Phase variable expression of capsular polysaccharide modifications allows <em>Campylobacter jejuni</em> to avoid bacteriophage infection in chickens

Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside. The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage–host co-evolution

From nanoliter to large-scale bioreactors: How integrated technologies bring antibody treatments to patients faster

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The inflammatory caspases coordinate mucosal restriction of Salmonella enterica serovar Typhimurium

We investigated the role of the inflammatory caspases during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Streptomycin-pretreated wildtype C57BL/6, Casp1/11 deficient (−/−), Casp1−/− and Casp11−/− mice were orally infected and S. Typhimurium burdens determined at 18h-7d post infection (p.i.). Increased cecal and luminal pathogen burdens were observed for all caspase-deficient mice as compared to wildtype, which correlated with increased intracellular S. Typhimurium loads in the crypt IECs. Interestingly, cecal pathology scores for all inflammatory caspase mice were decreased compared to wildtype mice, especially with regard to ‘epithelial integrity’ and ‘goblet cell loss’. To determine if the increased intracellular pathogen burdens were due to the loss of IEC-intrinsic inflammasomes, cell lines and enteroid monolayers derived from each genotype and infected. These studies revealed significantly increased intracellular burdens in caspase-deficient monolayers in concert with a marked decrease in IEC sloughing and cell death. In human epithelial monolayers, siRNA-depletion of caspase-4, a human ortholog of caspase-11, led to increased bacterial colonization as well as increased secretion of the proinflammatory cytokine, interleukin (IL)-18. Inflammatory caspase activity was measured in enteroid monolayers and peak activity in wildtype cells correlated with shedding, suggesting IEC-intrinsic inflammasome-based restriction of S. Typhimurium occurs through infected IEC expulsion. To examine the effect of inflammasome signaling on overall mucosal defense, mucus layer thickness was evaluated by immunofluorescence staining. At 18h p.i., wildtype tissues demonstrated a dramatic increase in mucus thickness while only a marginal increase was observed in caspase deficient mice. Also, expression of the antimicrobial lectins Reg3γ and β were attenuated in all caspase-deficient mice. Mucin release and Reg3γ and β induction has been previously linked to the cytokine IL-22. We detected higher IL-22 levels in infected wildtype mice and when IL-22 was neutralized, wildtype mice carried increased S. Typhimurium burdens and decreased infection-induced mucin secretion and Reg3γ and β induction. No differences were observed in Casp1/11−/− mice treated with neutralizing antibody or isotype control. These results thus indicate that the intestinal mucosa utilizes inflammasome signaling to coordinate multiple layers of innate defense at the gut surface to ultimately restrict enteric pathogen infections and systemic spread.Science, Faculty ofGraduat

Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes.

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella

Highly Sensitive, Flow Cytometry-Based Measurement of Intestinal Permeability in Models of Experimental ColitisSummary

Background &amp; Aims: Increased intestinal permeability is seen in a variety of inflammatory conditions such as enteric infections and inflammatory bowel disease. Because barrier function can provide a key biomarker of disease severity, it often is assayed in animal models. A common methodology involves gavaging mice with fluorescein isothiocyanate–conjugated dextran (FITC-D), followed by cardiac puncture to assay plasma fluorescence on a spectrophotometer. Although the FITC-D method is relatively simple, its sensitivity is limited and enables only a single measurement because the test requires killing the subject. Herein, we describe a novel flow cytometry–based method of intestinal permeability measurement based on detection of orally gavaged ovalbumin (OVA) that leaks out of the gut. Our approach uses minute blood volumes collected from the tail vein, permitting repeated testing of the same subject at multiple time points. By comparing this assay against the gold standard FITC-D method, we show the expanded utility of our OVA assay in measuring intestinal permeability. Methods: We directly compared our OVA assay against the FITC-D assay by co-administering both probes orally to the same animals and subsequently using their respective methodologies to measure intestinal permeability by detecting probe levels in the plasma. Permeability was assessed in mice genetically deficient in intestinal mucus production or glycosylation. In addition, wild-type mice undergoing dextran sodium sulfate–induced colitis or infected by the enteric bacterial pathogen Citrobacter rodentium also were tested. Results: The OVA assay showed very high efficacy in all animal models of intestinal barrier dysfunction tested. Besides identifying intestinal barrier dysfunction in mice with impaired mucin glycosylation, the assay also allowed for repeated tracking of intestinal permeability within the same animal over time, providing data that cannot be easily acquired with other currently applied methods. Conclusions: The OVA assay is a highly sensitive and effective method of measuring intestinal permeability in mouse models of barrier dysfunction and experimental colitis

Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens

Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11-deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human ortholog of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium-infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore, an epithelial cell-intrinsic noncanonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface

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

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

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

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