella marker vaccine

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