Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (P-ureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the P-ureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, Delta nikR and Delta arsS single mutants as well as a Delta arsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the P-ureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct cross-talk between HpArsRS and HpNikR at neutral pH has been demonstrated

ArsRS-Dependent Regulation of homB Contributes to Helicobacter pylori Biofilm Formation

One elusive area in the Helicobacter pylori field is an understanding of why some infections result in gastric cancer, yet others persist asymptomatically for the life-span of the individual. Even before the genomic era, the high level of intraspecies diversity of H. pylori was well recognized and became an intriguing area of investigation with respect to disease progression. Of interest in this regard is the unique repertoire of over 60 outer membrane proteins (OMPs), several of which have been associated with disease outcome. Of these OMPs, the association between HomB and disease outcome varies based on the population being studied. While the molecular roles for some of the disease-associated OMPs have been evaluated, little is known about the role that HomB plays in the H. pylori lifecycle. Thus, herein we investigated homB expression, regulation, and contribution to biofilm formation. We found that in H. pylori strain G27, homB was expressed at a relatively low level until stationary phase. Furthermore, homB expression was suppressed at low pH in an ArsRS-dependent manner; mutation of arsRS resulted in increased homB transcript at all tested time-points. ArsRS regulation of homB appeared to be direct as purified ArsR was able to specifically bind to the homB promoter. This regulation, combined with our previous finding that ArsRS mutations lead to enhanced biofilm formation, led us to test the hypothesis that homB contributes to biofilm formation by H. pylori. Indeed, subsequent biofilm analysis using a crystal-violet quantification assay and scanning electron microscopy (SEM) revealed that loss of homB from hyper-biofilm forming strains resulted in reversion to a biofilm phenotype that mimicked wild-type. Furthermore, expression of homB in trans from a promoter that negated ArsRS regulation led to enhanced biofilm formation even in strains in which the chromosomal copy of homB had been deleted. Thus, homB is necessary for hyper-biofilm formation of ArsRS mutant strains and aberrant regulation of this gene is sufficient to induce a hyper-biofilm phenotype. In summary, these data suggest that the ArsRS-dependent regulation of OMPs such as HomB may be one mechanism by which ArsRS dictates biofilm development in a pH responsive manner

Helicobacter pylori Biofilm Formation and Its Potential Role in Pathogenesis

Despite decades of effort, Helicobacter pylori infections remain difficult to treat. Over half of the world's population is infected by H. pylori, which is a major cause of duodenal and gastric ulcers as well as gastric cancer. During chronic infection, H. pylori localizes within the gastric mucosal layer, including deep within invaginations called glands; thanks to its impressive ability to survive despite the harsh acidic environment, it can persist for the host's lifetime. This ability to survive and persist in the stomach is associated with urease production, chemotactic motility, and the ability to adapt to the fluctuating environment. Additionally, biofilm formation has recently been suggested to play a role in colonization. Biofilms are surface-associated communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances. Biofilms pose a substantial health risk and are key contributors to many chronic and recurrent infections. This link between biofilm-associated bacteria and chronic infections likely results from an increased tolerance to conventional antibiotic treatments as well as immune system action. The role of this biofilm mode in antimicrobial treatment failure and H. pylori survival has yet to be determined. Furthermore, relatively little is known about the H. pylori biofilm structure or the genes associated with this mode of growth. In this review, therefore, we aim to highlight recent findings concerning H. pylori biofilms and the molecular mechanism of their formation. Additionally, we discuss the potential roles of biofilms in the failure of antibiotic treatment and in infection recurrence

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori.

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance

Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, nikR and arsS single mutants as well as a nikR/arsS double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct ‘cross-talk’ between HpArsRS and HpNikR at neutral p

<i>H</i>. <i>pylori</i> Δ<i>hydB</i> has no detectable hydrogenase activity and wild-type urease activity.

<p>(A) Cell lysates of the wild-type (WT) strain, urease mutant strain (Δ<i>ureB</i>), and hydrogenase mutant strain (Δ<i>hydB</i>) were used to measure hydrogenase activity using a methyl viologen assay. The rate at which H₂ was oxidized (in μmol/min) was obtained using the slope of absorbance at A_{578 nm}, which was normalized to the amount of total protein in the cell lysate (in μg), and normalized against the activity of the WT strain to obtain percent hydrogenase activity. Three biological replicates were tested for each strain, and the mean and standard deviation are graphed. (B) Cell lysates of the WT, Δ<i>ureB</i>, and Δ<i>hydB</i> strains were used to measure urease activity using ammonia production in a phenol-hypochlorite assay. The specific urease activity was normalized to the amount of total protein in the cell lysate (in μg), and normalized against the specific activity of the WT strain to obtain relative urease activity. Three biological replicates were tested for each strain, and the mean and standard deviation are graphed.</p

Strains, plasmids, and primers used in this study.

<p>Strains, plasmids, and primers used in this study.</p

The structure of HypA and its role in urease and hydrogenase maturation.

<p>(A) Representation of the NMR structure of <i>H</i>. <i>pylori</i> HypA (PDB: 2KDX) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183260#pone.0183260.ref025" target="_blank">25</a>] with the main chain colored in light grey and the metal binding sites in color to highlight the location of residues involved in metal coordination. Residues comprising the nickel-binding site (M1, H2, E3, and D40) are shown in green. Residues of the zinc-binding site (C74, C77, H79, C91, C94, and H95) are shown in cyan. The metal-binding oxygen (red), nitrogen (blue), and sulfur (yellow) atoms are shown as small spheres. The nickel atom representation in this figure (dotted green circle) was not resolved in the 2KDX structure, and the resolved zinc atom is shown as a dark grey sphere. The zinc-binding site adopts two pH-dependent conformations, as illustrated: Zn(Cys)₂(His)₂ at acidic pH, and Zn(Cys)₄ at neutral pH. (B) HypA contributes to the maturation of hydrogenase and urease through delivery of nickel (green circles). Urease requires nickel for activity, of which one of the downstream effects is acid resistance. In the absence of HypA, maturation of urease can still be accomplished through the addition of excess nickel (dashed line). Hydrogenase requires nickel for activity, but herein is shown not to contribute to <i>in vitro</i> acid resistance (red X). In the absence of HypA, maturation of hydrogenase cannot be accomplished through the addition of excess nickel.</p

Mutation of the metal coordination sites of HypA results in decreased hydrogenase activity.

<p>Cell lysates from the indicated <i>hypA</i> mutant strains, in addition to wild-type (WT) strain, urease mutant strain (Δ<i>ureB</i>), <i>hypA</i> mutant strain (<i>hypA</i>::<i>kan-sacB</i>), and <i>hypA</i> restorant (<i>hypA</i>-R) were utilized to determine hydrogenase activity using a methyl viologen assay. The rate at which H₂ was oxidized (in μmol/min) was obtained using the slope of absorbance at A_{578 nm}, which was normalized to the amount of total protein in the cell lysate (in μg), and normalized against the activity of the WT strain to obtain percent hydrogenase activity. The hydrogenase activities of <i>hypA</i> mutant strains with mutations found within the nickel-binding site (A) and within the zinc-binding site (B) are shown. Two biological replicates were tested in A, and three biological replicates were tested in B. The mean is graphed, with range (A) or standard deviation (B).</p