A. Marijke Keestra

Host-Derived Nitrate Boosts Growth of E. coli in the Inflamed Gut

E. coli kNOws How to Win The harmonious existence among the various microbial inhabitants of the gut is critical for good health. However, inflammation from injury or inflammatory bowel disease, can disrupt this balance and lead to the outgrowth of particular bacteria. The outgrowth of members of the Enterobacteriaceae family, which includes Escherichia coli, is often observed. Because E. coli are facultative rather an obligate anaerobes, Winter et al. (p. 708) postulated that they may be able to use by-products of reactive oxygen and nitrogen species, which are produced during inflammation, for anaerobic respiration, thereby edging out other fermenting bacteria. Indeed, in two mouse models of colitis and in a model of intestinal injury, various E. coli strains were able to use host-derived nitrate as an energy source and outcompete mutant strains unable to do this. During inflammation, Escherichia coli uses nitrate respiration to gain a growth advantage over other gut bacteria. Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient in inducible nitric oxide synthase did not support the growth of E. coli by nitrate respiration, suggesting that the nitrate generated during inflammation was host-derived. Thus, the inflammatory host response selectively enhances the growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.

Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Unique Properties of the Chicken TLR4/MD-2 Complex: Selective Lipopolysaccharide Activation of the MyD88-Dependent Pathway

During evolution, mammals have evolved a powerful innate immune response to LPS. Chickens are much more resistant to LPS-induced septic shock. Herein we report that chickens sense LPS via orthologs of mammalian TLR4 and myeloid differentiation protein-2 (MD-2) rather than the previously implicated chicken TLR2 isoform type 2 (chTLR2t2) receptor. Cloning and expression of recombinant chTLR4 and chMD-2 in HeLa 57A cells activated NF-κB at concentrations of LPS as low as 100 pg/ml. Differential pairing of chicken and mammalian TLR4 and MD-2 indicated that the protein interaction was species-specific in contrast to the formation of functional human and murine chimeric complexes. The chicken LPS receptor responded to a wide variety of LPS derivatives and to the synthetic lipid A compounds 406 and 506. The LPS specificity resembled the functionality of the murine rather than the human TLR4/MD-2 complex. Polymorphism in chTLR4 (Tyr383His and Gln611Arg) did not influence the LPS response. Interestingly, LPS consistently failed to activate the MyD88-independent induction of IFN-β in chicken cells, in contrast to the TLR3 agonist poly(I:C) that yielded a potent IFN-β response. These results suggest that chicken lack a functional LPS-specific TRAM-TRIF (TRIF-related adapter molecule/TIR-domain-containing adapter-inducing IFN-β) signaling pathway, which may explain their aberrant response to LPS compared with the mammalian species.

Salmonella enterica serotype Typhimurium ShdA is an outer membrane fibronectin-binding protein that is expressed in the intestine.

The shdA gene is the only determinant known to be required for persistence of Salmonella enterica serotype Typhimurium (S. Typhimurium) in the murine caecum and for efficient and prolonged shedding of the organism with the faeces. To study the biological activity of the ShdA protein, we examined its expression and binding activity. ShdA was not detected with anti‐ShdA antiserum in S. Typhimurium strain ATCC14028 grown in vitro, suggesting that this protein is not expressed under standard conditions of bacterial cultivation in the laboratory. However, in mice infected with S. Typhimurium, an immunofluorescence signal detected with anti‐ShdA antiserum co‐localized with that generated by anti‐O4 antiserum in thin sections from the caecum. Expression of the cloned shdA gene from the T7 promoter in vitro resulted in detection of ShdA in the outer membrane of S. Typhimurium and in binding of fibronectin to the bacterial surface. Binding of purified glutathione‐S‐transferase (GST)–ShdA fusion protein to fibronectin was dose dependent and could be partially inhibited by preincubation with antifibronectin antibodies. GST–ShdA bound to connective tissue and the basement membrane in thin sections from the murine caecum in situ. A similar labelling pattern was produced when thin sections of the murine caecum were stained with antifibronectin antiserum. Collectively, these data demonstrate that ShdA is a surface‐localized, fibronectin‐binding protein whose expression is induced in vivo in the murine caecum, a tissue in which a cognate receptor of this outer membrane protein is expressed.

The Central Leucine-Rich Repeat Region of Chicken TLR16 Dictates Unique Ligand Specificity and Species-Specific Interaction with TLR2

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.

Activation of Human and Chicken Toll-Like Receptors by Campylobacter spp.

ABSTRACT 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.

Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms

Responses to host amyloids and curli amyloid fibrils of Escherichia coli and Salmonella enterica serotype Typhimurium are mediated through Toll‐like receptor (TLR) 2. Here we show that TLR2 alone was not sufficient for mediating responses to curli. Instead, transfection experiments with human cervical cancer (HeLa) cells and antibody‐mediated inhibition of TLR signalling in human macrophage‐like (THP‐1) cells suggested that TLR2 interacts with TLR1 to recognize curli amyloid fibrils. TLR1/TLR2 also serves as a receptor for tri‐acylated lipoproteins, which are produced by E. coli and other Gram‐negative bacteria. Despite the presence of multiple TLR1/TLR2 ligands on intact bacterial cells, an inability to produce curli amyloid fibrils markedly reduced the ability of E. coli to induce TLR2‐dependent responses in vitro and in vivo. Collectively, our data suggest that curli amyloid fibrils from enterobacterial biofilms significantly contribute to TLR1/TLR2‐mediated host responses against intact bacterial cells.

Differential Activation of Human and Mouse Toll-Like Receptor 4 by the Adjuvant Candidate LpxL1 of Neisseria meningitidis

ABSTRACT Neisseria meningitidis LpxL1 lipopolysaccharide (LPS) bearing penta-acylated lipid A is considered a promising adjuvant candidate for inclusion in future N. meningitidis vaccines, as it elicits a markedly reduced endotoxic response in human macrophages relative to that in wild-type (hexa-acylated) LPS, while it is an equally effective adjuvant in mice. As dendritic cells (DC) and Toll-like receptors (TLR) are regarded as central mediators in the initiation of an immune response, here we evaluated the ability of LpxL1 LPS to mature and to activate human DC and examined its TLR4-/MD-2-activating properties. Unexpectedly, purified LpxL1 LPS displayed minimal human DC-stimulating properties compared to wild-type LPS. Although whole bacteria induced DC maturation and activation irrespective of their type of LPS, the LpxL1 mutant failed to activate the human recombinant TLR4/MD-2 complex expressed in HeLa cells. Similarly, purified LpxL1 LPS was unable to activate human TLR4/MD-2 and it even acted as an antagonist of wild-type LPS. Both wild-type and LpxL1 LPSs activated the murine TLR4/MD-2 complex, consistent with their abilities to induce maturation and activation of murine DC. Assays with cells transfected with different combinations of human and murine TLR4 and MD-2 indicated that TLR4 was a more-major determinant of the LPS response than MD-2. The species-specific activation of the TLR4/MD-2 complex by LpxL1 LPS may have an impact on the use of LpxL1 LPS as an adjuvant and the use of murine immunization models in human meningococcal vaccine development.

Cleavage and activation of a Toll-like receptor by microbial proteases

Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB–dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors.

A Salmonella Virulence Factor Activates the NOD1/NOD2 Signaling Pathway

ABSTRACT The invasion-associated type III secretion system (T3SS-1) of Salmonella enterica serotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasion in vitro but required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responses in vitro and in vivo. IMPORTANCE Salmonella enterica serotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol. Salmonella enterica serotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol.