Isolation andBiochemical andMolecular Analyses ofa Species-Specific Protein Antigen fromtheGastric Pathogen Helicobacter pylori

A protein ofMr26,000 whichwaspresent inlarge quantities inextracts ofcells ofHelicobacter pyloni was purified tohomogeneity byammoniumsulfate precipitation followed bygelfiltration andreversed-phase chromatography oranion-exchange chromatography. Theprotein appeared tobeassociated withthesoluble fraction ofthecell, andantibodies raised against theprotein werereactive withwhole-cell lysates ofavariety ofH.pylori strains inasimple immunodot blotassay. Thisreaction wasspecies specific. Protein sequence determination oftheaminoterminus andinternal cyanogen bromide fragments andaminoacidcomposition analysis wereperformed. Anoligonucleotide derived fromthese datawasusedtoclone afragment encoding mostofthecoding sequence. Expression inEscherichia coli wasdependent onvector promoters. TheDNA sequence ofthefragment wasdetermined. DNA probes derived fromthecloned fragment hybridized to genomic DNAofall H.pylon strains tested, butnottoDNAsofHelicobacter mustelae, Wolinella succinogenes, various Campylobacter species, andapanel ofgram-negative enteric bacteria. Theapparent uniqueness ofthis protein maybeexploited forthedevelopment ofspecies-specific diagnostics forthis gastric pathogen. Helicobacter (Campylobacter) pylori (11)isacurved or spiral gram-negative microaerophilic bacterium whichwas first isolated fromahumangastric biopsy specimen in1983 (46). Since this first isolation, ithasbecomeapparent that thisorganism maybeoneofthemostcommonbacterial pathogens ofhumans. Epidemiological evidence hasshown thatH.pylori colonizes theuppergastrointestinal tract of morethanoneintwoindividuals during their lifespan; in manyofthesepersons, theorganism isassociated with disease ofthegastrointestinal tract (12, 13,22).Indeed, H. pylori appears tobecausally associated withactive and chronic gastritis aswellaswithpeptic andduodenal ulcers (4,5,7,12,22)andmayalso beassociated withcarcinoma of thestomach (17, 21). Additional evidence forthepathogenic activity ofH.pylori hasbeenprovided bystudies with gnotobiotic andbarrier-born pigs(18, 20)andstudies with twohumanvolunteers (29, 35). Because oftheclinical importance ofthis pathogen and thelarge number oflaboratory identifications being routinely undertaken around theworld onadaily basis, there isaneed forthedevelopment ofrapid diagnostic testprotocols. Liberation of14Cfromlabeled ureabythepotent bacterial urease isreliable fordetection ofanactive H.pylori infection(30,33)buthasdisadvantages incost,time,and exposure toundesirable radiation. Cellextracts orpurified cell-surface components havebeenevaluated asantigens in thedevelopment ofserological tests forinfection (9,36). DNA-based approaches haveincluded restriction endonuclease analysis fortyping (28, 42)andthedevelopment of specific probes (6,34). We haveidentified a protein that appears tobeunique toH.pylori. We haveisolated this protein andcharacterized itbiochemically. Inthis paper we