Contribution of Efa1/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells

Enteropathogenic E. coli(EPEC) is an important diarrhoeal pathogen that induces characteristic lesions on the host intestine termed attaching and effacing (A/E) lesions. In this study we have examined the contribution of a large gene, efa1, which is present in all A/E pathogens, to the adherence phenotype of EPEC. An efa- derivative of EPEC JPN15 was constructed and this mutant was significantly less adherent to epithelial cells than the parent strain. The JPN15 efa- derivative was FAS-positive, produced EspA filaments and showed comparable levels of EspA secretion to JPN15. In addition, polyclonal antibodies raised to Efa1 partially inhibited the adherence of JPN15 to cultured epithelial cells. In further work, we showed that human and rabbit hosts infected with an A/E pathogen produced antibodies to Efa1 and we observed that the truncated form of efa1 present in EHEC O157:H7 was specific to that serotype. Generally efa1 was present in its entirety in the genomes of other A/E pathogens. Overall our data suggest that Efa1 has host cell binding activity, at least in tissue culture, and that it is produced during infection. These findings suggest that Efa1 may play a direct role in the pathogenesis of infections caused by A/E pathogens.     

Cytopathic effects of outer-membrane preparations of enteropathogenic Escherichia coli and co-expression of maltoporin with secretory virulence factor, EspB.

Enteropathogenic Escherichia coli (EPEC) is an important aetiological agent of persistent infantile diarrhoea. EPEC pathogenicity is not mediated through known toxins and the role played by outer-membrane proteins (OMPs) in the initial adherence of the bacterium, intimate attachment to epithelial cells and ultimately in the effacement of the intestinal epithelium is being pursued vigorously. In this study of the different cellular fractions of the bacterium investigated, only the outer-membrane fraction was able to disrupt HEp-2 cells. The outer-membrane fraction was also found to be cytotoxic and caused actin accumulation around the periphery of the host cells. To understand the role of OMPs in pathogenesis, protein profiles of outer-membrane preparations of wild-type and attenuated mutants lacking either the EPEC adherence factor (EAF) mega-plasmid or EPEC attaching and effacing gene A (eaeA) coding for a 94-kDa OMP, intimin or EPEC secretory protein gene B (espB) coding for a 34-kDa translocated signal transducing protein were compared and correlated with their cytopathic effects. A 43-kDa protein seen along with intimin in the outer membrane of EPEC was identified as maltoporin, an E. coli outer-membrane porin normally expressed only in response to maltose in the growth medium. In the case of EPEC, not only was this regulation lost, but also the expression of maltoporin was found to be tightly coupled to the expression of the secretory virulence factor EspB. Maltoporin per se is not toxic, as evidenced by the treatment of HEp-2 cells with the outer-membrane preparation of E. coli DH5a grown in the presence of maltose and the significance of this pathogenic adaptation is not clear. However, when maltoporin and possibly other unidentified proteins were not present as a component of the outer-membrane preparation, as in the outer-membrane preparation of an espB-negative strain, cellular disruption as well as actin accumulation proceeded at a very slow rate even though the cytotoxic effects were comparable to those of the wild-type EPEC strains.     

A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) organisms produce a characteristic histopathology in intestinal epithelial cells called attaching and effacing lesions. The eaeA gene is associated with attaching and effacing lesions and encodes intimin, a 94-kDa outer membrane protein. A 60-MDa plasmid, pMAR2, is essential for full virulence of EPEC strain E2348/69 (O127:H6). We have cloned sequences from pMAR2 that increase expression of the chromosomal eaeA gene as shown by increased alkaline phosphatase activity of an eaeA::TnphoA gene fusion, increased expression of the intimin protein, and increased production of eaeA mRNA. These sequences are called per for plasmid-encoded regulator. pMAR2-cured JPN15 containing cloned per sequences adheres to HEp-2 cells in greater numbers than JPN15 carrying the plasmid vector only. The cloned per sequences contain four open reading frames (ORFs) which have been designated perA through perD. Only perC can by itself activate expression of eaeA::TnphoA, although the levels of alkaline phosphatase activity seen with this ORF alone are considerably lower than those seen when all four ORFs are present. The molecular sizes of polypeptides predicted from perA, perB, perC, and perD ORFs are 24, 14.8, 10.5, and 9.4 kDa, respectively. The PerA predicted protein shares homology with members of the AraC family of bacterial regulators, but PerB, PerC, and PerD have no striking homology with previously described prokaryotic proteins. Our studies indicate that plasmid-encoded factors regulate the expression of eaeA and possibly genes encoding other outer membrane proteins and may be important for virulence of EPEC.     

Lack of correlation between haemagglutination and adherence to epithelial cells in Yersinia pseudotuberculosis

Yersinia pseudotuberculosis was examined for its haemagglutinating activity and adherence to cultured epithelial cells (HEp-2) in relation to possession of a virulence (VW) plasmid and to growth conditions. VW-lacking (VW-) bacteria were isolated from ten VW+ strains of each serovar which, after they were grown on CFA plates at 37 degrees C, agglutinated the erythrocytes from five different species. In contrast to the bacteria possessing the plasmid (VW+) half of the VW- bacteria, grown on CFA plates at 37 degrees C, did not agglutinate any of the erythrocytes used and the other half agglutinated only human erythrocytes. Furthermore, when grown on CFA plates at 25 degrees C, neither VW+ nor VW- bacteria showed a haemagglutinating activity. When the bacteria were grown in CFA broth, only two strains grown at 25 degrees C did not agglutinate any of the erythrocytes tested. The VW+ and VW- bacteria of the remaining strains, grown either at 25 degrees C or 37 degrees C, showed relatively high haemagglutinating activity. Adherence to HEp-2 cells did not correlate with haemagglutinating activity in Y. pseudotuberculosis; the VW+ bacteria grown at 37 degrees C adhered to HEp-2 cells more efficiently than either the VW- derivatives or the VW+ bacteria grown at 25 degrees C, regardless of the growth medium. These results indicate that some of the haemagglutinins detected on Y. pseudotuberculosis are not involved in the adherence to HEp-2 cells.     

Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.

The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.     

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.     

An endocytic process in HEp-2 cells induced by enteropathogenic Escherichia coli

Infection of HEp-2 cells by enteropathogenic Escherichia coli (EPEC) was examined by transmission and scanning electronmicroscopy. EPEC strains of serogroups O111:K58 and O55:K59 recently isolated from human patients did not exhibit enterotoxic activity, as judged by the Vero-cell and suckling-mouse assays, or invasive ability as judged by the Sereny test. These strains attached to and penetrated HEp-2 cells. Transmission electronmicroscopy showed bacteria in close contact with cell membranes 15 min after infection; later, intense swelling and budding of membranes and penetration of EPEC into the cell cytoplasm occurred. Intracellular bacteria were enclosed in membrane-bound vacuoles in the cell cytoplasm underlying localised adherence sites observed by light microscopy. Scanning electronmicroscopy showed morphologically altered membranes only at the sites of bacterial attachment. Bacteria inactivated by ultraviolet light were not internalised and cytochalasin B (greater than or equal to 10 mg/L) markedly inhibited uptake. These observations suggest that penetration of EPEC into HEp-2 cells occurs by an endocytic process in metabolically active bacteria.     

Signal transduction responses following adhesion of verocytotoxin-producing Escherichia coli.

Attaching and effacing adhesion to epithelial cells is a pathognomonic feature of infection by both enteropathogenic Escherichia coli (EPEC) and certain strains of verocytotoxin-producing E. coli (VTEC). EPEC adhesion to tissue culture epithelial cells results in activation of the phosphatidylinositol pathway, with elevated levels of inositol 1,4,5-triphosphate and cytosolic free calcium. In this report, we show that VTEC also activate this signal transduction pathway in infected epithelial cells. Specifically, increased levels of inositol 1,4,5-triphosphate and intracellular free calcium were observed in HEp-2 cells infected with VTEC of serotype O157:H7. VTEC of serotypes O157:H7 and O113:H21 also induced increases in intracellular calcium levels in the human intestinal crypt-like cell line T84, even with minimal or no attaching and effacing activity as monitored by transmission electron microscopy. In contrast to EPEC, VTEC failed to induce tyrosine phosphorylation of epithelial cell proteins in HEp-2 and T84 cells, as determined by indirect immunofluorescence microscopy. These findings suggest that signal transduction responses to VTEC, including elevated levels of inositol triphosphates and intracellular free calcium, are independent of formation of the attaching and effacing lesion. Our findings also show that VTEC pathogenesis may involve signal transduction pathways that are distinct from those induced by EPEC infection.     

Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells

Prebiotic oligosaccharides are thought to provide beneficial effects in the gastrointestinal tract of humans and animals by stimulating growth of selected members of the intestinal microflora. Another means by which prebiotic oligosaccharides may confer health benefits is via their antiadhesive activity. Specifically, these oligosaccharides may directly inhibit infections by enteric pathogens due to their ability to act as structural mimics of the pathogen binding sites that coat the surface of gastrointestinal epithelial cells. In this study, the ability of commercial prebiotics to inhibit attachment of microcolony-forming enteropathogenic Escherichia coli (EPEC) was investigated. The adherence of EPEC strain E2348/69 on HEp-2 and Caco-2 cells, in the presence of fructooligosaccharides, inulin, galactooligosaccharides (GOS), lactulose, and raffinose was determined by cultural enumeration and microscopy. Purified GOS exhibited the greatest adherence inhibition on both HEp-2 and Caco-2 cells, reducing the adherence of EPEC by 65 and 70%, respectively. In addition, the average number of bacteria per microcolony was significantly reduced from 14 to 4 when GOS was present. Adherence inhibition by GOS was dose dependent, reaching a maximum at 16 mg/ml. When GOS was added to adhered EPEC cells, no displacement was observed. The expression of BfpA, a bundle-forming-pilus protein involved in localized adherence, was not affected by GOS, indicating that adherence inhibition was not due to the absence of this adherence factor. In addition, GOS did not affect autoaggregation. These observations suggest that some prebiotic oligosaccharides may have antiadhesive activity and directly inhibit the adherence of pathogens to the host epithelial cell surface.     

Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells.

Plasmid-encoded adherence factors have been shown to be important for the full expression of enteropathogenic Escherichia coli (EPEC) pathogenicity and for EPEC adhesion to cultured HEp-2 cells. EPEC strain E2348 (O127) shows localized HEp-2 cell adhesion and possesses a 60-megadalton plasmid, pMAR2. When E2348 is cured of pMAR2 it loses the ability to adhere to HEp-2 cells, while nonadherent E. coli K-12 strains P678-54 and HB101 acquire HEp-2 adhesiveness after they gain the plasmid. By electron microscopy, E2348 was seen to adhere to HEp-2 cells in a manner that closely resembled EPEC adhesion to intestinal mucosa; bacteria were intimately attached to projections of the apical HEp-2 cell membrane and caused localized destruction of microvilli. The plasmid-containing K-12 strains, on the other hand, did not show intimate attachment and there was no modification of cell surface architecture. It is concluded that plasmid pMAR2 codes for an adhesin, possibly fimbrial in nature, that promotes HEp-2 adhesion but that other chromosomally encoded factors are required for EPEC to achieve the characteristic mode of intimate cell attachment.     

Serotype, antimicrobial resistance, and adherence properties of Escherichia coli strains associated with outbreaks of diarrheal illness in children in the United States.

Since most recorded outbreaks of diarrhea in U.S. infants attributed to Escherichia coli occurred before currently available pathogenicity assays existed, we examined the characteristics of nonenterotoxigenic E. coli strains isolated from 50 outbreaks of diarrheal disease in U.S. infants between 1934 and 1987. We assayed the strains for enteropathogenic E. coli (EPEC) serotype, localized adherence (LA) and diffuse adherence to tissue cultures, the presence of EPEC adherence factor genes, Shiga-like (Vero) toxin production, and antimicrobial resistance. EPEC serotypes were identified in 28 outbreaks (56%). LA to HeLa cells was found in 23 outbreak strains and correlated 100% with the EPEC adherence factor probe. LA was observed in 21 of 28 EPEC and 2 of 22 non-EPEC strains; however, 5 of 23 strains that were LA positive for HeLa cells did not adhere to HEp-2 or HL cells. One strain was diffuse adherence positive, and none was Shiga-like toxin positive. Multiple resistance was common in EPEC (64%), LA-positive (74%), and LA-positive EPEC (76%) strains but not in others (10%). EPEC serotypes or LA was found in 60% (n = 30) of the outbreak strains. The remaining E. coli strains may represent nonpathogenic normal flora, as-yet-undefined pathogens, or pathogens that have lost virulence-associated traits during storage or subculturing.     

Role of Lactosyl Glycan Sequences in Inhibiting Enteropathogenic Escherichia coli Attachment

Previously, we found that asialo-lactosamine sequences served as receptors for enteropathogenic Escherichia coli (EPEC) binding to Chinese hamster ovary (CHO) cells. In the present report, we have extended these earlier results by examining the ability of lactosamine- or fucosylated lactosamine-bovine serum albumin (BSA) glycoconjugates to inhibit EPEC, strain E2348/69, binding to HEp-2 cells. We found that, consistent with our previous findings with CHO cells, N-acetyllactosamine-BSA was the most effective inhibitor of EPEC localized adherence to HEp-2 cells, with Lewis X-BSA being the next best inhibitor. Further investigation revealed that coincubating EPEC E2348/69 with these BSA glycoconjugates alone caused a decrease in the expression of the bundle-forming pilus structural subunit (BfpA) and intimin by the bacteria. BfpA and intimin expression were reduced to the greatest extent by N-acetyllactosamine-BSA and Lewis X-BSA, respectively. These results suggest that the glycoconjugate inhibition of EPEC binding to HEp-2 cells might be achieved, wholly or in part, by an active mechanism that is distinct from simple competitive antagonism of receptor-adhesin interactions.     

Identification of secretory immunoglobulin A antibody targets from human milk in cultured cells infected with enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.     

人气管上皮细胞对绿脓杆菌抗菌作用观察

目的：探讨呼吸道上皮细胞与呼吸道致病菌之间的相互关系,观察呼吸道上皮细胞对绿脓杆菌的抗菌作用。方法：（1）贴壁生长的人呼吸道上皮细胞株HBE-16与绿脓杆菌标准株ATCC27853共孵育,庆大霉素杀死胞外菌,动态观察上皮细胞内活细菌数;（2）HBE-16细胞与绿脓杆菌共同悬浮于细胞培养基,在不同孵育时间点用平板菌落计数法计数活菌数。结果：绿脓杆菌不能在HBE-16细胞内生长,并被细胞逐渐清楚。悬浮状态下的HBE-16细胞对绿脓杆菌有一定杀菌作用。结论：呼吸道上皮细胞对胞内外绿脓杆菌的抗菌作用,可能是呼吸道抵抗细菌感染的一种天然免疫防御机制。     

Characterization of HEp-2 cell projection formation induced by diffusely adherentEscherichia coli

Diffusely adherentEscherichia coli(DAEC) are diarrheagenicE. coliwhose pathogenetic mechanisms are largely unknown. DAEC have been shown to induce an unusual phenotype upon adherence to HEp-2 cells in culture characterized by the induction of long thin membrane processes extending from the cell surface. In addition, DAEC have been shown to be protected from the bactericidal effects of gentamicin when incubated with HEp-2 cells. In our studies, we found that three DAEC strains induced formation of eukaryotic cell processes and were protected from gentamicin killing after a 3 h incubation. Pre-incubation of HEp-2 cells with colchicine or cytochalasin D prior to infection with DAEC strain C1845 resulted in decreased projection formation, suggesting that the effect was dependent upon microfilament and microtubule rearrangement. When the standard gentamicin protection assay was extended for an additional 3 h incubation in the presence of gentamicin, a greater number of DAEC survived gentamicin treatment, more eukaryotic projections were seen in association with the bacteria and the bacteria were actually observed to be «embedded» within these projections. Projection formation was not observed when the bacteria were separated from the cells by a permeable membrane or when the inoculum was inactivated by ultraviolet irradiation. Transposon TnphoAmutants of C1845 were screened for decreased gentamicin protection. All three mutants which were deficient in gentamicin protection demonstrated less projection formation. Insertion mutations affecting gentamicin protection were localized to both the chromosome (two) and a plasmid (one). Eukaryotic projections are a novel interaction of DAEC with epithelial cells, may play a role of the survival of the bacteria against host defenses and may contribute to DAEC pathogenesis. The effect is dependent upon epithelial cell contact and requires multiple bacterial genes.     

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.     

Efficient translocation of EspC into epithelial cells depends on enteropathogenic Escherichia coli and host cell contact

EspC is an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC). The pathogenic role of EspC in EPEC infection is unknown. We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4 degrees C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.     

Invasive Ability of an Escherichia coli Strain Isolated from the Ileal Mucosa of a Patient with Crohn’s Disease

Crohn's disease (CD) is an inflammatory bowel disease in which Escherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC) E. coli strains. Gentamicin protection assays showed that E. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0. 005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- or Shigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.     

Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells.

Enteropathogenic Escherichia coli (EPEC) strains have recently been shown to invade tissue culture cells. We describe a set of 22 Tn5 IS50L::phoA (TnphoA) insertion mutants of EPEC strain E2348-69 that are unable to invade HEp-2 cells. Each mutant was tested for the ability to adhere to and to induce the polymerization of actin in HEp-2 cells. Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was cloned. These studies resulted in the grouping of the mutants into five main categories. These include strains with plasmid and chromosomal insertions that alter adherence, chromosomal insertions that alter the ability to induce actin polymerization, and chromosomal insertions that do not affect adherence or actin polymerization. These studies indicate that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci.