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.     

Diffuse adherence of enteropathogenic Escherichia coli strains

For the identification and characterization of the factor(s) responsible for the diffuse adherence (DA) pattern of enteropathogenic Escherichia coli strains, E. coli strain 2787 isolated from a case of infantile diarrhoea was employed. A plasmid-derived 11-kb fragment was cloned into pBR322. The recombinant plasmid pIB6 was shown to confer the diffuse adherence phenotype on different E. coli K12 strains as well as pIB4, a plasmid with a 9.2-kb insert. The DNA fragment necessary for the expression of the DA phenotype could be reduced to 6.0 kb. Antiserum obtained against pIB4-encoded proteins recognized a surface-associated protein of about 100 kDa in Western blotting. The isolated 100-kDa protein was found to bind to HeLa cells. The antiserum against C600(pIB4) inhibits adherence of E. coli 2787 and C600(pIB6) to HeLa cells. For this reason, the protein is called adhesin involved in diffuse adherence (AIDA-I).     

Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells.

We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells. Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively). Prematurity and serious illness did not alter adherence in newborns. The increased susceptibility of premature newborns to E. coli K1 cannot be explained by enhanced epithelial cell adherence.     

Intimate adherence by enteropathogenic Escherichia coli modulates TLR5 localization and proinflammatory host response in intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease by altering enterocyte physiology and producing mucosal inflammation. Many details concerning the host response against EPEC remain unknown. We evaluated the role of EPEC virulence factors on the inflammatory response through an analysis of bacterial recognition, cell signalling, and cytokine production using an in vitro epithelial cell infection model. Interestingly, we found that EPEC infection recruits Toll-like receptor 5 (TLR5) to the cell surface. We confirmed that type 3 secretion system (T3SS) and flagellin (FliC) are necessary for efficient extracellular regulated kinases 1 and 2 (ERK1/2) activation and found that intimin could down-regulate this pathway. Besides flagellin, intimin was required to keep nuclear factor kappa B (NF-κB) activated during infection. EPEC infection activated tumour necrosis factor alpha (TNF-α) production and induced interleukin (IL)-1β and IL-8 release. Virulence factors such as intimin, T3SS, EspA and fliC were required for IL-1β secretion, whereas intimin and T3SS participated in IL-8 release. Flagellin was essential for late secretion of TNF-α and IL-8 and intimin stimulated cytokine secretion. Initial adherence limited TNF-α release, whereas late attachment sustained TNF-α secretion. We conclude that intimin modulates TLR5 activation and intimate adherence alters the proinflammatory response.     

Differential adherence of Shiga toxin-producing Escherichia coli harboring saa to epithelial cells

The majority of Shiga toxigenic Escherichia coli (STEC) strains isolated from severe STEC disease are those harboring the locus of enterocyte effacement (LEE), which encodes factors involved in adherence to epithelial cells. However, LEE-negative STEC are increasingly isolated from clinical cases. STEC autoagglutinating adhesin (Saa) is widely used as a marker of adhesin in the absence of LEE. In the present study, we compared the adherence of 32 saa-harboring STEC strains to cultured epithelial cells in the absence or presence of d-mannose. In the absence of d-mannose, 19 strains were adherent to HEp-2 and Caco-2 cells, while 12 were non-adherent. One strain showed detachment of epithelial cells. The adherence of 13 strains was sensitive to the presence of d-mannose. The saa mutant of strain T141, in which adherence was mannose resistant, did not show a significant decrease in adherence compared to the wild type, suggesting a Saa-independent mechanism of adherence. saa-harboring STEC exhibited differential binding properties to epithelial cells, which could not be attributed to the number of C-terminal repeats of Saa, or to the expression of Saa as detected by Western blotting. Our results suggest that multiple adherence mechanisms are present in saa-harboring STEC, implying a high degree of diversity in this group of STEC.     

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.     

Contribution of long polar fimbriae to the virulence of rabbit-specific enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major of cause of diarrhea among children in developing countries. Although EPEC is a human specific pathogen, some related strains are natural pathogens of animals, including laboratory-bred rabbits. We have identified two chromosomal loci in rabbit-specific EPEC (REPEC) O15:H- strain 83/39, which are predicted to encode long polar fimbriae (LPF). lpf(R154) was identical to a fimbrial gene cluster, lpf(O113), identified previously in enterohemorrhagic E. coli (EHEC) O113:H21. The second locus, lpf(R141), comprised a novel sequence with five predicted open reading frames, lpfA to lpfE, that encoded long fine fimbriae in nonfimbriated E. coli ORN103. The predicted products of lpf(R141) shared identity with components of the lpfABCC'DE gene cluster from EHEC O157:H7, and the fimbriae were similar in morphology and length to LPF from EHEC O157:H7. Interruption of lpf(R141) resulted in significant attenuation of REPEC 83/39 for rabbits with respect to the early stages of colonization and severity of diarrhea. However, there was no significant difference in the number of bacteria shed at later time points or in overall body weight and mortality rate of rabbits infected with lpf(R141) mutant strains or wild-type REPEC 83/39. Although rabbits infected with the lpf(R141) mutants did not develop severe diarrhea, there was evidence of attaching and effacing histopathology, which was indistinguishable in morphology, location, and extent compared to rabbits infected with wild-type REPEC 83/39. The results suggested that lpf(R141) contributes to the early stages of REPEC-mediated disease and that this is important for the development of severe diarrhea in susceptible animals.     

Enhancement of Escherichia coli adherence to epithelial cells derived from estrogen-stimulated rats.

The effect of exogenous estrogen administered to male and oophorectomized female rats was investigated with regard to in vitro adherence of eight uropathogenic strains of Escherichia coli to exfoliated bladder and vaginal epithelial cells. Uroepithelial cells obtained from estrogenized male and estrogenized oophorectomized female rats and vaginal cells obtained from estrogenized oophorectomized female rats demonstrated significantly enhanced (P less than 0.005) host cell avidity for E. coli attachment, irrespective of bacterial adhesin expressed, when compared with such cells from nonestrogenized male and female oophorectomized rats. These animal studies suggest that female reproductive hormones may contribute to urinary-tract infection in premenopausal females by enhancing susceptibility to E. coli colonization of uroepithelial cells.     

Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli.

Volunteer studies have shown that a 60-megadalton plasmid is required for full virulence of the human enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6). The plasmid, designated pMAR2, encodes localized adherence to HEp-2 cells in tissue culture via the adhesin known as the EPEC adherence factor (EAF). Using a DNA probe for the EAF, we have previously shown that these genes are specific for EPEC and are usually encoded on plasmids ranging from 55 to 65 megadaltons. In this study, Southern blot analysis and S1 nuclease homology determination reveal a high degree of sequence conservation among these plasmids, despite some variation in restriction maps. Phenotypic characterization of the prototype EAF plasmid pMAR2 reveals that the plasmid belongs to the group IncFII and is negative for alpha-hemolysin, colicin, and aerobactin synthesis, as well as biochemical markers and antibiotic resistance. Regions encoding adherence to HEp-2 cells were localized by Tn801 insertion mutagenesis. Adherence genes were then cloned as two distinct plasmid regions which confer the adherence phenotype only when complementing each other in trans.     

Cytoskeletal composition of attaching and effacing lesions associated with enteropathogenic Escherichia coli adherence to HeLa cells.

The cytoskeletal lesions associated with enteropathogenic Escherichia coli adhering to cultured HeLa epithelial cells were examined by immunofluorescence microscopy. The microfilament-associated proteins actin, alpha-actinin, talin, and ezrin were localized with adherent enteropathogenic E. coli, whereas tropomyosin, keratin and vimentin (intermediate filaments), tubulin (microtubules), and vinculin were not localized. These cytoskeletal structures differed significantly from those associated with Salmonella typhimurium internalization (invasion).     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits.

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

Isolation and characterization of the localized adherence factor of enteropathogenic Escherichia coli.

The binding factor of enteropathogenic Escherichia coli O111:H- responsible for localized adherence (LA) on HeLa cells was investigated. Inhibition of LA by carbohydrates and lectins showed that the reactive epitope on HeLa cells contains N-acetylgalactosamine units. Treatment of bacteria with EDTA for extraction of lipopolysaccharides eliminated these polymers as binding factors. Such treatment also caused a marked increase in adhesion suggesting steric hindrance by lipopolysaccharides of the LA factor binding capacity. Immunoblotting with rabbit antibodies showed a strong reaction with two components with approximate molecular sizes of 29 and 32 kilodaltons (kDa) present in the outer membrane preparations of bacteria. Both the absorbed rabbit immune serum and the outer membrane preparation of the bacteria inhibited bacterial adhesion by 100%. Outer membrane components were isolated from an N-acetylgalactosamine-agarose column by elution with KSCN, labeled with 125I, and immunoprecipitated with absorbed rabbit hyperimmune antiserum. The only component precipitated was the protein doublet at 29 to 32 kDa corresponding to the components detected by immunoblotting. The predominant component was always the 32-kDa polypeptide. We conclude that this component of the outer membrane is the best candidate for the LA factor in enteropathogenic E. coli.     

Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per. Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. ISEc13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.     

Effect of carbon source on localized adherence of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) strains attach to epithelial cells as discrete clusters of bacteria which are localized at a few sites on the cell surface. Previously, it was shown that this localized-adherence (LA) phenotype is induced by specific growth conditions. We found that wild-type EPEC attached to HEp-2 cells in an LA pattern when the bacteria were grown in Dulbecco's modified Eagle medium (DMEM) containing glucose as the carbon source. In contrast, bacteria incubated in DMEM containing galactose did not adhere to epithelial cells. The latter results were similar to those observed when JPN15, an LA-negative strain, was grown under conditions which promoted bacterial binding. The differences in attachment of wild-type EPEC were independent of the stage of log-phase growth of the cultures and of the number of CFU incubated with the HEp-2 monolayers. Expression of the adherence phenotype by organisms grown in glucose was associated with increased expression of intimin and bundle-forming pilin. In contrast, bacteria grown in medium containing galactose expressed these proteins at levels similar to those observed when JPN15 was grown in medium containing glucose.     

Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium.     

Interaction of enteropathogenic or enterohemorrhagic Escherichia coli with HeLa cells results in translocation of cortactin to the bacterial adherence site

Infection of cultured HeLa epithelial cells with enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic E. coli (EHEC) O157:H7 results in accumulation of cortactin under the adherent bacteria. Tyrosine phosphorylation of cortactin is not induced following HeLa cell infection with EHEC or EPEC, contrary to what has been reported to occur with Shigella flexneri.     

Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli.

Histopathological evidence suggests that the adherence of enteropathogenic Escherichia coli (EPEC) to the mucosa of the small bowel is an important step in pathogenesis. Several reports have shown that many EPEC isolates adhere to HEp-2 and HeLa cells in tissue cultures. In the HeLa cell assay, there are at least two distinct patterns of adherence: localized adherence, which is characterized by the formation of bacterial microcolonies, and diffuse adherence, in which bacteria cover the cell uniformly. We have found that these two patterns can be demonstrated in HEp-2 cells as well as in HeLa cells and that the results of the two assays are closely correlated. Using a DNA probe which is sensitive and specific for localized adherence to HEp-2 cells, we provide evidence that localized adherence and diffuse adherence by EPEC are due to at least two genetically distinct adhesions which confer phenotypic differences in both the morphology of HEp-2 cell adherence and in surface hydrophobicity. The two factors are each encoded on plasmids which vary in size from 55 to 70 megadaltons; one strain exhibiting localized adherence carried these genes on the chromosome.     

Quantitation of the adherence of an enteropathogenic Escherichia coli to isolated rabbit intestinal brush borders.

Two assays were developed to quantitate the adherence of an Escherichia coli strain (RDEC-1) known to colonize the mucosal surface of the small intestine of rabbits to brush borders isolated from rabbit intestinal epithelial cells. In the first assay, the mean adherence per rabbit brush border was determined by counting the number of organisms adhering to each of 40 brush borders under phase microscopy. The mean adherence of RDEC-1 (11.5 +/- 0.7 per rabbit brush border) was significantly greater than adherence of two nonpathogenic strains: HS (2.7 +/- 0.4 per rabbit brush border) and 640 (0.8 +/- 0.1 per rabbit brush border). A similar distinction between the adherence of RDEC-1 and the control (nonadherent) organisms could be made more rapidly by determining the percentage of the total number of brush borders which had 10 or more adherent organisms; this second assay was used to define the optimum conditions for adherence. Maximum adherence was seen within 15 min. Adherence was temperature dependent, with adherence after 1 min at 37 degrees C being fourfold greater than that at 4 degrees C. The pH optimum for adherence was between 6.5 and 7.0, and adherence was abolished below pH 5.0. With the first, more sensitive assay, the effect of electrolytes and a number of hexoses and hexosamines on adherence was analyzed. RDEC-1 adherence was inhibited at high ionic strengths; however, adherence was not influenced at moderately high concentrations (20 mg/ml) by either d-mannose or l-fucose, in contrast to the case for other reported enteric pathogens. These two quantitative in vitro assays for adherence produce consistent results and have been used to partially characterize the adherence of RDEC-1 to rabbit brush borders.     

Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.     

Inhibitory effect of substituted aromatic hydrocarbons on adherence of Escherichia coli to human epithelial cells.

Adherence of Escherichia coli to epithelial cells is postulated to be a necessary step in the pathogenesis of urinary tract infections. Investigations have focused on the role of carbohydrates in adherence because of the ability of mannose to inhibit adherence; however, there have been several reports of an additional hydrophobic receptor. Here we describe the inhibition of adherence mediated by substituted aromatic hydrocarbon compounds and the relationship of our findings to support the role of a postulated hydrophobic-binding domain.