A novel DNA-binding protein plays an important role in Helicobacter pylori stress tolerance and survival in the host

Abstract

The gastric pathogen Helicobacter pylori must combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus of hp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A Δhp119 strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version of hp119 at a separate chromosomal locus. The mutant strain was approximately 10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all contained H. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the Δhp119 mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the Δhp119 mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role in Helicobacter pylori stress tolerance and survival in the host.

FIG 1

DNA binding activity of HP119. Plasmid pGEMT DNA (6 nM, 3 kb) was incubated with various amounts (60, 600, and 6,000 nM) of purified HP0119 protein. As a negative control, pGEMT DNA was incubated with 600 or 6,000 nM BSA protein. Different forms of plasmid DNA (OC, open circular; L, linear; SC, supercoiled) are labeled on the left. The protein-DNA complex was visualized by ethidium bromide staining after agarose gel (1%) electrophoresis.

FIG 2

Survival of H. pylori cells upon exposure to air. H. pylori cell suspensions in PBS were incubated at 37°C under normal atmospheric conditions (21% partial-pressure O₂). Samples were removed at the times indicated on the x axis and were used for plate count determinations after incubation in a 5% oxygen environment. The data are the means from three experiments, with standard deviations as indicated. Symbols: square, wild type; triangle, hp119::cat; circle, hp119 complementation strain. Based on statistical analysis (Student t test), the cell survival differences between the WT and the mutant strains are significant (P < 0.01) for all the data points except for the 2-h time point.

FIG 3

Mouse colonization results of H. pylori strains. The mice were inoculated with H. pylori at a dose of 1.5 × 10⁸ viable cells per animal. Colonization of H. pylori in mouse stomachs was examined 3 weeks after the inoculation. Data are presented as a scatter plot (log scale) of CFU per gram of stomach as determined by plate counts. Each point represents the CFU count from one mouse stomach, and the horizontal bars represent the geometric means of the colonization numbers for each group. The detection limit of the assay is 500 CFU/g stomach, corresponding to a log₁₀ (CFU/g) of 2.7.

FIG 4

Comparative 2D PAGE protein profiles of H. pylori WT and Δhp119 mutant strains grown under oxygen stress. Proteins of the WT strain were labeled with Cy3 (green), and proteins from Δhp119 mutant strain were labeled with Cy5 (red) (see Materials and Methods).