pH-regulated gene expression of the gastric pathogen Helicobacter pylori

Abstract

Colonization by the gastric pathogen Helicobacter pylori has been shown to be intricately linked to the development of gastritis, ulcers, and gastric malignancy. Little is known about mechanisms employed by the bacterium that help it adapt to the hostile environment of the human stomach. In an effort to extend our knowledge of these mechanisms, we utilized spotted-DNA microarrays to characterize the response of H. pylori to low pH. Expression of approximately 7% of the bacterial genome was reproducibly altered by shift to low pH. Analysis of the differentially expressed genes led to the discovery that acid exposure leads to profound changes in motility of H. pylori, as a larger percentage of acid-exposed bacterial cells displayed motility and moved at significantly higher speeds. In contrast to previous publications, we found that expression of the bacterial virulence gene cagA was strongly repressed by acid exposure. Furthermore, this transcriptional repression was reflected at the level of protein accumulation in the H. pylori cell.

FIG. 1.

Microarray analysis of the low-pH response of H. pylori. Cluster diagram showing the expression profile of the 118 genes meeting the filter criteria as explained in Materials and Methods after shift to pH 5.0. Two independent experiments are depicted, and relative expression patterns are shown for each time point. Red indicates an increase in expression, while green indicates reduced expression. Representative genes are listed and their relative location indicated by an arrow. A complete list of the regulated factors and the relative changes in expression can be found in Table 1 and as supplementary information at http://falkow.stanford.edu/whatwedo/supplementarydata/.

FIG. 2.

The effect of acidic pH on H. pylori motility. A culture of H. pylori was split such that equal portions were suspended at pH 7.0 or 5.0. The response to the different pH conditions was monitored by video microscopy, and the speed of motile bacteria was determined as described in Materials and Methods. Each point represents one motile bacterium at the indicated pH, and the speed of that bacterium is indicated on the y axis. The average speed for each condition is indicated. The statistical significance of the differences in speed between the two sets of pH conditions was determined by using a two-tailed Mann-Whitney test, which revealed a P of 0.0002.

FIG. 3.

The effect of low pH on CagA accumulation. Total protein was harvested from G27 shifted to pH 5.0 brucella broth plus 10% FBS at the times indicated. An equal amount of protein from each point was separated on a 6% polyacrylamide gel, transferred to nitrocellulose membranes, and probed with an anti-CagA specific antibody. The relative change (n-fold) from the zero time point is indicated underneath each lane.

FIG. 4.

Growth curve analysis of wild-type B128 and its ansB derivative. Brucella broth cultures supplemented with 10% FBS were started from overnight cultures of each strain. Strains were grown in microaerophilic conditions with shaking as described in Materials and Methods, and samples were taken for OD₆₀₀ measurements at the indicated times.

FIG. 5.

ansB mutants of H. pylori are defective for in vivo competition. Equal concentrations of wild-type and ansB strains were mixed and were used to infect Mongolian gerbils. Colonizing bacteria were recovered after approximately 2.5 to 3 weeks, and the number of CFU of each strain was determined by differential plating. Each circle represents an infected animal, and open circles represent animals for which no ansB derivatives were isolated. Data for two independent experiments are depicted, and the geometric means for each (0.004 and 0.005) are indicated by a black bar.