Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-κB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.

Toll-like receptors (TLRs) activate antimicrobial gene expression in response to detection of specific bacterial products. Relatively little is known about TLR5, the only TLR thought to be preferentially expressed by epithelial cells, beyond that it confers activation of the transcription factor NF-κB in a MyD-88 dependent manner in response to flagellin. Because TLRs, in general, are also thought to signal through members of the MAPK family, we examined flagellin-induced MAPK activation (via examining its phosphorylation status) and its subsequent role in expression of the chemokine IL-8 in polarized intestinal epithelia. Flagellin, like other proinflammatory stimuli (TNF-α, Salmonella typhimurium), activated p38 MAPK in a TLR5-dependent manner, whereas aflagellate bacteria or EGF did not activate this kinase. Although ERK1 and -2 were also observed to be activated in response to flagellin, their activation was not restricted to proinflammatory stimuli because they were also potently activated by aflagellate bacteria ( S. typhimurium or Escherichia coli) and EGF (neither of which activate NF-κB in these cells). Pharmacological inhibition of p38 MAPK (by SB-203580) potently (IC50 = 10 nM) reduced expression of IL-8 protein (maximal inhibition, 75%) but had no effect on NF-κB activation, only slightly attenuated upregulation of IL-8 mRNA levels in response to flagellin, and did not effect IL-8 mRNA stability. Together, these results indicate that epithelial TLR5 mediates p38 activation and subsequently regulates flagellin-induced IL-8 expression independently of NF-κB, probably by influencing IL-8 mRNA translation.

Helicobacter pylori colonizes the human stomach for decades unless pharmacologically eradicated. We hypothesized that this flagellated pathogen escapes immune clearance, in part, by avoiding detection by the flagellin receptor Toll-like receptor 5 (TLR5). In contrast to other gram-negative microbes, H. pylori did not release flagellin. Furthermore, recombinant H. pylori flagellin (FlaA) was significantly less potent (1000-fold) than Salmonella typhimurium flagellin in activating TLR5-mediated interleukin (IL)-8 secretion. TLR5 can mediate flagellin-induced IL-8 secretion via p38 mitogen-activated protein kinase signaling; however, compared with potent induction by S. typhimurium flagellin, H. pylori FlaA-dependent p38 activation was substantially attenuated. In addition, disruption of H. pylori flaA decreased motility but had no effect on H. pylori-induced IL-8 secretion, which indicates that H. pylori flagellin plays no role in activating epithelial orchestration of inflammation. We conclude that H. pylori evades TLR5-mediated detection, which may contribute to its longterm persistence in individual hosts.Approximately 50% of the world's population is colonized by Helicobacter pylori, and, although the majority of these infections are asymptomatic, long-term interactions between H. pylori and humans significantly increase the risk for peptic ulcer disease and distal gastric adenocarcinoma [1,2]. Regardless of clinical outcome, the vast majority of colonized persons never eliminate H. pylori, unless a targeted antibiotic regimen is employed; thus, a signature feature of H. pylori-induced gastritis is its capacity to persist for decades. This is in marked contrast to inflammatory reactions in-

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IB␣/ NF-B signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor ␣-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.The epithelial cells lining the gastrointestinal tract form a highly specialized barrier that separates two very distinct environments, thus maintaining the delicate balance between the gut lumen and the underlying tissue (1, 2). As part of its barrier function, the intestinal epithelium is able to detect surface attached enteric pathogens like Salmonella enterica, serovar Typhimurium (S. typhimurium), and orchestrate a proinflammatory response. This multifaceted response involves the rapid secretion of chemoattractants, the development of a chemotactic gradient in the surrounding subepithelial matrix (3-5), and the migration of neutrophils to the site of infection, ultimately triggering secretory diarrhea (6 -11). The concurrent apical release of a soluble factor, designated PEEC (for pathogenelicited epithelial chemoattractant), aids in directing the final polymorphonuclear leukocyte (PMN) movement across the epithelial tight junctions into the gut lumen (12). In addition to IL-8, 1 a variety of other chemokines are secreted in response to S. typhimurium attachment (13-15). Salmonella-induced secretion of IL-8 is known to require the activation of the nuclear transcription factor 17). In quiescent cells, the NF-B heterodimer (p50/p65) is held in a stable, inactive, cytoplasmic complex with IB␣ molecules until activated by an as yet undefined Salmonella...

The genome of Vibrio cholerae contains five flagellin genes that encode proteins (FlaA-E) of 39 -41 kDa with 61-82% identity among them. Although the existing live oral attenuated vaccine strains against cholera are protective in humans, there is an intrinsic residual cytotoxic and inflammatory component associated with these candidate vaccine strains. Bacterial flagellins are known to be potent inducers of proinflammatory molecules via activation of Toll-like receptor 5. Here we found that purified flagella from wild type V. cholerae 395 induced significant release of interleukin (IL)-8 from cultured HT-29 human colonic epithelial cells. Furthermore we found that filtered supernatants of KKV90, a ⌬flaA isogenic strain unable to produce flagella, were still able to activate production of IL-8 albeit to significantly lower levels than the wild type, suggesting that other activators of proinflammatory molecules were still present in these supernatants. Cholera remains a devastating bacterial cause of human morbidity and mortality in some areas of the world (1). The disease is produced by Vibrio cholerae, a Gram-negative curved rod that colonizes the human intestine where it secretes the potent cholera toxin (CT), 1 which ultimately stimulates cellular adenylate cyclase to cause massive intestinal fluid loss leading to profuse watery diarrhea. CT is the major V. cholerae virulence factor, and it is encoded by the ctxA and ctxB genes carried on the transmissible prophage CTX⌽ (2). V. cholerae produces an array of virulence factors, which are coordinately regulated by the transcriptional activator ToxR (3). In turn, ToxR activates ToxT, a second transcriptional regulator that activates the expression of CT and the toxincoregulated pilus (TCP) (4, 5). TCP is considered the most important intestinal colonization factor of V. cholerae (6).In addition to CT, the accessory cholera toxin (Ace) (7) and the zonula occludens toxin (Zot) (7, 8) were reported as potential cytotoxic factors, but these proteins were later demonstrated to be components of a filamentous bacteriophage (2). Several in vitro studies have shown that V. cholerae secretes other cytotoxic factors such as the hemagglutinin/ protease (HAP), hemolysin (Hly), and repeats-in-toxin (RTX) (9 -11). These cytotoxic factors may cause tissue damage by different mechanisms that could contribute to proinflammatory responses. However, only RTX mutants have been demonstrated in a murine pulmonary cholera model to show less severe pathology and decreased serum levels of proinflammatory IL-6 and murine macrophage inflammatory protein-2, suggesting that RTX participates in the severity of acute inflammatory responses (12).Research on cholera vaccines has focused largely on oral formulations that stimulate the mucosal immune system thereby mimicking natural infection (13). Through the years, different formulations of cholera vaccines have been proposed that include formalin or heat-killed bacteria alone or in combination with CT B-subunit. As new putative virulence factors ...