Multiple Acid Sensors Control Helicobacter pylori Colonization of the Stomach.

Helicobacter pylori's ability to respond to environmental cues in the stomach is integral to its survival. By directly visualizing H. pylori swimming behavior when encountering a microscopic gradient consisting of the repellent acid and attractant urea, we found that H. pylori is able to simultaneously detect both signals, and its response depends on the magnitudes of the individual signals. By testing for the bacteria's response to a pure acid gradient, we discovered that the chemoreceptors TlpA and TlpD are each independent acid sensors. They enable H. pylori to respond to and escape from increases in hydrogen ion concentration near 100 nanomolar. TlpD also mediates attraction to basic pH, a response dampened by another chemoreceptor TlpB. H. pylori mutants lacking both TlpA and TlpD (ΔtlpAD) are unable to sense acid and are defective in establishing colonization in the murine stomach. However, blocking acid production in the stomach with omeprazole rescues ΔtlpAD's colonization defect. We used 3D confocal microscopy to determine how acid blockade affects the distribution of H. pylori in the stomach. We found that stomach acid controls not only the overall bacterial density, but also the microscopic distribution of bacteria that colonize the epithelium deep in the gastric glands. In omeprazole treated animals, bacterial abundance is increased in the antral glands, and gland colonization range is extended to the corpus. Our findings indicate that H. pylori has evolved at least two independent receptors capable of detecting acid gradients, allowing not only survival in the stomach, but also controlling the interaction of the bacteria with the epithelium

The density of gland-associated <i>H</i>. <i>pylori</i> in the antrum of the stomach is influenced by gastric acidity.

<p>(<b>A-D</b>) 3D confocal immunofluorescence reconstructions of mice infected with wild-type (WT) or Δ<i>tlpAD H</i>. <i>pylori</i> followed by treatment with omeprazole or no treatment. <i>H</i>. <i>pylori</i> are stained in green and actin in red. Scale bar is 100μm for all 4 panels. (<b>E</b>) Quantification of the number of bacteria per gland in the antrum of animals infected with WT or Δ<i>tlpAD</i> treated with omeprazole or untreated. The antral glands of seven animals in each cohort were analyzed for bacterial colonization by 3D volumetric analysis. The number of bacteria in each individual gland is plotted as a scatter point. * <i>P</i> < 0.05, **** <i>P</i> < 0.0001 (Mann Whitney test). The panels on the right show an example of the volumetric measurement of bacteria in gastric glands. 3D confocal reconstructions of glands stained with anti–<i>H pylori</i> (<i>green</i>), phalloidin (<i>red</i>), and DAPI (<i>blue</i>) were generated. Bacterial microcolony signals are identified by fluorescence intensity and size using Volocity software and the identified voxels marked in <i>gray</i> (<i>bottom panels</i>) are measured. The number of bacteria per gland is calculated based on the average voxel volume of 1 bacterium. Scale bar is 10μm for all 4 panels.</p

<i>H</i>. <i>pylori</i> expresses two chemoreceptors that rapidly sense acid gradients.

<p>(<b>A</b>) Quantification of the responses of wild-type (WT) <i>H</i>. <i>pylori</i> vs. Δ<i>tlpA</i>, Δ<i>tlpB</i>, Δ<i>tlpC</i>, Δ<i>tlpD</i> to a 100 mM hydrochloric acid gradient. Temporal data of motility responses were collected by videomicroscopy and quantified as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006118#ppat.1006118.g001" target="_blank">Fig 1C</a>. The difference in bacterial density within the field of view at two time points, four seconds before the acid gradient (pre) and ten seconds after exposure to the acid gradient (post) is represented by bar graphs. (<b>B</b>) Quantification of the responses of WT <i>H</i>. <i>pylori</i> vs. Δ<i>cheW</i>, Δ<i>tlpAD</i>, Δ<i>tlpAB</i>, Δ<i>tlpAC</i>, Δ<i>tlpBC</i>, Δ<i>tlpBD</i>, Δ<i>tlpCD</i> to a 100 mM hydrochloric acid gradient. Each bar represents the percent of bacteria in the field of view at four seconds pre-exposure and 10 seconds post-exposure to the gradient. (<b>C</b>) Still images of bacterial motility traces (lasting 1.5 seconds) of WT, Δ<i>tlpAD</i>, and Δ<i>tlpB H</i>. <i>pylori</i> within the field of view before and after exposure to a gradient of 5 mM urea and 50 mM hydrochloric acid. (n = 3 movies per condition). Bars represent the mean. Error bars represent s.d. NS indicates no statistical significance, **** <i>P</i> < 0.0001 (2-way repeated measures ANOVA).</p

<i>H</i>. <i>pylori</i> can respond to acidic and basic changes in pH with nanomolar sensitivity.

<p>(<b>A</b>) Quantification of the responses of wild-type (WT) <i>H</i>. <i>pylori</i> to phosphate buffer solutions with pH ranging from 4.2 to 7.1. The addition (+) or depletion (-) of hydrogen ions relative to the hydrogen ion concentration in the culture medium (pH 6.7) is indicated next to the pH of the buffer solution introduced. Each point represents the percent of swimming bacteria remaining in the field of view at each time point in the digitized video microscopy movie frames. Points for one movie are plotted per condition. Time zero is defined as the moment the needle is introduced and the gradient is initiated. (<b>B</b>) Still images of bacterial motility traces (lasting 5 seconds) of WT <i>H</i>. <i>pylori</i> before (panels in left column) and after injection of a phosphate buffer solution with pH 7.1, 6.5 or 6.6 (panels in right column). The pH of the culture medium was 6.7. The positions of the needle tips are marked in yellow.</p

<i>H</i>. <i>pylori</i> attraction to basic changes in pH is mediated through TlpD.

<p>Still images of bacterial motility traces (lasting 5 seconds) of wild-type <i>H</i>. <i>pylori</i> vs. Δ<i>tlpA</i> vs. Δ<i>tlpD</i> vs. Δ<i>tlpAD</i> before (panels in left column) and after injection of a phosphate buffer solution with pH 9.2 (panels in right column). The pH of the culture medium was 6.7. The positions of the needle tips are marked in yellow.</p

TlpA and TlpD are important in establishing colonization in acidic conditions in a murine model of gastric infection. Acid suppression rescues Δ<i>tlpAD</i> colonization defect.

<p>Recovered colony forming units (CFU) of <i>H</i>. <i>pylori</i> per gram of stomach tissue after a 2-week infection with wild-type (WT) <i>H</i>. <i>pylori</i>, Δ<i>tlpA H</i>. <i>pylori</i>, Δ<i>tlpD H</i>. <i>pylori</i>, or Δ<i>tlpAD H</i>. <i>pylori</i> (n = 10 mice for WT <i>H</i>. <i>pylori</i>, Δ<i>tlpAD H</i>. <i>pylori</i>, and Δ<i>tlpD H</i>. <i>pylori</i>; n = 7 for Δ<i>tlpA H</i>. <i>pylori</i>). For the experimental groups, omeprazole was administered in the drinking water 3 days prior to infection and throughout the course of the infection (purple), or for 1 week beginning 1 week after infection with <i>H</i>. <i>pylori</i> (blue). Blue dotted line indicates the limit of detection. Bars represent the geometric mean. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, **** <i>P</i> < 0.0001 (Mann Whitney test).</p

TlpD is more sensitive to a hydrochloric acid gradient than TlpA.

<p>Still images of bacterial motility traces (lasting 1.5 seconds) of wild-type <i>H</i>. <i>pylori</i> vs. Δ<i>tlpB</i> vs. Δ<i>tlpA</i> vs. Δ<i>tlpD</i> before (panels in left column) and after exposure to a gradient of either 25 mM (panels in middle column) or 50 mM hydrochloric acid (panels in right column). The positions of the needle tips are marked in yellow.</p

The dCache Chemoreceptor TlpA of Helicobacter pylori Binds Multiple Attractant and Antagonistic Ligands via Distinct Sites.

The Helicobacter pylori chemoreceptor TlpA plays a role in dampening host inflammation during chronic stomach colonization. TlpA has a periplasmic dCache_1 domain, a structure that is capable of sensing many ligands; however, the only characterized TlpA signals are arginine, bicarbonate, and acid. To increase our understanding of TlpA's sensing profile, we screened for diverse TlpA ligands using ligand binding arrays. TlpA bound seven ligands with affinities in the low- to middle-micromolar ranges. Three of these ligands, arginine, fumarate, and cysteine, were TlpA-dependent chemoattractants, while the others elicited no response. Molecular docking experiments, site-directed point mutants, and competition surface plasmon resonance binding assays suggested that TlpA binds ligands via both the membrane-distal and -proximal dCache_1 binding pockets. Surprisingly, one of the nonactive ligands, glucosamine, acted as a chemotaxis antagonist, preventing the chemotaxis response to chemoattractant ligands, and acted to block the binding of ligands irrespective of whether they bound the membrane-distal or -proximal dCache_1 subdomains. In total, these results suggest that TlpA senses multiple attractant ligands as well as antagonist ones, an emerging theme in chemotaxis systems. IMPORTANCE Numerous chemotactic bacterial pathogens depend on the ability to sense a diverse array of signals through chemoreceptors to achieve successful colonization and virulence within their host. The signals sensed by chemoreceptors, however, are not always fully understood. This is the case for TlpA, a dCache_1 chemoreceptor of H. pylori that enables the bacterium to induce less inflammation during chronic infections. H. pylori causes a significant global disease burden, which is driven by the development of gastric inflammation. Accordingly, it is essential to understand the processes by which H. pylori modulates host inflammation. This work uncovers the signals that TlpA can sense and highlights the underappreciated ability to regulate chemotactic responses by antagonistic chemoreceptor ligands, which is an emerging theme among other chemotactic systems