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

A Novel Mouse Model of <i>Campylobacter jejuni</i> Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection

<div><p><i>Campylobacter jejuni</i> is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how <i>C. jejuni</i> colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, <i>C. jejuni</i> typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal <i>C. jejuni</i> colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (<i>Sigirr^−/−</i>), a negative regulator of MyD88-dependent signaling led to heavy and widespread <i>C. jejuni</i> colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. <i>C. jejuni</i> heavily colonized the cecal and colonic crypts of <i>Sigirr^−/−</i> mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established <i>C. jejuni</i> pathogenicity factors, capsular polysaccharides (<i>kpsM</i>) and motility/flagella (<i>flaA</i>). We also explored the basis for the inflammatory response elicited by <i>C. jejuni</i> in <i>Sigirr^−/−</i> mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by <i>C. jejuni</i>. Despite heavy colonization, <i>Tlr4^−/−/Sigirr^−/−</i> mice were largely unresponsive to infection by <i>C. jejuni</i>, whereas <i>Tlr2^−/−/Sigirr^−/−</i> mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the <i>C. jejuni</i> capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of <i>Sigirr^−/−</i> mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to <i>C. jejuni</i>.</p></div

Colonization of WT and <i>Sigirr−/−</i> mice by <i>C. jejuni</i> 81–176, 3 and 7 DPI.

<p>(A) High numbers (∼10⁹ CFUs/g) of <i>C. jejuni</i> were recovered at both 3 and 7 DPI from the ceca of infected mice that were pre-treated with 5 mg of vancomycin. No statistically significant differences in numbers were found between WT and <i>Sigirr^−/−</i> mice as indicated by a t-test, p>0.05. n = 10 or 11 WT mice, and 12 or 13 <i>Sigirr^−/−</i> mice for 3 and 7 DPI respectively. (B) H&E stained, formalin-fixed histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice 3 and 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment for a total of 6–9 mice per group. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. WT (B6) mice did not exhibit any significant signs of inflammation, while <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice showed a significant increase relative to the uninfected <i>Sigirr^−/−</i> control, both 3 and 7 DPI. <i>Tlr4^−/−/Sigirr^−/−</i> mice showed a statistically significant increase, relative to control mice at 3 DPI only, but even at 3 DPI, were significantly less than either <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test (NS p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Immunofluorescence staining of <i>C. jejuni</i> cecal colonization <i>in vivo</i>.

<p>Formalin-fixed tissue sections of ceca obtained from <i>C. jejuni</i>-infected WT and <i>Sigirr^−/−</i> mice 7 DPI at ×200 magnification. Cell nuclei are stained with DAPI (blue), epithelial cells are outlined with antibodies specific to β-actin (green), and <i>C. jejuni</i> (red) are clearly visible around the edge of the lumen and into the cecal crypts.</p

TLR2 and 4 reporter assays.

<p>HEK-Blue hTLR2 (A) and HEK-Blue hTLR4 (B) reporter cell lines were exposed for 4 hrs to either live and heat-killed wildtype <i>C. jejuni</i> 81–176, <i>ΔkpsM</i> or <i>ΔkpsM+kpsM</i>. The wild-type <i>C. jejuni</i> stimulates both TLR2 and TLR4 in a dose-dependent fashion. The <i>ΔkpsM</i> mutant significantly increased the signaling by both TLR2 and TLR4, as indicated by the assay, with the increase in stimulation also being in a dose-dependent manner, except for the TLR4 assay where the readers were near the maximum for both the 20 and 200 MOI readings. The complemented <i>ΔkpsM+kpsM</i> strain completely restored the wild-type phenotype with TLR2 and mostly restored the phenotype with TLR4. Values represent the mean of three independent experiments and statistical significance was determined by a two-way ANOVA with a Bonferroni post-test. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Colonization and pathology of <i>Sigirr^−/−</i> and TLR-deficient mice by <i>C. jejuni ΔkpsM in vivo</i>.

<p>(A) H&E stained histological sections of ceca recovered from <i>Sigirr^−/−</i>, <i>Tlr2^−/−/Sigirr^−/−</i> and <i>Tlr4^−/−/Sigirr^−/−</i> mice, colonized with <i>C. jejuni ΔkpsM</i> 7 DPI, at 100× magnification. Very severe inflammation is evident in the <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice, however once again, no significant pathology was evident in the <i>Tlr4^−/−/Sigirr^−/−</i> mice. (B) Immunofluorescence of <i>Sigirr^−/−</i> mice infected by <i>C. jejuni ΔkpsM</i>, 7 DPI. Sections are stained for DAPI (blue), β-actin (green), and <i>C. jejuni</i> (red). <i>Sigirr^−/−</i> mice exhibit significant neutrophil infiltration, hyperplasia, and <i>C. jejuni ΔkpsM</i> is clearly visible in large masses within the cecal crypts. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. Only <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice at 7 DPI showed a significant increase in pathology (****p<0.0001), relative to the uninfected <i>Sigirr^−/−</i> control. The <i>Tlr2^−/−/Sigirr^−/−</i> mice also exhibited statistically significantly higher inflammation at 7 DPI relative to <i>Sigirr^−/−</i> mice also at 7 DPI (**p<0.001). In contrast, none of the mouse strains at 3 DPI showed any statistically significant increase in pathology, relative to control mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test.</p

Immunofluorescent staining of intracellular <i>C. jejuni in vivo</i>.

<p>(A) Intracellular <i>C. jejuni</i> are visible in the cecal epithelium. <i>C. jejuni</i> (red), are visible against the β-actin (green) and the nuclei (DAPI, blue) of the cecal epithelium of a <i>Sigirr^−/−</i> mouse, ×1000 magnification. (B) Confocal image of <i>C. jejuni</i> (red) present within epithelial cells of the colon of a <i>Sigirr^−/−</i> mouse, highlighted against the Cytokeratin 19 of the cytoskeleton (green) and the nuclei (blue), with the z-stack cross-section indicating the <i>C. jejuni</i> within the cell. (C) Cross-section of a Z-stack, of a colonic epithelial cell of a <i>Sigirr^−/−</i> mouse. The internalized <i>C. jejuni</i> (red) are clearly visible within the cytoplasm of the cell, as outlined by the β-actin (green) along the edge of the cell. (D) Internalized <i>C. jejuni</i> (red) co-localize with LAMP-1 positive (green) vesicles present within epithelial cells of a <i>Sigirr^−/−</i> mouse colon.</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>ΔkpsM</i> and <i>ΔflaA</i>.

<p>Colonization of WT and <i>Sigirr^−/−</i> mice by <i>ΔkpsM</i> (A) and <i>ΔflaA</i> (B) and their respective complemented strains (<i>ΔkpsM</i>+<i>kpsM</i> and <i>ΔflaA</i>+<i>flaA</i>), at both 3 and 7 DPI. The <i>ΔkpsM</i> mutant exhibited reduced colonization at 3 DPI only, while the <i>ΔflaA</i> mutant was unable to colonize at either 3 or 7 DPI. The complemented <i>ΔflaA+flaA</i> colonized at high numbers, similar to wild-type. Statistical significance was determined by a Mann-Whitney test, ***p<0.001. n = 7–10 mice for the <i>ΔkpsM</i> mutant and complement, n = 5–7 mice for the <i>ΔflaA</i> mutant and complement. (C) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i> at ×100 magnification. No noticeable inflammation was evident in either WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i>. (D) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔkpsM</i> 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. WT mice did not exhibit signs of inflammation when infected with <i>C. jejuni ΔkpsM</i>, however <i>Sigirr^−/−</i> mice exhibited signs of severe inflammation at 7 DPI.</p

A Bacterial Cell Shape-Determining Inhibitor

<i>Helicobacter pylori</i> and <i>Campylobacter jejuni</i> are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The <i>H. pylori</i> metalloprotease Csd4 and its <i>C. jejuni</i> homologue Pgp1 cleave the amide bond between <i>meso</i>-diaminopimelate and <i>iso</i>-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either <i>csd4</i> or <i>pgp1</i> results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-<i>iso</i>-d-Glu-<i>meso</i>-Dap, with a <i>K</i>_i value of 1.5 μM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the “promoted-water pathway” mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with <i>H. pylori</i> at millimolar concentrations. A diminished, yet observable, effect on the morphology of <i>C. jejuni</i> was also apparent. Cell straightening was more pronounced with an acapsular <i>C. jejuni</i> mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials against these human pathogens

Cytokine production in infected mice.

<p>(A–H) RT-qPCR conducted on RNA extracted from the ceca of uninfected control or infected mice. Controls are the pooled results of 9, vancomycin pre-treated, but uninfected mice, euthanized 3 days post-treatment. All infected mice represent the average results of 3 independent experiments, each of which include the pooled RNA of 2–3 mice, for 6–9 mice total for each mouse strain, euthanized either 3 or 7 DPI. Statistical significance was determined using a One way ANOVA with a Bonferroni post-test. * p<0.05 relative to WT (B6) or <i>Sigirr^−/−</i> uninfected control mice. ** p<0.05 relative to the infected WT (B6) mice euthanized on the same DPI in addition to the uninfected control mice.</p