<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

Image_3_ArsRS-Dependent Regulation of homB Contributes to Helicobacter pylori Biofilm Formation.PDF

<p>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.</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

Hydrogenase activity, urease activity, survival at pH 2.3 with urea, and dissociation constants.^a

<p>Hydrogenase activity, urease activity, survival at pH 2.3 with urea, and dissociation constants.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183260#t002fn001" target="_blank">^a</a></p

The Δ<i>hydABCDE</i> strain of <i>H</i>. <i>pylori</i> 26695 is not attenuated for acid survival.

<p>The wild-type (WT) strain, urease mutant strain (Δ<i>ureAB</i>), and hydrogenase mutant strain (Δ<i>hydABCDE</i>) were incubated for 1 hr in PBS adjusted to pH 6.0 (A and B) or to pH 2.3 (C and D), in the absence (A and C) or presence (B and D) of 5 mM urea. The number of colony-forming units (CFU) was measured at 0 min (T₀) and at 60 min (T₆₀), and percent survival was calculated as CFU at T₆₀ divided by CFU at T₀. Data from individual biological replicates are shown as points, with the bar plotted at the mean. Open symbols indicate that no bacteria were recovered and thus, the CFU are plotted as a function of the limit of detection (1000 CFU/mL). Three biological replicates were performed. For panels A-C, a one-way ANOVA followed by Dunnett’s test for multiple comparisons was performed; the comparison was made only to WT. In panel D, the same statistical tests were performed on the log-transformed data. **** = p < 0.0001.</p

The Δ<i>hydB</i> strain of <i>H</i>. <i>pylori</i> G27 is not attenuated for acid survival.

<p>The wild-type (WT) strain, urease mutant strain (Δ<i>ureB</i>), and hydrogenase mutant strain (Δ<i>hydB</i>) were incubated for 1 hr in PBS adjusted to pH 6.0 (A and B) or to pH 2.3 (C and D), in the absence (A and C) or presence (B and D) of 5 mM urea. The number of colony-forming units (CFU) was measured at 0 min (T₀) and at 60 min (T₆₀), and percent survival was calculated as CFU at T₆₀ divided by CFU at T₀. Data from individual biological replicates are shown as points, with the bar plotted at the mean. Open symbols indicate that no bacteria were recovered and thus, the CFU are plotted as a function of the limit of detection (100 CFU/mL). Three biological replicates were performed. For panels A-C, a one-way ANOVA followed by Dunnett’s test for multiple comparisons was performed; the comparison was made only to WT. In panel D, the same statistical tests were performed on the log-transformed data. **** = p < 0.0001.</p

Image_2_ArsRS-Dependent Regulation of homB Contributes to Helicobacter pylori Biofilm Formation.PDF

<p>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.</p

Outline of <i>bab</i> genotyping by PCR.

<p>(Top) Schematic representation of the three loci where the <i>bab</i> genes are generally detected: Locus A, B and C. The annealing positions (arrows) and names of each locus-specific forward are shown. (Bottom) Annealing positions (arrows) and names of <i>bab</i>-specific reverse primer are indicated with their respective <i>bab</i> gene: <i>babA</i> depicted by the black box, <i>babB</i> depicted by the white box, <i>babC</i> depicted by the grey box. Primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137078#pone.0137078.t001" target="_blank">Table 1</a>, and a full explanation of the genotyping scheme can be found in the Materials and Methods.</p