Data_Sheet_1_Multiple Campylobacter jejuni proteins affecting the peptidoglycan structure and the degree of helical cell curvature.PDF

Campylobacter jejuni is a Gram-negative helical bacterium. Its helical morphology, maintained by the peptidoglycan (PG) layer, plays a key role in its transmission in the environment, colonization, and pathogenic properties. The previously characterized PG hydrolases Pgp1 and Pgp2 are important for generating C. jejuni helical morphology, with deletion mutants being rod-shaped and showing alterations in their PG muropeptide profiles in comparison to the wild type. Homology searches and bioinformatics were used to identify additional gene products involved in C. jejuni morphogenesis: the putative bactofilin 1104 and the M23 peptidase domain-containing proteins 0166, 1105, and 1228. Deletions in the corresponding genes resulted in varying curved rod morphologies with changes in their PG muropeptide profiles. All changes in the mutants complemented except 1104. Overexpression of 1104 and 1105 also resulted in changes in the morphology and in the muropeptide profiles, suggesting that the dose of these two gene products influences these characteristics. The related helical ε-Proteobacterium Helicobacter pylori has characterized homologs of C. jejuni 1104, 1105, and 1228 proteins, yet deletion of the homologous genes in H. pylori had differing effects on H. pylori PG muropeptide profiles and/or morphology compared to the C. jejuni deletion mutants. It is therefore apparent that even related organisms with similar morphologies and homologous proteins can have diverse PG biosynthetic pathways, highlighting the importance of studying PG biosynthesis in related organisms.</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

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

Summary of muropeptide composition of PG in mutant strains.

a<p>Percentages calculated as per <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat.1002603-Glauner1" target="_blank">[54]</a>. Underlined values differ by more than 2 standard deviations from that of wild-type; underlined and bold values differ by more than 5 standard deviations from that of wild-type.</p>b<p>Calculated from 6 independent samples.</p>c<p>As previously reported <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat.1002603-Sycuro1" target="_blank">[13]</a>.</p>d<p>Shape of each strain: h-helical rod, c-curved rod, s-straight rod, v-variable (“c” shape, curved rod, straight rod).</p

Summary of muropeptide composition of <i>C. jejuni</i> wild-type 81-176, Δ<i>pgp1</i> mutant, Δ<i>pgp1</i> complement (Δ<i>pgp1</i>c), and <i>pgp1</i> overexpression (81-176+<i>pgp1</i>) strains, and the resultant Δ<i>pgp1</i> PG profiles of Pgp1 activity assays consisting of Δ<i>pgp1</i> PG incubated without enzyme, with Pgp1 in the presence of ZnCl₂, and with Pgp1 without ZnCl₂ but with EDTA.

1<p>Numbers represent the percent area of each muropeptide from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002602#ppat.1002602.s006" target="_blank">Table S4</a> calculated to give a total of 100%. Values indicated with an asterisk (*) represent an equal to or greater than 20% difference in comparison to wild-type 81-176 or Δ<i>pgp1</i> PG to which no enzyme was added; bolded asterisked values (<b>*</b>) indicate a greater than 30% change.</p>2<p>Values for the percentage of O-acetylated species do not represent the true level of PG O-acetylation in these strains, as most O-acetyl groups are lost in the standard alkaline muropeptide reduction procedure used in this study.</p

Pgp1 has metal-dependent DL-carboxypeptidase activity on Δ<i>pgp1</i> PG, cleaving monomeric tripeptide disaccharides to dipeptides.

<p><b>A,</b> SDS-PAGE analysis of affinity purified Pgp1 with a predicted molecular weight of 67.9 kDa, indicated by an arrow. HPLC chromatograms of <b>B,</b> purified Δ<i>pgp1</i> PG; <b>C,</b> Δ<i>pgp1</i> PG incubated with purified Pgp1 and ZnCl₂; and <b>D,</b> Δ<i>pgp1</i> PG with purified Pgp1 and EDTA. Peaks corresponding to monomeric disaccharide dipeptides and tripeptides are indicated. <b>E,</b> a schematic diagram of the Pgp1 cleavage site indicated with an arrow. G, N-acetylglucosamine; M, reduced N-acetylmuramic acid; L-Ala, L-alanine; D-iGlu, D-isoglutamic acid; <i>meso-</i>DAP, <i>meso-</i>diaminopimelic acid.</p

The role of cell shape and PG composition on host related phenotypes.

<p><b>A,</b> Δ<i>pgp1</i> is defective for chick colonization. Each point represents the log CFU/g cecal contents of an individual chick 6 days following infection with 10⁴ CFUs of the indicated <i>C. jejuni</i> strains. The geometric mean is denoted by a black bar. <b>B–D,</b> to assay the ability of <i>C. jejuni</i> wild type and Δ<i>pgp1</i> PG to activate Nod proteins, human embryonic kidney cells (HEK293T) were co-transfected with either <i>C. jejuni</i> 81-176 or Δ<i>pgp1</i> PG at 0.1 µg/mL, vectors for a NF-κB luciferase reporter, and either human Nod1 (<b>B,</b> hNod1), mouse Nod1 (<b>C,</b> mNod1) or human Nod2 (<b>D,</b> hNod2). Nod activation was determined by measuring the activity of the NF-κB luciferase reporter in comparison to the non-stimulated (NS) negative control. Positive controls used were TriDAP, FK565, and MDP. Data represent the mean ± SEM of three independent experiments. <b>E,</b> Deletion of <i>pgp1</i> increases IL-8 secretion in the INT407 epithelial cell line. Quantification of IL-8 levels was performed by ELISA. Data represent the mean ± SEM of three independent experiments. The asterisk (*) indicates a statistically significant difference using the unpaired Student's t-test, with * or *** indicating <i>p</i><0.05 or <i>p</i><0.001, respectively.</p