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The impact of bacterial members of the microbiota on the development of colorectal cancer (CRC) has become clear in recent years. However, exactly how bacteria contribute to the development of cancer is often still up for debate. The impact of bacteria-derived metabolites, which can influence the development of CRC either in a promoting or inhibiting manner, is undeniable. Here, we discuss the effects of the most well-studied bacteria-derived metabolites associated with CRC, including secondary bile acids, short-chain fatty acids, trimethylamine-N-oxide and indoles. We show that the effects of individual metabolites on CRC development are often nuanced and dose- and location-dependent. In the coming years, the array of metabolites involved in CRC development will undoubtedly increase further, which will emphasize the need to focus on causation and mechanisms and the clearly defined roles of bacterial species within the microbiota.

PurposeAlthough elective surgery is generally safe, some procedures remain associated with an increased risk of complications. Improved preoperative risk stratification and earlier recognition of these complications may ameliorate postoperative recovery and improve long-term outcomes. The perioperative longitudinal study of complications and long-term outcomes (PLUTO) cohort aims to establish a comprehensive biorepository that will facilitate research in this field. In this profile paper, we will discuss its design rationale and opportunities for future studies.ParticipantsPatients undergoing elective intermediate to high-risk non-cardiac surgery are eligible for enrolment. For the first seven postoperative days, participants are subjected to daily bedside visits by dedicated observers, who adjudicate clinical events and perform non-invasive physiological measurements (including handheld spirometry and single-channel electroencephalography). Blood samples and microbiome specimens are collected at preselected time points. Primary study outcomes are the postoperative occurrence of nosocomial infections, major adverse cardiac events, pulmonary complications, acute kidney injury and delirium/acute encephalopathy. Secondary outcomes include mortality and quality of life, as well as the long-term occurrence of psychopathology, cognitive dysfunction and chronic pain.Findings to dateEnrolment of the first participant occurred early 2020. During the inception phase of the project (first 2 years), 431 patients were eligible of whom 297 patients consented to participate (69%). Observed event rate was 42% overall, with the most frequent complication being infection.Future plansThe main purpose of the PLUTO biorepository is to provide a framework for research in the field of perioperative medicine and anaesthesiology, by storing high-quality clinical data and biomaterials for future studies. In addition, PLUTO aims to establish a logistical platform for conducting embedded clinical trials.Trial registration numberNCT05331118.

The impacts of individual commensal microbes on immunity and disease can differ dramatically depending on the surrounding microbial context; however, the specific bacterial combinations that dictate divergent immunological outcomes remain largely undefined. Here, we characterize an immunostimulatory Allobaculum species from an inflammatory bowel disease patient that exacerbates colitis in gnotobiotic mice. Allobaculum inversely associates with the taxonomically divergent immunostimulatory species Akkermansia muciniphila in human-microbiota-associated mice and human cohorts. Co-colonization with A. muciniphila ameliorates Allobaculum-induced intestinal epithelial cell activation and colitis in mice, whereas Allobaculum blunts the A.muciniphila-specific systemic antibody response and reprograms the immunological milieu in mesenteric lymph nodes by blocking A.muciniphila-induced dendritic cell activation and T cell expansion. These studies thus identify a pairwise reciprocal interaction between human gut bacteria that dictates divergent immunological outcomes. Furthermore, they establish a generalizable framework to define the contextual cues contributing to the "incomplete penetrance" of microbial impacts on human disease.

Background & aims: It is currently unclear whether reported changes in the gut microbiome are cause or consequence of inflammatory bowel disease (IBD). Therefore, we studied the gut microbiome of IBD-discordant and -concordant twin pairs, which offers the unique opportunity to assess individuals at increased risk of developing IBD, namely healthy cotwins from IBD-discordant twin pairs. Methods: Fecal samples were obtained from 99 twins (belonging to 51 twin pairs), 495 healthy age-, sex-, and body mass index–matched controls, and 99 unrelated patients with IBD. Whole-genome metagenomic shotgun sequencing was performed. Taxonomic and functional (pathways) composition was compared among healthy cotwins, IBD-twins, unrelated patients with IBD, and healthy controls with multivariable (ie, adjusted for potential confounding) generalized linear models. Results: No significant differences were observed in the relative abundance of species and pathways between healthy cotwins and their IBD-twins (false discovery rate

The immunopathogenesis of inflammatory bowel disease (IBD) has been attributed to a combination of host genetics and intestinal dysbiosis. Previous work in a small cohort of IBD patients suggested that pro-inflammatory bacterial taxa are highly coated with secretory immunoglobulin IgA. Using bacterial fluorescence-activated cell sorting coupled with 16S rRNA gene sequencing (IgA-SEQ), we profiled IgA coating of intestinal microbiota in a large cohort of IBD patients and identified bacteria associated with disease and treatment. Forty-three bacterial taxa displayed significantly higher IgA coating in IBD compared with controls, including 8 taxa exhibiting differential IgA coating but similar relative abundance. Patients treated with anti-TNF-α therapies exhibited dramatically altered microbiota-specific IgA responses compared with controls. Furthermore, increased IgA coating of Oscillospira was associated with a delay in time to surgery. These results demonstrate that investigating IgA responses to microbiota can uncover potential disease-modifying taxa and reveal improved biomarkers of clinical course in IBD.

Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.

Sound evidence supports a role for interleukin‐17 (IL‐17) ‐producing γδ T cells and IL‐17‐producing helper T (Th17) cells in intestinal homeostasis, especially in intestinal barrier integrity. In the present study, we aimed to evaluate the role of IL‐17 cytokine in the regulation of intestinal immunity and obesity‐induced metabolic syndrome (MetS) in an experimental murine model. C57BL/6 wild‐type (WT) mice and mice lacking the IL‐17 cytokine receptor (IL‐17RA(−/−)) were fed either a control diet (CD) or a high‐fat diet (HFD) for 9 weeks. Our data demonstrate that IL‐17RA(−/−) mice are protected against obesity, but develop hyperglycemia, hyperinsulinemia and insulin resistance. In parallel, HFD‐fed IL‐17RA(−/−) mice display intense inflammation in the ileum compared with WT mice on the HFD. IL‐17RA(−/−) mice fed the HFD exhibit impaired neutrophil migration to the intestinal mucosa and reduced gene expression of the CXCL‐1 chemokine and CXCR‐2 receptor in the ileum. Interestingly, the populations of neutrophils (CD11b(+) Ly6G(+)) and anti‐inflammatory macrophages (CD11b(+) CX3CR1(+)) are increased in the mesenteric lymph nodes of these mice. IL‐17RA(−/−) mice on the HFD also display increased commensal bacterial translocation into the bloodstream and elevated lipopolysaccharide (LPS) levels in the visceral adipose tissue (VAT). Metagenomic analysis of bacterial 16S gene revealed increased Proteobacteria and Bacteroidetes phyla, the main representatives of Gram‐negative bacteria, and reduced Akkermansia muciniphila in the fecal samples of IL‐17RA(−/−) mice fed the HFD. Together, these data indicate that the IL‐17/IL‐17R axis drives intestinal neutrophil migration, limits gut dysbiosis and attenuates LPS translocation to VAT, resulting in protection to MetS.

Stressed gut epithelium gets some relief Immunoglobulin A (IgA) is the most abundantly expressed antibody isotype and can be found at various mucosal surfaces in the body, including the gastrointestinal (GI) tract. IgA is polyreactive and can coat and restrain both commensal bacteria and enteric pathogens. Grootjans et al. found that endoplasmic reticulum (ER) stress in the intestinal epithelial cells of mice induced the T cell– and microbiota-independent expansion of peritoneal B1b cells, which secrete IgA. Similarly, human subjects homozygous for a variant of an autophagy gene ( ATG16L1 ) known to cause ER stress showed increased numbers of GI IgA + cells compared with controls. Thus, epithelial ER stress serves as an advantageous “eustress” response that can functionally antagonize its well-characterized role in promoting inflammation. Science , this issue p. 993

SummaryThe mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify a mechanism by which mitochondria and downstream proapoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a proinflammatory type of cell death.

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Inflammasome activation is important for the development of an effective host defense against many pathogens, including RNA viruses. However, the mechanism by which the inflammasome recognizes RNA viruses and its role in rabies virus (RABV) pathogenicity and immunogenicity remain poorly defined. To determine the function of the inflammasome in response to RABV infection, we infected murine bone marrow-derived dendritic cells (BMDCs) with RABV. Our results indicate that the infection of BMDCs with RABV induces both the production of pro-interleukin-1β (pro-IL-1β) and its processing, resulting in the secretion of active IL-1β through activation of the NLRP3-, ASC-, and caspase-1-dependent inflammasome. As previously shown for the induction of type I interferon by RABV, the induction of pro-IL-1β also depends upon IPS-1. We demonstrate that both the production of pro-IL-1β and activation of the inflammasome require viral replication. We also demonstrate that increased viral replication in BMDCs derived from IFNAR-deficient mice resulted in significantly more IL-1β release. Additionally, IL-1 receptor-deficient mice show an increase in RABV pathogenicity. Taken together, these results indicate an important role of the inflammasome in innate immune recognition of RABV.

Streptozotocin causes T1D by inducing the translocation of intestinal bacteria into pancreatic lymph nodes and driving the development of pathogenic Th1 and Th17 cells through NOD2 receptor., Type 1 diabetes (T1D) is an autoimmune disease that is triggered by both genetic and environmental factors, resulting in the destruction of pancreatic β cells. The disruption of the intestinal epithelial barrier and consequent escape of microbial products may be one of these environmental triggers. However, the immune receptors that are activated in this context remain elusive. We show here that during streptozotocin (STZ)-induced T1D, the nucleotide-binding oligomerization domain containing 2 (NOD2), but not NOD1, participates in the pathogenesis of the disease by inducing T helper 1 (Th1) and Th17 cells in the pancreatic LNs (PLNs) and pancreas. Additionally, STZ-injected wild-type (WT) diabetic mice displayed an altered gut microbiota compared with vehicle-injected WT mice, together with the translocation of bacteria to the PLNs. Interestingly, WT mice treated with broad-spectrum antibiotics (Abx) were fully protected from STZ-induced T1D, which correlated with the abrogation of bacterial translocation to the PLNs. Notably, when Abx-treated STZ-injected WT mice received the NOD2 ligand muramyl dipeptide, both hyperglycemia and the proinflammatory immune response were restored. Our results demonstrate that the recognition of bacterial products by NOD2 inside the PLNs contributes to T1D development, establishing a new putative target for intervention during the early stages of the disease.

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food-borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti-sigma factor proteins: it represses transcription of sigma(28)-dependent genes, forms a complex with sigma factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook-basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the sigma(28)- and the sigma(54)-dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence sigma(28)-dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-κB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys (Pam3CSK4), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6–16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.

The Gram-negative bacterium Campylobacter is the leading cause of bacterial entero-colitis in humans and is associated with the occurrence of life-threatening auto-immune based neurological disorders. Chickens, which are often heavily colonized with Campylobacter without signs of pathology, are considered the most important source for human infection. Although vaccination is a well established and effective method to combat various microbes in poultry, a commercial vaccine against Campylobacter has not yet been developed. For the development of such a vaccine, three main challenges can be identified: (1) the identification of novel cross-protection-inducing antigens, (2) the induction of a rapid, potent immune response, and (3) the development of novel adjuvants to further stimulate immunity against Campylobacter. The rapidly emerging knowledge of the biology of Campylobacter in combination with the recent advances in the fields of molecular vaccinology and immunology provide the required setting for the development of an effective vaccine against Campylobacter in poultry.

Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin.

The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA , the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA , ratB , sivI , and sivH . DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genus Salmonella . Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA , sivI , and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.

SummarySpecific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow-cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA-SEQ) and show that high IgA coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA-SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with predefined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.

Nucleotide oligomerization domain (Nod)-like Receptors (NLRs) are cytosolic sensors that mediate the activation of Caspase-1 and the subsequent processing and secretion of the pro-inflammatory cytokines IL-1β and IL-18, as well as an inflammatory cell death termed pyroptosis. While a multitude of bacteria have been shown to activate one or more NLRs under in vitro conditions, the exact impact of NLR activation during the course of colonization, both of pathogenic and commensal nature, is less understood. In this review, we will focus on the role of intestinal NLRs during the various stages of infection with common gastrointestinal bacterial pathogens, as well as NLR function in controlling and shaping the microbiota.

Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1β but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia in the steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.

Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.

Campylobacter infection in humans is accompanied by severe inflammation of the intestinal mucosa, in contrast to colonization of chicken. The basis for the differential host response is unknown. Toll-like receptors (TLRs) sense and respond to microbes in the body and participate in the induction of an inflammatory response. Thus far, the interaction of Campylobacter with chicken TLRs has not been studied. Here, we investigated the potential of four Campylobacter strains to activate human TLR1/2/6, TLR4, TLR5, and TLR9 and chicken TLR2t2/16, TLR4, TLR5, and TLR21. Live bacteria showed no or very limited potential to activate TLR2, TLR4, and TLR5 of both the human and chicken species, with minor but significant differences between Campylobacter strains. In contrast, lysed bacteria induced strong NF-κB activation through human TLR1/2/6 and TLR4 and chicken TLR2t2/16 and TLR4 but not via TLR5 of either species. Interestingly, C. jejuni induced TLR4-mediated beta interferon in human but not chicken cells. Furthermore, isolated chromosomal Campylobacter DNA was unable to activate human TLR9 in our system, whereas chicken TLR21 was activated by DNA from all of the campylobacters tested. Our data are the first comparison of TLR-induced immune responses in humans and chickens. The results suggest that differences in bacterial cell wall integrity and in TLR responses to Campylobacter LOS and/or DNA may contribute to the distinct clinical manifestation between the species.

Campylobacter jejuni is the principal bacterial foodborne pathogen. A major challenge still is to identify the virulence strategies exploited by C. jejuni. Recent genomics, proteomics, and metabolomics approaches indicate that C. jejuni displays extensive inter- and intrastrain variation. The diverse behavior enables bacterial adaptation to different environmental conditions and directs interactions with the gut mucosa. Here, we report recent progress in understanding the molecular mechanisms and functional consequences of the phenotype diversity. The results suggest that C. jejuni actively penetrates the intestinal mucus layer, secretes proteins mainly via its flagellar apparatus, is engulfed by intestinal cells, and can disrupt the integrity of the epithelial lining. C. jejuni stimulates the proinflammatory pathway and the production of a large repertoire of cytokines, chemokines, and innate effector molecules. Novel experimental infection models suggest that the activation of the innate immune response is important for the development of intestinal pathology.

Campylobacter jejuni is the principal bacterial foodborne pathogen. A major challenge still is to identify the virulence strategies exploited by C. jejuni. Recent genomics, proteomics, and metabolomics approaches indicate that C. jejuni displays extensive inter- and intrastrain variation. The diverse behavior enables bacterial adaptation to different environmental conditions and directs interactions with the gut mucosa. Here, we report recent progress in understanding the molecular mechanisms and functional consequences of the phenotype diversity. The results suggest that C. jejuni actively penetrates the intestinal mucus layer, secretes proteins mainly via its flagellar apparatus, is engulfed by intestinal cells, and can disrupt the integrity of the epithelial lining. C. jejuni stimulates the proinflammatory pathway and the production of a large repertoire of cytokines, chemokines, and innate effector molecules. Novel experimental infection models suggest that the activation of the innate immune response is important for the development of intestinal pathology.

Mammalian Toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and activates the innate immune system. The avian TLR repertoire exhibits considerable functional diversity compared to mammalian TLRs and evidence of a functional TLR5 in the avian species is lacking. In the present study we cloned and successfully expressed chicken TLR5 (chTLR5) in HeLa cells, as indicated by laser confocal microscopy. Infection of chTLR5 transfected cells with Salmonella enterica serovar Enteritidis activated NF-kappaB in a dose- and flagellin-dependent fashion. Similar NF-kappaB activation was observed with recombinant bacterial flagellin. Targeted mutagenesis of the proline residue at position 737 in the chTLR5-TIR domain was detrimental to chTLR5 function, confirming that the observed effects were conferred via chTLR5 and the MyD88 signaling pathway. Comparison of human, mouse and chicken TLR5 activation by flagellin of S. enterica serovar Typhimurium revealed that chTLR5 consistently yielded stronger responses than human but not mouse TLR5. This species-specific reactivity was not observed with flagellin of serovar Enteritidis. The species-specific TLR5 response was nullified after targeted mutagenesis of a single amino acid (Q89A) in serovar Typhimurium flagellin, while L415A and N100A substitutions had no effect. These results show that chickens express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. The finding that single amino acid substitutions in bacterial flagellin can alter the species-specific TLR5 response may influence the host range and susceptibility of infection.

Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin.

Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori . However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tet r ) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Genetic transformation of the Tet s strain 26695 with smaller overlapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the Tet r strain 181, the Tet s strain 26695, and four Tet r 26695 transformants showed that a single triple-base-pair substitution, AGA 926-928 →TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tet r H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tet r strain 181.