Distinct pathologies of <i>H. pylori</i>–induced disease.

<p>(A) Duodenal ulcer disease correlates with high inflammation in the antrum (red bursts), lower levels of inflammation in the corpus, and high acid secretion (+). (B) Gastric ulcer or adenocarcinoma correlates with increased inflammation in the corpus, low acid secretion, and multifocal atrophy (wavy lines).</p

Mechanisms that create genetic diversity in <i>H. pylori</i>.

<p>Colored arrows represent different genes, and the correspondingly colored triangles, rectangles, and circles represent the proteins encoded by these genes. Diversification mechanisms (right side of figure) include spontaneous point mutations, slipped-strand mispairing, and intragenomic recombination. Allelic changes involving nonsynonymous point mutations and mosaic genes resulting from intragenomic recombination can alter the function and/or the antigenic epitopes of the encoded protein. Gene expression can also be regulated by gene conversion resulting from intragenomic recombination, and phase variation mediated by slipped-strand mispairing. Reassortment of genes (left side of figure) by natural transformation with exogenous DNA also contributes to genetic diversity. Natural transformation with DNA from a superinfecting strain, for example, can introduce new genes and new alleles of already present genes (horizontal gene transfer). Similarly, natural transformation with DNA from a variant clone of the same strain can further propagate an advantageous allele acquired by within-genome diversification.</p

Wzk can substitute for MurJ in <i>E</i>. <i>coli</i>.

<p>(A) Summary of complementation experiments. The loss of <i>murJ</i> in <i>E</i>. <i>coli</i> can only be accomplished with a plasmid encoding wild-type <i>wzk</i>. The <i>wzkE525A</i> allele, which carries a mutation in the Walker B motif that is required for ATPase activity, cannot complement a Δ<i>murJ</i> allele. (B) Growth of <i>E</i>. <i>coli</i> strain NR3647 [MG1655 Δ<i>lacIZYA</i>::<i>FRT</i> Δ<i>murJ</i>::<i>kan</i> (pIH23)] in LB broth supplemented with 40 μM IPTG as determined by OD₆₀₀. The data represents the average and standard deviation of six independent cultures. (C) <i>E</i>. <i>coli</i> strain NR2920 (MG1655 Δ<i>lacIYZA</i>::<i>FRT</i> Δ<i>murJ</i>::<i>kan</i> (pRC7KanMurJ, pIH23) expressing both <i>murJ</i> and <i>wzk</i> exhibits the rod-shape cellular morphology typical of wild-type <i>E</i>. <i>coli</i> cells (MurJ⁺ Wzk⁺ panel) under a 100X phase-contrast objective. Δ<i>murJ</i> cells complemented with <i>wzk</i> (Wzk⁺ panels) exhibit morphology characteristic of cells with PG defects: larger size, aberrant morphology, and lysis (marked with white arrow heads).</p

Generation of enzymatically inactive Wzk variant.

<p>(A) Expression of different <i>H</i>. <i>pylori</i> Wzk variants was induced by IPTG in <i>E</i>. <i>coli</i> DH5α for 4 h. Cell lysates from normalized cultures were separated on SDS-PAGE, followed by immunoblotting to detect the expression levels of Wzk mutants relative to wild type enzyme. Among the different Wzk variants, WzkE525A displayed expression levels comparable to that of wild type. (B) WzkE525A flippase activity is diminished <i>in vivo</i>. Both AcrA from <i>C</i>. <i>jejuni</i> and the accessory gene cluster required for its <i>N</i>-glycosylation were reconstituted in <i>E</i>. <i>coli</i>, with the exception of the native flippase. Flippase activity of Wzk and its E525A variant was tested by monitoring the glycosylation levels of <i>C</i>. <i>jejuni</i> AcrA via immunoblotting. Monoclonal anti-histidine was used to detect the expression of histidine-tagged AcrA (green), while the <i>C</i>. <i>jejuni</i> glycan was detected by the rabbit polyclonal anti-<i>C</i>. <i>jejuni</i> glycan (red). The glycosylated form of AcrA (G) is marked by the colocalization of both signals (yellow). Unglycosylated form of AcrA is marked U.</p

MurJ and Wzk translocate Und-PP linked saccharides across the cytoplasmic membrane.

<p>Nucleotide-linked sugars (not shown) are used to build the PG precursor lipid II and the O-antigen precursor Und-PP-O-antigen on the membrane-bound lipid carrier Und-P. The first Und-P glycosyltransferase in each respective pathway are MraY (not shown) and WecA. After each Und-PP-linked intermediate is synthesized in the inner leaflet of the cytoplasmic membrane, flippases MurJ and Wzk translocate them across the bilayer. At the periplasmic leaflet of the cytoplasmic membrane, the disaccharide-pentapeptide component of lipid II is used to build glycan chains that are crosslinked into the preexisting PG matrix, while the O polysaccharide portion of Und-PP-O-antigen is transferred onto LPS molecules by the WaaL ligase (not shown). After these steps, the lipid carrier is recycled (green dotted arrows). The O polysaccharide is composed of <i>N</i>-acetyl glucosamine (GlcNAc, grey hexagon labeled G), galactose (green squares), and fucose (orange triangles); the disaccharide in lipid II is composed of <i>N</i>-acetyl muramic acid (MurNAc, black hexagon labeled M) and GlcNAc (grey hexagon labeled G), while the pentapeptide is composed of L-Ala (light blue circle), D-Glu (green circle), <i>meso</i>-2,6-diaminopimelic acid (<i>meso</i>-A₂pm, red circle) and two D-Ala (dark blue circle) residues.</p

Current understanding of muropeptide modification in <i>H. pylori</i>.

<p>This schematic shows peptide modification activities that can generate the muropeptides observed in the <i>H. pylori</i> sacculus. Known <i>H. pylori</i> proteins demonstrated (Csd3, Csd4) or predicted (Csd1, Csd2) to perform these activities are indicated. CPase, carboxypeptidase; EPase, endopeptidase.</p

<i>H. pylori</i> cell shape mutant morphologies and associated loci identified in a visual screen.

<p>The transposon insertion site and orientation (indicated by the spelling of the transposon's selectable marker, chloramphenicol acetyltransferase (<i>cat</i>)), is shown for each straight rod shape mutant identified in the screen. A) HPG27_353 (<i>csd4</i>) shape locus. B–E) Phase contrast (B, D) and transmission electron microscopy (TEM) (C, E) images of wild-type (B–C) and <i>csd4</i> mutant cells (D–E). F) HPG27_1195 (<i>csd5</i>) shape locus. G–H) Phase contrast (G) and TEM (H) images of <i>csd5</i> mutant cells. Strains used: NSH57, LSH18, LSH31, LSH36.</p

Assessment of the straight rod <i>H. pylori</i>'s colonization and pro-inflammatory potential.

<p>A) One week C57BL/6 mouse competition data compiled from three independent experiments. Data are plotted as a competitive index: [CFU/mL_MUT∶CFU/mL_{WT/Complement} stomach output]/[CFU/mL_MUT∶CFU/mL_{WT/Complement} inoculum] with each data point representing a single mouse. Black points indicate mice from which only one strain was recovered. Strains used: LSH100, LSH122, LSH124. B–D) Survival at low pH (B), in the presence of polymyxin B (C), or in high salt (D). Data comprise two independent experiments of four replicates per strain and condition (mean ± SD). Strains used: NSH57, LSH18. E) IL-8 production during infection of AGS gastric epithelial cells. Culture supernatants of triplicate wells were assayed for IL-8 using a commercial ELISA assay after infection at a multiplicity of infection of 10 (mean ± SD). Shown are data from one of three independent experiments with similar results. Strains used: NSH57, LSH13, LSH18.</p

Functional analyses of Csd4 enzymatic activity and its role in shape determination.

<p>A) SDS-PAGE depicting steps in the purification of His-tagged <i>H. pylori</i> Csd4 protein from <i>E. coli</i> cells. Induced protein was purified using a Ni-NTA agarose column as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat.1002603.s001" target="_blank">Text S1</a>. WC, whole cell lysate; CL, cleared lysate; MW, molecular weight; FT, flow through. Positions of the 20 kDa and 50 kDa molecular weight markers are indicated. B) HPLC analysis of muropeptides released from purified <i>csd4</i> mutant (LSH122) PG treated with purified His-tagged Csd4 protein in the presence of Zn²⁺ or EDTA, or without protein. In the presence of Zn²⁺ but not EDTA, Csd4 trimmed the monomeric tripeptides to dipeptides, indicative of the protein having DL-carboxypeptidase activity. C–D) Muropeptides detected before and after incubation of Csd4 with purified disaccharide tripeptide (C) and disaccharide tetrapeptide substrates (D). Data indicate Csd4 cleaves tripeptide, but not tetrapeptide. E–F) Scatter plot arraying the wild-type, <i>csd4</i> deletion, <i>csd4</i> point, and <i>csd5</i> deletion mutant populations by length (x-axis, µm) and cell curvature (y-axis, arbitrary units). Each contour depicts the morphology of a single cell captured from a 1000× phase contrast image using CellTool software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat.1002603-Sycuro1" target="_blank">[13]</a>. The software algorithmically determines each cell's length along its two-dimensional central axis as well as the degree of cell body curvature (excluding the poles). 200–300 cells were analyzed for each strain. E) Smooth histograms displaying kernel density estimates of each strain's cell curvature (x-axis). Bootstrapped Kolmogorov–Smirnov statistical comparisons of population cell curvature distributions yielded p-values<0.001 for all pairwise comparisons with the exception of <i>csd4</i> vs. <i>csd4E222A</i>, p = 0.19. Strains used: NSH57, LSH18, LSH31, LSH146.</p

Motility of <i>H. pylori</i> cell shape mutants in soft agar and viscous polymer solutions.

<p>A, D) Motility phenotype of indicated strains in soft agar (mean halo diameter ± SD in 0.3% soft agar after four days). Data shown are from one experiment of 17–22 stabs/strain and are consistent with the findings from replicate experiments. Contours representative of each strain's average cell shape (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat-1002603-g003" target="_blank">Figure 3</a> legend) are shown below panel D and are superimposed on a grid to highlight the slight differences in cell curvature that correlate with motility. p-values were generated using one-way ANOVA with the Bonferroni correction for multiple comparisons. B–C) Velocity of wild-type and the <i>csd4</i> mutant in broth containing porcine mucus (B) and methylcellulose (C). Data shown are the mean ± SD from measurements of 9–30 cells/strain/condition. No statistically significant differences between wild-type and the <i>csd4</i> mutant were observed in any condition (p>0.2, Student's t-test with equal variances). Strains used: A) LSH100, LSH122, LSH123; B–C) NSH57, LSH18; D) LSH100, LSH134, NSH152a, LSH146, NSH153a, NSH160a.</p