The gastric pathogen Helicobacter pylori is one of a number of bacteria which bind specifically to gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro at neutral pH. Since this organism encounters an acid pH during initial infection of the stomach, we have monitored the effect of pH on receptor binding specificity and found induction of specific binding to sulfoglycolipids (sulfatide) following brief treatment at low pH. We have previously shown that heat shock proteins (hsps) bind to sulfatide, and the suspicion that this was a stress-induced response is supported by the fact that a similar change in H. pylori binding specificity was observed if the organisms were briefly exposed to heat shock treatment. Following the stress stimulus, the change in glycolipid binding specificity was prevented by the inclusion of inhibitors of protein synthesis or by incubation with anti-hsp antibodies. Expression of hsps in the surface extract and surface reactivity with anti-hsp antibodies correlated with the change in glycolipid binding specificity. Despite the presence of high levels of H. pylori cell surface urease activity, which may neutralize the microenvironmental pH, the acid-induced change in binding specificity was enhanced in the presence of urea. These studies suggest that cell surface hsps mediate sulfatide recognition by this organism under stress conditions. A binary receptor model is proposed for gastric colonization by H. pylori.

Adhesion of Helicobacter pylori was reported previously to be morphologically identical to "attaching and effacing" Escherichia coli. Therefore, the aim of the present study was to define the adhesion phenotype of H. pylori LC-11 to HEp-2, KATO-III, HEL, and CHO tissue culture cells. By using both staining of F-actin with fluorescein-labeled phalloidin and ultrastructural analysis, diffuse bacterial adhesion to discrete microvillusdenuded regions of the plasma membrane was observed in each of the infected cell lines. However, strain LC-11 did not induce formation of F-actin adhesion pedestals on the eukaryotic cells. H. pyloni was negative by colony blot hybridization with an E. coli attaching and effacing gene probe. Elevations in inositol triphosphates followed infection of HEp-2 cells with H. pylori (405% of control values + 147%; P < 0.05). To correlate the observed histopathology with expression of the H. pylori phosphatidylethanolamine receptor, a thin-layer chromatography overlay-binding assay was used to identify receptors in each of the cell lines. H. pylori adhered to eukaryotic cells regardless of the presence (HEp-2, KATO-III, and CHO cells) or absence (HEL cells) of the lipid receptor as detected under the assay conditions. However, in comparison to cell lines that possess the phosphatidylethanolamine receptor, HEL cells demonstrated less quantitative H. pylori binding. These findings suggest that mechanisms distinct from E. coli enteropathogens underlie the adhesion of H. pylori to mucosal surfaces. In addition to the phosphatidylethanolamine H. pylori receptor, another host factor(s) likely mediates the attachment of H. pyloyi to human eukaryotic cells.

We have previously shown that Helicobacter pylori specifically binds to a glycerolipid species preferentially found in the antrum of the human stomach. We now show by high-pressure liquid chromatographic analysis that this species is a form of phosphatidylethanolamine and that H. pylori specifically binds to bona fide phosphatidylethanolamine as detected by a thin-layer chromatogram overlay procedure. Considerable variation in the binding of H. pyloyi to phosphatidylethanolamine from different sources was observed, however, suggesting the importance of the nature of the long-chain hydrophobic moiety. A similar binding specificity was shown by exoenzyme S from Pseudonwnas aeruginosa, consistent with our hypothesis that that an exoenzyme S-like adhesin is responsible for the binding of H. pylori to its lipid receptors.Colonization of the human stomach with Helicobacter pylori has been implicated as the major etiological agent in the development of gastritis and possibly subsequent duodenal ulcer (3, 6, 27) and gastric carcinoma (17,19,20). Because of the known tropism of this organism for colonization of gastric epithelium, even when mislocated elsewhere in the gastrointestinal tract (31), we previously investigated the presence of gastric membrane species able to specifically bind to this organism, as monitored by the solid-phase thin-layer chromatography (TLC) overlay procedure (13). We isolated a charged glycerolipid species from the antrum of human stomach and from human erythrocytes which was specifically recognized by this organism, using this procedure.Exoenzyme S is an ADP-ribosyltransferase and is one of the virulence factors of Pseudomonas aeruginosa involved in the pathogenesis of this organism (30). We have recently found (14) that this enzyme also specifically binds to the H. pylori receptor glycerolipid.We now report on the partial structural characterization of this receptor species.MATERUILS AND METHODS Phosphatidylethanolamine (PE) from various tissues; phosphatidic acid, phosphatidylserine, and phosphatidylcholine from egg yolk; and phosphatidylglycerol from bovine brain were purchased from Sigma. Plastic-backed silica gel (SIL G) TLC plates were from Brinkman Inc. Goat antirabbit-horseradish peroxidase conjugate was from Bio-Rad.Purification of H. pylori receptor. The Helicobacter glycerolid receptor was purified as previously described from pooled human erythrocytes (13). Briefly, the cells were extracted with chloroform-methanol (2:1, vol/vol), and lower-phase lipids were separated by silicic acid chromatography. The column was eluted in sequence with chloroform, acetone-methanol (3:1), and finally methanol. The methanol fraction was reapplied on a fresh column which was eluted with a linear polarity gradient of chloroform methanol (10:1 to 2:1). * Corresponding author.Receptor binding. H. pylori was grown on Skirrow's medium and transferred to brucella broth supplemented with 10% fetal calf serum before binding (13). Only highly motile cultures were used for binding experiments. Purified exoen...

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-833Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined.Fluoroquinolones are potent antibacterial agents, derived from nalidixic acid, that inhibit DNA gyrase (11,12). Active DNA gyrase is a tetramer composed of two A and two B subunits, with molecular masses of 97 and 90 kDa for each monomer, respectively (17, 26), encoded by the gyrA and gyrB genes (10,12 DNA gyrase activity can be selectively inhibited by quinolones (8,25). Apparently, quinolones bind to the gyrase-DNA complex at the site of cleaved DNA, where they interact with the single-stranded DNA region (20,25). It has been proposed that magnesium ions are needed for quinolone-DNA binding and possibly for formation of the quinolone-gyrase-cleavable DNA complex (30). Single point mutations in gyrA that confer resistance to quinolone drugs have been described for a variety of microorganisms (14, 21-23, 31, 32, 36, 38). These mutations occur in a conserved region of the N-terminal domain of the A subunit close to the catalytic Tyr-122 site, termed the quinolone resistance-determining region (QRDR).In E. coli, replacement of Ser-83 by Leu or Trp confers high-level resistance to quinolones. Mutations in other residues also result in resistance phenotypes (29,39).Fluoroquinolone resistance is rarely found among Salmonella species. A clinical isolate of Salmonella typhimurium in which resistance to ciprofloxacin was associated with alterations in both gyrase subunits has recently been described (15). The exact locations and genetic changes associated with these mutations have not been defined.In this study, we analyzed the DNA sequences of gyrA fragments from spontaneous resistant mutants of S. typhimurium Su694 and their corresponding fluoroquinolone susceptibilities. Strain Su694 is a derivative of S. typhimurium LT2 (described elsewhere [2]). Spontaneous mutants were obtained by plating an overnight culture of Su694 on nalidixic acid-or pefloxacin-containing agar. The quinolone concentrations used were 10 and 20 times the MIC for Su694 (9). Several individual colonies were collected from these plates to purify DNA and amplify an 800-bp gyrA fragment containing the QRDR. Primers for PCR amplification were designed on the basis of the known E. coli gyrA sequence (35) and correspond to nucleotides 93 to 110 and 874 to 890 for forward and reverse reactions, respectively.PCR amplifications were performed according to the manufacturer's instructions, with 100 ng of DNA template and final concentrations of 2 mM MgCl 2 , 200 M (each) deoxynucleotide triphosphate, 10 pmol of each primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer Cet...

No abstract