The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogen Helicobacter pylori fails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains of H. pylori inhibit NO production by activated macrophages at physiologic concentrations of L-arginine, the common substrate for iNOS and arginase. Inactivation of the gene rocF, encoding constitutively expressed arginase in H. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show that L-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficient H. pylori. The substantially higher levels of NO generated by macrophages cocultured with rocF-deficient H. pylori resulted in efficient killing of the bacteria, whereas wild-type H. pylori exhibited no loss of survival under these conditions. Killing of the arginase-deficient H. pylori was NO-dependent, because peritoneal macrophages from iNOS ؊/؊ mice failed to affect the survival of the rocF mutant. Thus, bacterial arginase allows H. pylori to evade the immune response by down-regulating eukaryotic NO production. Helicobacter pylori is a Gram-negative microaerophilic bacterium, which selectively colonizes the human stomach. Current prevalence of H. pylori is Ϸ40% of the population in the U.S. (1) and substantially higher in underdeveloped regions. H. pylori causes chronic gastritis, peptic ulcers, and gastric carcinoma and lymphoma, leading to its classification as a Class I carcinogen (2). Despite inciting substantial acute and chronic immune and inflammatory responses, H. pylori infection generally persists for the life of the host. Understanding how the bacterium evades the host response remains a critical issue in managing the public health burden of this infection.Nitric oxide (NO) is a central component of innate immunity and an effective antimicrobial agent (3). This activity is especially marked for intracellular pathogens such as Mycobacterium tuberculosis (4) and Leishmania major (5), which are killed by an NO-dependent mechanism. Reactive nitrogen intermediates can also effectively kill extracellular parasites (6, 7) and bacteria such as Escherichia coli (8). Chemical sources of NO and peroxynitrite have a direct toxic effect on H. pylori (9, 10). However, the effect of cell-derived NO on H. pylori has not been investigated. The survival of H. pylori, despite marked induction of inducible NO synthase (iNOS) in macrophages (11) and gastric tissues (12), suggests that the bacterium has developed mechanisms to avoid NO-dependent killing.Arginases are a primordial enzyme family, which are highly conserved across kingdoms (13). Mammalian arginases compete with NO synthases for the common substrate L-arginine (14), hydrolyzing the amino acid to urea and L-ornithine. Therefore, arginases can regulate cellular NO production (15, 16) and counteract the biological effects of NO (7,17). H....

The ulcer-causing pathogen Helicobacter pylori uses 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-type H. pylori. Infection by completely nonchemotactic mutants (cheW or cheY) resulted in significantly less inflammation after both 3 and 6 months of infection. Mutants lacking either the TlpA or TlpB H. pylori chemotaxis 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 nonchemotactic cheY or cheW mutants 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 which H. pylori uses chemotaxis to guide it to a productive interaction with the stomach epithelium.

Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant 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 the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, 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 the tlpB-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 in H. pylori pathogenesis.

Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only ϳ10 3 CFU/ml. All H. pylori strains (n ‫؍‬ 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), ␤-cyclodextrin (200 g/ml), or cholesterol (50 g/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 g/ml), ␤-cyclodextrin (200 g/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or ␤-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification of H. pylori from clinical specimens.

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