R. Paul Wilson

Simian immunodeficiency virus–induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut

Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor–deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

SipA, SopA, SopB, SopD, and SopE2 Contribute to Salmonella enterica Serotype Typhimurium Invasion of Epithelial Cells

ABSTRACT The centisome 63 type III secretion system (T3SS-1) encoded by Salmonella pathogenicity island 1 (SPI1) mediates invasion of epithelial cells by Salmonella enterica serotype Typhimurium. Characterization of mutants lacking individual genes has revealed that T3SS-1 secreted proteins (effectors) SopE2 and SopB are required for invasion while the SipA protein accelerates entry into cells. Here we have revisited the question of which T3SS-1 effectors contribute to the invasion of epithelial cells by complementing a strain lacking all of the effector genes that are required to cause diarrhea in a calf (a sipA sopABDE2 mutant). Introduction of either the cloned sipA, the cloned sopB, or the cloned sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for nonpolarized HT-29 cells. However, a contribution of sopA or sopD to invasion was not apparent when invasion assays were performed with the nonpolarized colon carcinoma cell lines T84 and HT-29. In contrast, introduction of either the sopA, the sopB, the sopD, or the sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells. Furthermore, introduction of a plasmid carrying sipA and sopB increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells significantly compared to the introduction of plasmids carrying only sipA or sopB. We conclude that SipA, SopA, SopB, SopD, and SopE2 contribute to S. enterica serotype Typhimurium invasion of epithelial cells in vitro.

The Vi Capsular Antigen of Salmonella enterica Serotype Typhi Reduces Toll-Like Receptor-Dependent Interleukin-8 Expression in the Intestinal Mucosa

ABSTRACT Human infections with nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that in S. enterica serotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.

CsgA is a pathogen‐associated molecular pattern of Salmonella enterica serotype Typhimurium that is recognized by Toll‐like receptor 2

Knowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen‐associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant (csgBA) was attenuated in its ability to elicit fluid accumulation and GROα mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL‐8 production than the wild type in human macrophage‐like cells. Purified thin curled fimbriae induced IL‐8 expression in human embryonic kidney (HEK293) cells transfected with Toll‐like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione‐S‐transferase (GST) elicited IL‐8 production in HEK293 cells transfected with TLR2/CD14. Proteinase K treatment abrogated IL‐8 production elicited in these cells by GST–CsgA, but not by synthetic lipoprotein. GST–CsgA elicited more IL‐6 production than GST in bone marrow‐derived macrophages from TLR2+/+ mice, while there was no difference in IL‐6 secretion between GST–CsgA and GST in macrophages from TLR2–/– mice. These data suggested that CsgA is a PAMP that is recognized by TLR2.

The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella

The viaB locus enables Salmonella enterica serotype Typhi to reduce Toll‐like receptor (TLR) dependent cytokine production in tissue culture models. This DNA region contains genes involved in the regulation (tviA), biosynthesis (tviBCDE) and export (vexABCDE) of the Vi capsule. Expression of the Vi capsule in S. Typhimurium, but not expression of the TviA regulatory protein, reduced tumour necrosis factor‐alpha (TNF‐α) and IL‐6 production by murine bone‐marrow derived macrophages. Production of TNF‐α and IL‐6 was dependent on expression of TLR4 as stimulation of macrophages from TLR4−/− mice with S. Typhimurium did not result in expression of these cytokines. Intraperitoneal infection of mice with S. Typhimurium induced expression of TNF‐α and inducible nitric oxide synthase (iNOS) in the liver. Introduction of the cloned viaB region into S. Typhimurium reduced TNF‐α and iNOS expression to levels observed after infection with a S. Typhimurium msbB mutant. In contrast, no differences in TNF‐α expression between the S. Typhimurium wild type and strains expressing the Vi‐capsule or carrying a mutation in msbB were observed after infection of TLR4−/− mice. We conclude that the Vi capsule prevents both in vitro and in vivo recognition of S. Typhimurium lipopolysaccharide by TLR4.

Capsule-mediated immune evasion: a new hypothesis explaining aspects of typhoid fever pathogenesis.

The genus Salmonella contains a group of closely related organisms that are pathogenic for humans and other vertebrates. The human disease manifestations caused most frequently by Salmonella serotypes worldwide are typhoid fever and gastroenteritis (reviewed in reference [102][1]). Both illnesses

Responses to amyloids of microbial and host origin are mediated through toll-like receptor 2.

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimers disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.

The Capsule Encoding the viaB Locus Reduces Interleukin-17 Expression and Mucosal Innate Responses in the Bovine Intestinal Mucosa during Infection with Salmonella enterica Serotype Typhi

ABSTRACT The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica serotype Typhi. Wild-type serotype Typhi induces less CXC chemokine production in tissue culture models than does an isogenic viaB mutant. Here we investigated the in vivo relevance of these observations by determining whether the presence of the viaB region prevents inflammation in two animal models of gastroenteritis. Unlike S. enterica serotype Typhimurium, serotype Typhi or a serotype Typhi viaB mutant did not elicit marked inflammatory changes in the streptomycin-pretreated mouse model. In contrast, infection of bovine ligated ileal loops with a serotype Typhi viaB mutant resulted in more fluid accumulation and higher expression of the chemokine growth-related oncogene alpha (GROα) and interleukin-17 (IL-17) than did infection with the serotype Typhi wild type. There was a marked upregulation of IL-17 expression in both the bovine ligated ileal loop model and the streptomycin-pretreated mouse model, suggesting that this cytokine is an important component of the inflammatory response to infection with Salmonella serotypes. Introduction of the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GROα and IL-17 expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in reducing intestinal inflammation in vivo.

The Salmonella enterica serotype Typhi regulator TviA reduces interleukin‐8 production in intestinal epithelial cells by repressing flagellin secretion

Unlike non‐typhoidal Salmonella serotypes, S. enterica serotype Typhi does not elicit neutrophilic infiltrates in the human intestinal mucosa. The Vi capsule‐encoding tviABCDEvexABCDE operon (viaB locus) is a S. Typhi‐specific DNA region preventing production of interleukin (IL)‐8 during infection of intestinal epithelial cells. We elucidated the mechanism by which the viaB locus reduces IL‐8 production in human colonic epithelial (T84) cells. A S. Typhi tviABCDEvexABCDE deletion mutant, but not a tviBCDEvexABCDE deletion mutant, elicited increased IL‐8 production, which could be reduced to wild‐type levels by introducing the cloned tviA regulatory gene. Thus, IL‐8 expression in T84 cells was modulated by the TviA regulatory protein, but not by the Vi capsular antigen. Consistent with previous reports, IL‐8 secretion by T84 cells was dependent on the presence of the flagellin protein FliC. TviA reduced expression of flhDC::lacZ and fliC::lacZ transcriptional fusions and secretion of FliC in S. Typhi. Introduction of tviA into S. enterica serotype Typhimurium reduced flagellin secretion and IL‐8 expression. In conclusion, the viaB locus reduces IL‐8 production in T84 cells by a TviA‐mediated repression of flagellin secretion. Our data suggest that changes in flagella gene regulation played an important role during evolution of the human‐adapted S. Typhi.

Contribution of Flagellin Pattern Recognition to Intestinal Inflammation during Salmonella enterica Serotype Typhimurium Infection

ABSTRACT Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3α and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.