The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogenHelicobacter pylorifails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains ofH. pyloriinhibit NO production by activated macrophages at physiologic concentrations ofl-arginine, the common substrate for iNOS and arginase. Inactivation of the generocF, encoding constitutively expressed arginase inH. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show thatl-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficientH. pylori. The substantially higher levels of NO generated by macrophages cocultured withrocF-deficientH. pyloriresulted in efficient killing of the bacteria, whereas wild-typeH. pyloriexhibited no loss of survival under these conditions. Killing of the arginase-deficientH. pyloriwas NO-dependent, because peritoneal macrophages from iNOS^−/−mice failed to affect the survival of therocFmutant. Thus, bacterial arginase allowsH. pylorito evade the immune response by down-regulating eukaryotic NO production.

The human gastric pathogen Helicobacter pylori steals host cholesterol, modifies it by glycosylation, and incorporates the glycosylated cholesterol onto its surface via a cholesterol glucosyltransferase, encoded by cgt . The impact of cholesterol on H. pylori antimicrobial resistance is unknown. H. pylori strain 26695 was cultured in Ham's F12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 12 antibiotics, six antifungals, and seven antimicrobial peptides (including LL-37 cathelicidin and human alpha and beta defensins). Of 25 agents tested, cholesterol-grown H. pylori cells were substantially more resistant (over 100-fold) to nine agents than were H. pylori cells grown without cholesterol. These nine agents included eight antibiotics and LL-37. H. pylori was susceptible to the antifungal drug pimaricin regardless of cholesterol presence in the culture medium. A cgt mutant retained cholesterol-dependent resistance to most antimicrobials but displayed increased susceptibility to colistin, suggesting an involvement of lipid A. Mutation of lpxE , encoding lipid A1-phosphatase, led to loss of cholesterol-dependent resistance to polymyxin B and colistin but not other antimicrobials tested. The cgt mutant was severely attenuated in gerbils, indicating that glycosylation is essential in vivo . These findings suggest that cholesterol plays a vital role in virulence and contributes to the intrinsic antibiotic resistance of H. pylori .

Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli . An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.

Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3ζ-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(−), depleted l-arginine and caused a decrease in CD3ζ expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.

The ulcer-causing pathogenHelicobacter pyloriuses directed motility, or chemotaxis, to both colonize the stomach and promote disease development. Previous work showed that mutants lacking the TlpB chemoreceptor, one of the receptors predicted to drive chemotaxis, led to less inflammation in the gerbil stomach than did the wild type. Here we expanded these findings and examined the effects on inflammation of completely nonchemotactic mutants and mutants lacking each chemoreceptor. Of note, all mutants colonized mice to the same levels as did wild-typeH. pylori. Infection by completely nonchemotactic mutants (cheWorcheY) resulted in significantly less inflammation after both 3 and 6 months of infection. Mutants lacking either the TlpA or TlpBH. pylorichemotaxis receptors also had alterations in inflammation severity, while mutants lacking either of the other two chemoreceptors (TlpC and HylB) behaved like the wild type. Fully nonchemotactic and chemoreceptor mutants adhered to cultured gastric epithelial cells and caused cellular release of the chemokine interleukin-8 in vitro similar to the release caused by the wild type. The situation appeared to be different in the stomach. Using silver-stained histological sections, we found that nonchemotacticcheYorcheWmutants were less likely than the wild type to be intimately associated with the cells of the gastric mucosa, although there was not a strict correlation between intimate association and inflammation. Because others have shown that in vivo adherence promotes inflammation, we propose a model in whichH. pyloriuses chemotaxis to guide it to a productive interaction with the stomach epithelium.

Helicobacter pyloricauses disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical forH. pylorito colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in theH. pylorichemotaxis pathway,cheYandtlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. ThecheYmutation was complemented with a wild-type copy ofcheYinserted into the chromosomalrdxAgene. ThecheYmutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While thecheYmutant completely failed to colonize gerbil stomachs, thetlpBmutant colonized at levels similar to those of the wild type. With thetlpBmutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in thetlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved inH. pyloripathogenesis.