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ABSTRACTPersistent colonization of mucosal surfaces by bacteria in the mammalian host requires concerted expression of colonization factors, depending on the environmental conditions. Helicobacter hepaticusis a urease-positive pathogen that colonizes the intestinal and hepatobiliary tracts of rodents. Here it is reported that urease expression of H. hepaticusis iron repressed by the transcriptional regulator Fur. Iron restriction of growth medium resulted in a doubling of urease activity in wild-type H. hepaticusstrain ATCC 51449 and was accompanied by increased levels of urease subunit proteins and ureAmRNA. Insertional inactivation of the furgene abolished iron-responsive repression of urease activity, whereas inactivation of the perRgene did not affect iron-responsive regulation of urease activity. The iron-responsive promoter element was identified directly upstream of the H. hepaticus ureAgene. Recombinant H. hepaticusFur protein bound to this ureApromoter region in a metal-dependent matter, and binding resulted in the protection of a 41-bp, Fur box-containing operator sequence located at positions −35 to −75 upstream of the transcription start site. In conclusion, H. hepaticusFur controls urease expression at the transcriptional level in response to iron availability. This represents a novel type of urease regulation in ureolytic bacteria and extends the already diverse regulatory repertoire of the Fur protein.

ABSTRACTThe transition metal nickel plays an important role in gastric colonization and persistence of the important human pathogen Helicobacter pylori, as it is the cofactor of the abundantly produced acid resistance factor urease. Nickel uptake through the inner membrane is mediated by the NixA protein, and the expression of NixA is controlled by the NikR regulatory protein. Here we report that NikR also controls the nickel-responsive expression of the FecA3 (HP1400) and FrpB4 (HP1512) outer membrane proteins (OMPs), as well as the nickel-responsive expression of an ExbB-ExbD-TonB system, which may function in energization of outer membrane transport. Transcription and expression of the frpB4and fecA3genes were repressed by nickel in wild-type H. pylori26695, but they were independent of nickel and derepressed in an isogenic nikRmutant. Both the frpB4and fecA3genes were transcribed from a promoter directly upstream of their start codon. Regulation by NikR was mediated via nickel-dependent binding to specific operators overlapping either the +1 or −10 sequence in the frpB4and fecA3promoters, respectively, and these operators contained sequences resembling the proposed H. pyloriNikR recognition sequence (TATWATT-N11-AATWATA). Transcription of the HP1339-1340-1341 operon encoding the ExbB2-ExbD2-TonB2 complex was also regulated by nickel and NikR, but not by Fur and iron. In conclusion, H. pyloriNikR controls nickel-responsive expression of the HP1400 (FecA3) and HP1512 (FrpB4) OMPs. We hypothesize that these two NikR-regulated OMPs may participate in the uptake of complexed nickel ions and that this process is energized by the NikR-regulated ExbB2-ExbD2-TonB2 system, another example of the specific adaptation of H. pylorito the gastric lifestyle.

ABSTRACTMaintaining metal homeostasis is crucial for the adaptation of Helicobacter pylorito the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pyloriinfection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylorimetal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylorireference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC(HP0971), cznB(HP0970), and cznA(HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznCand cznAmutants but not the cznBmutant displayed an 8- to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznCmutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABCare essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pyloriin the gastric environment.

ABSTRACTAlthough the adaptive mechanisms allowing the gastric pathogen Helicobacter pylorito survive acid shocks have been well documented, the mechanisms allowing growth at mildly acidic conditions (pH ∼5.5) are still poorly understood. Here we demonstrate that H. pyloristrain 26695 increases the transcription and activity of its urease, amidase, and formamidase enzymes four- to ninefold in response to growth at pH 5.5. Supplementation of growth medium with NiCl2resulted in a similar induction of urease activity (at low NiCl2concentration) and amidase activity (at ≥500 μM NiCl2) but did not affect formamidase activity. Mutation of the furgene, which encodes an iron-responsive repressor of both amidases, resulted in a constitutively high level of amidase and formamidase activity at either pH but did not affect urease activity at pH 7.0 or pH 5.5. In contrast, mutation of the nikRgene, encoding the nickel-responsive activator of urease expression, resulted in a significant reduction of acid-responsive induction of amidase and formamidase activity. Finally, acid-responsive repression of furtranscription was absent in the H. pylori nikRmutant, whereas transcription of the nikRgene itself was increased at pH 5.5 in wild-type H. pylori. We hypothesize that H. pyloriuses a repressor cascade to respond to low pH, with NikR initiating the response directly via the urease operon and indirectly via the members of the Fur regulon.

ABSTRACTGenetic analysis of two Helicobacter pyloristrains isolated from a single gastric biopsy showed evidence of extensive horizontal gene transfer. Several large recombinations were identified in the rdxAgene, which is involved in metronidazole resistance.

ABSTRACTThe reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pyloriferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylorito multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfrgene restricted growth of H. pyloriunder these conditions. The lowered total iron content in the pfrmutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoBmutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfrtranscription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pyloriFur to the pfrpromoter region. The functions of H. pyloriPfr in iron metabolism are essential for survival in the gastric mucosa, as the pfrmutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfrphenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylorito survive in its hostile natural environment.

ABSTRACTThe only known niche of the human pathogen Helicobacter pyloriis the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylorigenome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella entericaserovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pyloriFur homologue in acid resistance. Isogenic furmutants were generated in three H. pyloristrains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the furmutants was severely impaired. No differences were observed between the survival of the furmutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the furmutant at acidic pH. This indicates that the phenotype of the furmutant at low pH was not due to increased iron sensitivity. Transcription of furwas repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.

ABSTRACTUpon contact with host cells, the intracellular pathogenSalmonella entericaserovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. entericaserovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonellagene designated sspJthat is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104to 107bacteria in C3H/HeN and 101to 104bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJcomplementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

ABSTRACTThe nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of H. pylori, expression of urease genes is thought to be constitutive. Here it is demonstrated that H. pyloriregulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 μM NiCl2resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptionalureA::lacZfusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of H. pyloriurease, a phenomenon likely to be of importance during the colonization and persistence of H. pyloriin the gastric mucosa.

ABSTRACTThe lipopolysaccharide (LPS) of Helicobacter pyloriexpresses the Lewis x (Lex) and/or Leyantigen. We have shown previously that H. pyloriLPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lexplus Leyor nonfucosylated polylactosamine. H. pylorihas two α3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pyloriLPS phase variation and demonstrate that the on or off status of α3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the α3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of α3-fucosyltransferase knockout mutants. The data also show that the two α3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futAand futBto designate the orthologs of the H. pylori26695 α3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the α3-fucosylation in H. pyloriprecedes α3-fucosyltransferase, an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.

ABSTRACTSalmonella typhimuriuminfection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimuriumgenes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1,000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimuriumgenome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonellaspp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of theSalmonellagenome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.