Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

ABSTRACT Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins. The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins.

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A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01210-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6064-6072 ◽

Cited By ~ 21

Author(s):

Rémi Porte ◽

Delphine Fougeron ◽

Natalia Muñoz-Wolf ◽

Julien Tabareau ◽

Anne-France Georgel ◽

...

Keyword(s):

Influenza Virus ◽

Combination Treatment ◽

Intranasal Administration ◽

Pathogenic Bacteria ◽

Bacterial Load ◽

Pneumococcal Infection ◽

Toll Like Receptor ◽

Content Type ◽

Toll Like Receptor 5 ◽

Stimulation Of

ABSTRACTProphylactic intranasal administration of the Toll-like receptor 5 (TLR5) agonist flagellin protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might improve the therapeutic index of antibiotics for the treatment ofStreptococcus pneumoniaerespiratory infections in mice. Intranasal administration of flagellin was combined with either oral administration of amoxicillin or intraperitoneal injection of trimethoprim-sulfamethoxazole to treatS. pneumoniae-infected animals. Compared with standalone treatments, the combination of antibiotic and flagellin resulted in a lower bacterial load in the lungs and greater protection againstS. pneumoniaedissemination and was associated with an early increase in neutrophil infiltration in the airways. The antibiotic-flagellin combination treatment was, however, not associated with any exacerbation of inflammation. Moreover, combination treatment was more efficacious than standalone antibiotic treatments in the context of post-influenza virus pneumococcal infection. Lastly, TLR5 signaling was shown to be mandatory for the efficacy of the combined antibacterial therapy. This report is the first to show that combining antibiotic treatment with the stimulation of mucosal innate immunity is a potent antibacterial strategy against pneumonia.

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Transcriptional and Pathological Host Responses to Coinfection with Virulent or Attenuated Mycoplasma gallisepticum and Low-Pathogenic Avian Influenza A Virus in Chickens

Infection and Immunity ◽

10.1128/iai.00607-19 ◽

2019 ◽

Vol 88 (1) ◽

Cited By ~ 5

Author(s):

J. A. Canter ◽

E. R. Tulman ◽

J. Beaudet ◽

D.-H. Lee ◽

M. May ◽

...

Keyword(s):

Avian Influenza ◽

Mycoplasma Gallisepticum ◽

Host Responses ◽

Ciliary Activity ◽

Toll Like Receptor ◽

Transcriptional Responses ◽

Content Type ◽

Pathogenic Avian Influenza ◽

Expression Of Genes ◽

Low Pathogenic Avian Influenza

ABSTRACT The avian pathogen Mycoplasma gallisepticum, the etiological agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a copathogen such as low-pathogenic avian influenza virus (LPAIV). To further investigate the intricacies of this copathogenesis, chickens were monoinfected or coinfected with either virulent M. gallisepticum strain Rlow or LPAIV H3N8 (A/duck/Ukraine/1963), with assessment of tracheal histopathology, pathogen load, and transcriptomic host responses to infection by RNA sequencing. Chickens coinfected with M. gallisepticum Rlow followed by LPAIV H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening than chickens infected with LPAIV H3N8 alone and greater viral loads than chickens infected first with H3N8 and subsequently with M. gallisepticum Rlow. Recovery of live M. gallisepticum was significantly higher in chickens infected first with LPAIV H3N8 and then with M. gallisepticum Rlow, compared to chickens given a mock infection followed by M. gallisepticum Rlow. The transcriptional responses to monoinfection and coinfection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as Toll-like receptor 15, Toll-like receptor 21, and matrix metallopeptidase 1. Pathway and gene ontology analyses of these differentially expressed genes suggest that coinfection with virulent M. gallisepticum and LPAIV induces decreases in the expression of genes related to ciliary activity in vivo and alters multiple immune-related signaling cascades. These data aid in the understanding of the relationship between M. gallisepticum and LPAIV during copathogenesis in the natural host and may contribute to further understanding of copathogen infections of humans and other animals.

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Campylobacter jejuni Disrupts Protective Toll-Like Receptor 9 Signaling in Colonic Epithelial Cells and Increases the Severity of Dextran Sulfate Sodium-Induced Colitis in Mice

Infection and Immunity ◽

10.1128/iai.06066-11 ◽

2012 ◽

Vol 80 (4) ◽

pp. 1563-1571 ◽

Cited By ~ 34

Author(s):

Jennifer R. O'Hara ◽

Troy D. Feener ◽

Carrie D. Fischer ◽

Andre G. Buret

Keyword(s):

Campylobacter Jejuni ◽

Dextran Sulfate ◽

Dextran Sulfate Sodium ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Toll Like Receptor ◽

Intestinal Epithelial Barrier ◽

Content Type ◽

Tlr9 Agonist ◽

Dss Colitis

ABSTRACTInflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with a dysregulated immune response to commensal bacteria in susceptible individuals. The relapse of IBD may occur following an infection withCampylobacter jejuni. Apical epithelial Toll-like receptor 9 (TLR9) activation by bacterial DNA is reported to maintain colonic homeostasis. We investigated whether a priorC. jejuniinfection disrupts epithelial TLR9 signaling and increases the severity of disease in a model of mild dextran sulfate sodium (DSS) colitis in mice. In a further attempt to identify mechanisms, T84 monolayers were treated withC. jejunifollowed by a TLR9 agonist. Transepithelial resistance (TER) and dextran flux across confluent monolayers were monitored. Immunohistochemistry, Western blotting, and flow cytometry were used to examine TLR9 expression. Mice colonized byC. jejunilacked any detectable pathology; however, in response to low levels of DSS, mice previously exposed toC. jejuniexhibited significantly reduced weight gain and increased occult blood and histological damage scores. Infected mice treated with DSS also demonstrated a significant reduction in levels of the anti-inflammatory cytokine interleukin-25.In vitrostudies indicated that apical application of a TLR9 agonist enhances intestinal epithelial barrier function and that this response is lost inC. jejuni-infected monolayers. Furthermore, infected cells secreted significantly more CXCL8 following the basolateral application of a TLR9 agonist. Surface TLR9 expression was reduced inC. jejuni-infected monolayers subsequently exposed to a TLR9 agonist. In conclusion, infection byC. jejunidisrupts TLR9-induced reinforcement of the intestinal epithelial barrier, and colonization byC. jejuniincreases the severity of mild DSS colitis.

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EptC of Campylobacter jejuni Mediates Phenotypes Involved in Host Interactions and Virulence

Infection and Immunity ◽

10.1128/iai.01046-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 430-440 ◽

Cited By ~ 40

Author(s):

Thomas W. Cullen ◽

John P. O'Brien ◽

David R. Hendrixson ◽

David K. Giles ◽

Rhonda I. Hobb ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Lipid A ◽

Inner Core ◽

Complex Surface ◽

Surface Structures ◽

Avian Host ◽

Toll Like Receptor ◽

Core Oligosaccharide ◽

Gene Encoding ◽

Content Type

ABSTRACTCampylobacter jejuniis a natural commensal of the avian intestinal tract. However, the bacterium is also the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Like many bacterial pathogens,C. jejuniassembles complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work inC. jejuniidentified a gene encoding a novel phosphoethanolamine (pEtN) transferase, EptC (Cj0256), that plays a promiscuous role in modifying the flagellar rod protein, FlgG; the lipid A domain of lipooligosaccharide (LOS); and severalN-linked glycans. In this work, we report that EptC catalyzes the addition of pEtN to the first heptose sugar of the inner core oligosaccharide of LOS, a fourth enzymatic target. We also examine the role pEtN modification plays in circumventing detection and/or killing by host defenses. Specifically, we show that modification ofC. jejunilipid A with pEtN results in increased recognition by the human Toll-like receptor 4–myeloid differentiation factor 2 (hTLR4-MD2) complex, along with providing resistance to relevant mammalian and avian antimicrobial peptides (i.e., defensins). We also confirm the inability of aberrant forms of LOS to activate Toll-like receptor 2 (TLR2). Most exciting, we demonstrate that strains lackingeptCshow decreased commensal colonization of chick ceca and reduced colonization of BALB/cByJ mice compared to wild-type strains. Our results indicate that modification of surface structures with pEtN by EptC is key to its ability to promote commensalism in an avian host and to survive in the mammalian gastrointestinal environment.

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Role of Cecal Microbiota in the Differential Resistance of Inbred Chicken Lines to Colonization by Campylobacter jejuni

Applied and Environmental Microbiology ◽

10.1128/aem.02607-19 ◽

2020 ◽

Vol 86 (7) ◽

Author(s):

Cosmin Chintoan-Uta ◽

Trong Wisedchanwet ◽

Laura Glendinning ◽

Abi Bremner ◽

Androniki Psifidi ◽

...

Keyword(s):

Gut Microbiota ◽

Microbial Communities ◽

Campylobacter Jejuni ◽

Bacterial Infections ◽

Differential Resistance ◽

Gut Bacteria ◽

Content Type ◽

Significant Difference ◽

Cecal Microbiota

ABSTRACT Campylobacteriosis is the leading foodborne bacterial diarrheal illness in many countries, with up to 80% of human cases attributed to the avian reservoir. The only control strategies currently available are stringent on-farm biosecurity and carcass treatments. Heritable differences in the resistance of chicken lines to Campylobacter colonization have been reported and resistance-associated quantitative trait loci are emerging, although their impact on colonization appears modest. Recent studies indicated a protective role of the microbiota against colonization by Campylobacter in chickens. Furthermore, in murine models, differences in resistance to bacterial infections can be partially transferred between lines by transplantation of gut microbiota. In this study, we investigated whether heritable differences in colonization of inbred chicken lines by Campylobacter jejuni are associated with differences in cecal microbiota. We performed homologous and heterologous cecal microbiota transplants between line 61 (resistant) and line N (susceptible) by orally administering cecal contents collected from 3-week-old donors to day-of-hatch chicks. Recipient birds were challenged (day 21) with C. jejuni 11168H. In birds given homologous microbiota, the differential resistance of lines to C. jejuni colonization was reproduced. Contrary to our hypothesis, transfer of cecal microbiota from line 61 to line N significantly increased C. jejuni colonization. No significant difference in the overall composition of the cecal microbial communities of the two lines was identified, although line-specific differences for specific operational taxonomic units were identified. Our data suggest that while heritable differences in avian resistance to Campylobacter colonization exist, these are not explained by significant variation in the cecal microbiota. IMPORTANCE Campylobacter is a leading cause of foodborne diarrheal disease worldwide. Poultry are a key source of human infections, but there are currently few effective measures against Campylobacter in poultry during production. One option to control Campylobacter may be to alter the composition of microbial communities in the avian intestines by introducing beneficial bacteria, which exclude the harmful ones. We previously described two inbred chicken lines which differ in resistance to intestinal colonization by Campylobacter. Here, we investigated the composition of the microbial communities in the gut of these lines and whether transferring gut bacteria between the resistant and susceptible lines alters their resistance to Campylobacter. No major differences in microbial populations were found, and resistance or susceptibility to colonization was not conferred by transferring gut bacteria between lines. The data suggest that gut microbiota did not play a role in resistance to Campylobacter colonization, at least in the lines used.

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