Elizabeth A. Marcus

Acid-Adaptive Genes of Helicobacter pylori

ABSTRACT Helicobacter pylori is the only neutralophile that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms. One of the adaptive mechanisms is increased buffering due to expression of an acid-activated inner membrane urea channel, UreI, and a neutral pH-optimum intrabacterial urease. To delineate other possible adaptive mechanisms, changes in gene expression in response to acid exposure were examined using genomic microarrays of H. pylori exposed to different levels of external pH (7.4, 6.2, 5.5, and 4.5) for 30 min in the absence and presence of 5 mM urea. Gene expression was correlated with intrabacterial pH measured using 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein and compared to that observed with exposure to 42°C for 30 min. Microarrays containing the 1,534 open reading frames of H. pylori strain 26695 were hybridized with cDNAs from control (pH 7.4; labeled with Cy3) and acidic (labeled with Cy5) conditions. The intrabacterial pH was 8.1 at pH 7.4, fell to 5.3 at pH 4.5, and rose to 6.2 with urea. About 200 genes were up-regulated and ∼100 genes were down-regulated at pH 4.5 in the absence of urea, and about half that number changed in the presence of urea. These genes included pH-homeostatic, transcriptional regulatory, motility, cell envelope, and pathogenicity genes. The up-regulation of some pH-homeostatic genes was confirmed by real-time PCR. There was little overlap with the genes induced by temperature stress. These results suggest that H. pylori has evolved multifaceted acid-adaptive mechanisms enabling it to colonize the stomach that may be novel targets for eliminating infection.

The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

The role of the periplasmic alpha-carbonic anhydrase (alpha-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since alpha-CA catalyzes the conversion of CO2 to HCO3-, the role of CO2 in periplasmic buffering was studied using an alpha-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that alpha-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the alpha-CA knockout. In the mutant or in the presence of acetazolamide, there was an approximately 3 log10 decrease in acid survival. In acid, absence of alpha-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the alpha-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of alpha-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3- by the periplasmic alpha-CA.

Expression of the Helicobacter pylori ureI Gene Is Required for Acidic pH Activation of Cytoplasmic Urease

ABSTRACT ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we producedH. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determinedureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastricHelicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacterspecies. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

Gene expression in vivo shows that Helicobacter pylori colonizes an acidic niche on the gastric surface

Helicobacter pylori is a gastric-dwelling pathogen responsible, with acid secretion, for peptic ulcer and a 20-fold increase in the risk of gastric cancer. Several transcriptomes have been described after short-term exposure to acidity in vitro, but there are no data identifying the effects of chronic gastric exposure on bacterial gene expression. Comparison of the in vivo to the in vitro transcriptome at pH 7.4 identified several groups of genes of known function that increased expression >2-fold, and three of these respond both to acidity in vitro and to gastric infection. Almost all known acid acclimation genes are highly up-regulated. These include ureA, ureB, and rocF and the pH-gated urea channel, ureI. There is also up-regulation of two groups of motility and chemotaxis genes and for pathogenicity island genes, especially cagA, a predictor for pathogenicity. Most of these genes interact with HP0166, the response element of the pH-sensing two-component histidine kinase, HP0165/HP0166, ArsRS. Based on the pH profile of survival of ureI deletion mutants in vitro and their inability to survive in gastric acidity, the habitat of the organism at the gastric surface is acidic with a pH ≤ 4.0. Hence, the pH of the habitat of H. pylori on the surface of the stomach largely determines the regulation of these specific groups of genes.

Genes of Helicobacter pylori Regulated by Attachment to AGS Cells

ABSTRACT Reciprocal interactions between Helicobacter pylori and cells of the gastric epithelium to which it adheres may affect colonization. Changes in gene expression of H. pylori induced by adhesion to AGS gastric cancer cells by coculture were compared to changes in gene expression of H. pylori cultured without AGS cells by using cDNA filter macroarrays. Adhesion was quantitatively verified by confocal microscopy of green fluorescent protein-expressing bacteria. Four experiments showed that 22 and 21 H. pylori genes were consistently up- and down-regulated, respectively. The up-regulated genes included pathogenicity island, motility, outer membrane protein, and translational genes. The σ28 factor antagonist flgM, flgG, the stress response gene, flaA, omp11, and the superoxide dismutase gene (sodB) were down-regulated. The up-regulation of cag3, flgB, tonB, rho, and deaD was confirmed by quantitative PCR, and the up-regulation of lpxD, omp6, secG, fabH, HP1285, HP0222, and HP0836 was confirmed by reverse transcription (RT)-PCR. The down-regulation of flaA, sodB, and HP0874 was confirmed by quantitative PCR, and the down-regulation of omp11 was confirmed by RT-PCR. The alteration of gene expression in H. pylori after adhesion to gastric cells in vitro suggests that changes in motility, outer membrane composition, and stress responses, among other changes, may be involved in gastric colonization.

Cell Lysis is Responsible for the Appearance of Extracellular Urease in Helicobacter pylori

Helicobacter pylori is a neutralophilic bacterium that colonizes the acidic human gastric surface using the neutralizing capacity of a constitutively produced urease. Urease is present both in the cytoplasm and bound to the outside surface of the bacteria. The origin of the surface urease continues to be controversial. This study provides additional evidence that the origin of surface urease is cell lysis, not secretion.

Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells.

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection.

Cytoplasmic Histidine Kinase (HP0244)-Regulated Assembly of Urease with UreI, a Channel for Urea and Its Metabolites, CO2, NH3, and NH4+, Is Necessary for Acid Survival of Helicobacter pylori

Helicobacter pylori colonizes the normal human stomach by maintaining both periplasmic and cytoplasmic pH close to neutral in the presence of gastric acidity. Urease activity, urea flux through the pH-gated urea channel, UreI, and periplasmic alpha-carbonic anhydrase are essential for colonization. Exposure to pH 4.5 for up to 180 min activates total bacterial urease threefold. Within 30 min at pH 4.5, the urease structural subunits, UreA and UreB, and the Ni(2+) insertion protein, UreE, are recruited to UreI at the inner membrane. Formation of this complex and urease activation depend on expression of the cytoplasmic sensor histidine kinase, HP0244. Its deletion abolishes urease activation and assembly, impairs cytoplasmic and periplasmic pH homeostasis, and depolarizes the cells, with an approximately 7-log loss of survival at pH 2.5, even in 10 mM urea. Associated with this assembly, UreI is able to transport NH(3), NH(4)(+), and CO(2), as shown by changes in cytoplasmic pH following exposure to NH(4)Cl or CO(2). To be able to colonize cells in the presence of the highly variable pH of the stomach, the organism expresses two pH-sensor histidine kinases, one, HP0165, responding to a moderate fall in periplasmic pH and the other, HP0244, responding to cytoplasmic acidification at a more acidic medium pH. Assembly of a pH-regulatory complex of active urease with UreI provides an advantage for periplasmic buffering.

Involvement of the HP0165-HP0166 Two-Component System in Expression of Some Acidic-pH-Upregulated Genes of Helicobacter pylori

About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His(6) or a mutated response regulator, HP0166-D52N-His(6), that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His(6)). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His(6). Seven promoters for genes encoding alpha-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His(6). Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His(6), but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).

The effects of varying acidity on Helicobacter pylori growth and the bactericidal efficacy of ampicillin.

Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens.