Helicobacter pylori infection elicits an aggressive inflammatory response that the bacterium is able to resist by virtue of its well-adapted antioxidant defence mechanisms. Catalase (KatA) appears to be a key enzyme in this resistance. Upstream of katA, a low-affinity ferric uptake regulator (Fur)-box has been identified. Downstream of katA, an ORF (HP0874) with no known function has also been identified. Non-polar isogenic mutants of katA, fur and HP0874 were constructed by allelic exchange. The impact of these mutations on the catalase activities and bacterial viability following exposure to hydrogen peroxide was studied. Concurrently, the effect of variation in the iron content of the media used to grow the cells was determined. The data showed that catalasedeficient isolates of H. pylori were hypersensitive to hydrogen peroxide, whereas wild-type cells could resist " "100 mM hydrogen peroxide. Fur-deficient mutants and cells grown on low-iron-containing medium showed a distinct reduction in catalase activity and increased sensitivity to hydrogen peroxide. The data suggest a direct or indirect effect of Fur and iron on the activity of catalase. HP0874-deficient mutants showed no reduction in catalase activity but showed an increased sensitivity to hydrogen peroxide. That is, the protein encoded by HP0874 appears to have a role in resistance to hydrogen peroxide not directly related to catalase activity. This is the first report of a functional relationship of the product of this ORF. There is evidence of protein-protein interaction between KatA and the product encoded by HP0874, and the name ' KatA-associated protein ' (KapA) is proposed.

Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (n ‫؍‬ 75) and BALB/c (n ‫؍‬ 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 (P ‫؍‬ 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j (n ‫؍‬ 35) and BALB/c (n ‫؍‬ 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.

The efficacy of an orogastric vaccine comprised of purified Helicobacter pylori catalase plus the mucosal adjuvant cholera toxin (CT) was examined with both the Helicobacter felis and H. pylori mouse models with BALB/c mice. Native H. pylori catalase (200 g) plus CT was initially used as a vaccine antigen in the H. felis mouse model and protected 80% (8 of 10) of the challenged animals, while all control animals were infected (20 of 20). In a follow-up experiment, recombinant H. pylori catalase plus CT was used for immunization, and groups of mice were challenged with the Sydney strain of H. pylori. Immunization with recombinant catalase protected a significant proportion (9 of 10) of the mice from H. pylori challenge, indicating that this enzyme should be considered as a candidate for a future vaccine. This study provides the first available data on the efficacy of protective immunization with the new Sydney strain of H. pylori in a mouse model. These data also provide indirect evidence that proteins which are normally intracellular, such as catalase, may be present on the surface of H. pylori and thus may provide targets for immunization.

Helicobacter pylori-induced gastritis is an essential precursor lesion for the development of peptic ulcers or gastric adenocarcinoma. We demonstrate that nonresponsiveness to H. pylori SS1 infection is dominantly inherited in mice. F 1 hybrid crosses between a nonresponder mouse and three responder strains all possessed the nonresponder phenotype. Secretion of interleukin-10 but not gamma interferon was associated with nonresponsiveness to infection.

The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions. Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate. The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products. The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner. We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK. IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed.Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not. Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.The narK gene in Escherichia coli, located at 27 min on the chromosome, is near the structural genes for nitrate reductase (narGHJI) plus two regulatory genes that are involved in nitrate-dependent gene expression, narX and narL (1,30,40). The narK gene was recently sequenced, and a role in nitrate transport was suggested on the basis of its similarity in protein sequence to a family of cytoplasmic membrane transport proteins (31). This role is supported by observations that nitrate uptake and metabolism are altered in narK mutants (9,17,31).In a previous study, narK expression was examined under anaerobic conditions and found to be impaired in strains defective in either narL or fnr (40). However, the effect of oxygen and nitrate on narK expression was not examined nor was the effect of varying other nutritional conditions. To address these questions, narK-lacZ fusions were constructed and inserted into the chromosome in single copy, and their expression was analyzed under a variety of conditions. The role of the narX, narQ, chlD, chlE, chU, fiur, fis, and himA gene products on narK-lacZ expression was documented as was the effect of limiting iron and molybdenum availability. The conditions optimal for narK expression are consistent with a role for NarK in nitrate transport and/or metabolism. MATERIALS AND METHODSBacterial strains, bacteriophages, and plasmids. The genotypes and origins of the E. coli K-12 strains, bacteriophage, and plasmids used are given in Table 1. Strains TK100 (hiNMA) and TK101 (fis) were constructed by P1 transduction of the respective mutations from SD1 and SD6 into MC4100/ XTS8 (7,35). By using the same approach, the TK102 (narQ), TK103 [A(narXL) narQ], and TK104 (fur) strains were constructed (Table 1).Construction of narK-acZ gene fusions. Phage M13IS4 was constructed by inserting a ...