Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells

The type III secretion system (TTSS) of enteropathogenic Escherichia coli (EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells. However, the identity of the effector molecule directly involved in this event is unknown. In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells. Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells. In contrast, supernatants of EPEC fliC mutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of the fliC gene in the mutant lacking flagella. Purified flagella from E. coli K-12, EPEC serotypes H6 and H34, and enterohemorrhagic E. coli serotype H7 all induced IL-8 release in T84 cells. Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody. Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin. Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction.     

Effect of zinc in enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5′-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5′-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5′-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5′-nucleotidase in vitro than the nucleotide analog α,β-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than α,β-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5′-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.     

Mouse model of enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in humans. EPEC infection of cultured intestinal epithelial cells induces attaching and effacing (A/E) lesions, alters intestinal ion transport, increases paracellular permeability, and stimulates inflammation. The lack of a small-animal model has restricted in vivo studies examining EPEC-host interactions. The aim of this study was to characterize the C57BL/6J mouse as a model of EPEC infection. We have shown that EPEC can adhere to and colonize the intestinal epithelium of C57BL/6J mice. Animal weight and water intake were not altered during 10 days of EPEC infection. The proximal colon of infected mice contained semisolid stool, with stool pellets forming only in the distal colon. In contrast, the entire colon of control mice contained formed stool. Microvillous effacement and actin rearrangement, characteristic of A/E lesions, were seen in the intestine of infected mice but not control mice. Histological assessment revealed increased numbers of lamina propria neutrophils with occasional crypt abscesses, intraepithelial lymphocytes, and goblet cells in the intestine of EPEC-infected mice. Altogether, these data suggest that the C57BL/6J mouse is susceptible to infection by EPEC and will provide a suitable in vivo model for studying the consequences of EPEC infection.     

Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits

Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.     

Role of EspB in experimental human enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.     

Flagellin-dependent and -independent inflammatory responses following infection by enteropathogenic Escherichia coli and Citrobacter rodentium

Enteropathogenic Escherichia coli (EPEC) and the murine pathogen Citrobacter rodentium belong to the attaching and effacing (A/E) family of bacterial pathogens. These noninvasive bacteria infect intestinal enterocytes using a type 3 secretion system (T3SS), leading to diarrheal disease and intestinal inflammation. While flagellin, the secreted product of the EPEC fliC gene, causes the release of interleukin 8 (IL-8) from epithelial cells, it is unclear whether A/E bacteria also trigger epithelial inflammatory responses that are FliC independent. The aims of this study were to characterize the FliC dependence or independence of epithelial inflammatory responses to direct infection by EPEC or C. rodentium. Following infection of Caco-2 intestinal epithelial cells by wild-type and DeltafliC EPEC, a rapid activation of several proinflammatory genes, including those encoding IL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 3alpha (MIP3alpha), and beta-defensin 2, occurred in a FliC-dependent manner. These responses were accompanied by mitogen-activated protein kinase activation, as well as the Toll-like receptor 5 (TLR5)-dependent activation of NF-kappaB. At later infection time points, a subset of these proinflammatory genes (IL-8 and MIP3alpha) was also induced in cells infected with DeltafliC EPEC. The nonmotile A/E pathogen C. rodentium also triggered similar innate responses through a TLR5-independent but partially NF-kappaB-dependent mechanism. Moreover, the EPEC FliC-independent responses were increased in the absence of the locus of enterocyte effacement-encoded T3SS, suggesting that translocated bacterial effectors suppress rather than cause the FliC-independent inflammatory response. Thus, we demonstrate that infection of intestinal epithelial cells by A/E pathogens can trigger an array of proinflammatory responses from epithelial cells through both FliC-dependent and -independent pathways, expanding our understanding of the innate epithelial response to infection by these pathogens.     

The Afa/Dr adhesins of diffusely adhering Escherichia coli stimulate interleukin-8 secretion,activate mitogen-activated protein kinases,and promote polymorphonuclear transepithelial migration in T84 polarized epithelial cells

Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.     

An ultrastructural study of enteropathogenic Escherichia coli infection in human infants

Over the past 2 years, we have studied and treated 18 infants with protracted diarrhea due to an enteropathogenic Escherichia coli serogroup 0119. All patients had persistent stool escretion and jejunal overgrowth with this pathogenic E. coli. Jejunal biopsy revealed atrophy of villi with a chronic inflammatory cell infiltrate in the lamina propria. E. coli 0119 adhered to the luminal surface of enterocytes. Electron microscopy showed disappearance of glycocalyx and microvilli at the areas of bacterial adherence. Intracellular damage was indicated by dilatation of rough endoplasmic reticulum, mitochondrial changes, and cytoplasmic pallor. Similar changes in histology and ultrastructure occurred in ileal epithelial cells. Glandular crypt epithelium showed prominent subnuclear vacuolation and separation of lateral intercellular junctions throughout the small intestine. Rectal mucosal biopsy showed mucus depletion and irregular atrophy of the epithelium, with E. coli 0119 adherent to the luminal surface. Ultrasuctural damage paralleled that in the small intestine. E. coli 0119 causes damage to epithelial cells throughout the infant intestinal tract. This damage leads to atrophy of villi and a marked reduction in absorptive surface area, resulting in protracted diarrhea.     

Involvement of the mannose receptor and p38 mitogen-activated protein kinase signaling pathway of the microdomain of the integral membrane protein after enteropathogenic Escherichia coli infection

The microdomain of the integral membrane protein (MIMP) has been shown to adhere to mucin and to antagonize the adhesion of enteropathogenic Escherichia coli (EPEC) to epithelial cells; however, the mechanism has not been fully elucidated. In this study, we further identified the receptor of MIMP on NCM460 cells and investigated the mechanism (the p38 mitogen-activated protein kinase [MAPK] pathway) following the interaction of MIMP and its corresponding receptor, mannose receptor. We first identified the target receptor of MIMP on the surfaces of NCM460 cells using immunoprecipitation-mass spectrometry technology. We also verified the mannose receptor and examined the degradation and activation of the p38 MAPK signaling pathway. The results indicated that MIMP adhered to NCM460 cells by binding to the mannose receptor and inhibited the phosphorylation of p38 MAPK stimulated after EPEC infection via inhibition of the Toll-like receptor 5 pathway. These findings indicated that MIMPs relieve the injury of NCM460 cells after enteropathogenic E. coli infection through the mannose receptor and inhibition of the p38 MAPK signaling pathway, both of which may therefore be potential therapeutic targets for intestinal diseases, such as inflammatory bowel disease.     

Role of mitogen-activated protein kinases in activation-induced apoptosis of T cells

A member of the mitogen-activated protein (MAP) kinase family, Jun N-terminal kinase (JNK), has been implicated in regulating apoptosis in various cell types. We have investigated the requirement for another type of MAP kinase, extracellular signal-regulated protein kinase (ERK) in activation-induced cell death (AICD) of T cells. AICD is the process by which recently activated T cells undergo apoptosis when restimulated through the T-cell antigen receptor. Here we show that both JNK and ERK are activated rapidly upon T-cell receptor (TCR) ligation prior to the onset of AICD. A chemical inhibitor of ERK activation, PD 098059, inhibits ERK activation and apoptosis, while JNK activation is not inhibited. This suggests that JNK activation is not sufficient for apoptosis. TCR cross-linking induces expression of the apoptosis-inducing factor, Fas ligand (FasL), and its expression correlates with ERK activation. In addition, apoptosis induced by direct ligation of the Fas receptor by anti-Fas antibody is not associated with ERK activation and is not inhibited by PD 098059. These data suggest that ERK activation is an early event during T-cell apoptosis induced by antigen-receptor ligation, and is not involved in apoptosis per se but in the expression of FasL. MAP kinase family members may be similarly involved in inducing apoptosis signals in other cell types.     

Host cell death due to enteropathogenic Escherichia coli has features of apoptosis

Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen.     

Adherence of enterohemorrhagic Escherichia coli strains to a human colonic epithelial cell line (T84).

Enterohemorrhagic Escherichia coli (EHEC) produce Shiga-like toxins and attach to certain tissue culture cells. T84 cells are human colonic carcinoma cells. Unlike previously studied cell lines, T84 cells grown on collagen-coated surfaces polarize and produce tight junctions and desmosomes, forming a colonic epithelial cell layer in vitro. The purpose of this study was to examine the attachment of EHEC strains to the T84 cell line as a possibly more relevant in vitro model of EHEC adherence. Twelve EHEC strains were grown overnight in Penassay broth, suspended in minimal essential medium with and without 0.5% mannose, and incubated for 1 to 3 h with 5- to 7-day-old T84 cell monolayers grown on collagen-coated coverslips. The bacteria were removed, and attachment was quantitated microscopically. For both E. coli O157:H7 and other EHEC serotypes, there were marked differences in adherence between strains (range of 152 to 3 bacteria per oil immersion field). Mannose partially inhibited the adherence of some EHEC strains. Adherence to the T84 cells appeared to be related to the amount of pili present and not to the serotype. Electron micrographs showed that a highly adherent strain (strain 43-12) tended to form microcolonies in the area of tight junctions on the T84 cell monolayers. In addition, the attachment of these EHEC strains to T84 cells correlated with their ability to adhere to isolated rabbit colonocytes (r = 0.91, P = 0.00004; without mannose) (r = 0.60, P = 0.04; with mannose). These data show that there are EHEC strain-related differences in adherence which can be demonstrated in a human-derived colonic epithelial cell line (T84) and that these cells can be used to study EHEC adherence.     

T cell responses to bacterial infection.

Many exciting advances in our understanding of T cell mediated immunity to bacterial infection have occurred in the past several years. T cell responses have been more fully characterized, due in part to the development of MHC class I tetramers. The importance of cytokines and various effector molecules in defense against infection has come to light. Finally, intracellular bacteria are being exploited to deliver antigens and DNA in an effort to induce immunity to pathogens.     

Cross-reactive protection against enterohemorrhagic Escherichia coli infection by enteropathogenic E. coli in a mouse model

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC (E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.     

封闭枯否细胞对LPS诱导激活丝裂原活化蛋白激酶的影响

目的： 利用小鼠LPS血症模型，探讨封闭枯否细胞（KCs）对LPS诱导MAPK信号转导通路的影响。方法： 雄性昆明种小鼠在注射LPS（5 mg/kg）前48 h及24 h，分别静脉注射GdCl₃（10 mg/kg）或等量的生理盐水，于LPS或生理盐水注射后30 min，分别取出肝脏或分离KCs；体外培养小鼠KCs经GdCl₃（100 μmol/L）预处理1 h，加入含LPS（100 μg/L）的DMEM培养基继续孵育30 min，分别检测肝脏及体内外KCs ERK1/2、p38MAPK蛋白表达和磷酸化水平，及GdCl₃对KCs吞噬、分泌功能的影响。结果： GdCl₃可抑制LPS 诱导KCs活化和TNF-α分泌，但不能抑制LPS诱导KCs或肝脏ERK1/2、p38MAPK磷酸化，也不能影响ERK1/2、p38MAPK蛋白表达，KCs 经GdCl₃单独短时间处理（1 h），可使其少量分泌TNF-α。结论： 封闭KCs并不能通过调节细胞内ERK1/2、p38MAPK信号转导途径，抑制LPS诱导的KCs活化及TNF-α释放，而可能是通过其它信号转导途径（JNK、NF-кB、GPCR等）间的相互作用减轻肝损伤。