Effacement Of Microvilli Research Articles

Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins.

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions.

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR: four of these nine were eaeB positive. The 5' end, but not the 3' end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.

Distinct binding properties of eaeA-negative verocytotoxin-producing Escherichia coli of serotype O113:H21.

Infection of humans with verotoxin-producing Escherichia coli (VTEC) O113:H21 is associated with clinical features comparable to those associated with infection with attaching and effacing VTEC strains including those of serotype O157:H7. We have shown previously that the adhesion phenotype of VTEC O157:H7 is influenced by the presence of a homolog of the chromosomal eaeA (for E. coli attaching and effacing) gene. In contrast, by colony blot hybridization, VTEC O113:H21 is negative for the eaeA gene. Therefore, the aim of this study was to define the adhesion phenotype of VTEC O113:H21 strain CL-15 to both cultured epithelial cells (HEp-2) and rabbit intestine in vivo. Under transmission electron microscopy, areas of microvillus effacement were observed in regions directly beneath the organism in CL-15-infected cells both in vitro and in vivo. However, F-actin adhesion pedestals on the host plasma membrane were absent. Failure of CL-15 to induce polymerization of actin was confirmed by using staining of F-actin with fluorescein-labeled phalloidin. Under indirect immunofluorescence microscopy, CL-15-infected HEp-2 cells also failed to demonstrate the recruitment of another cytoskeletal element, alpha-actinin, below foci of bacterial adhesion. In contrast, VTEC O157:H7 infection of HEp-2 cells was associated with increased alpha-actinin immunofluorescence. These findings suggest that bacterial factors distinct from those of EaeA are necessary for the adhesion phenotype of VTEC O113:H21.

The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model.

The eaeA gene of enteropathogenic Escherichia coli (EPEC) is necessary for intimate attachment to epithelial cells in vitro. Enterohemorrhagic E. coli (EHEC) strains also possess an eae gene and are capable of intimate attachment and microvillus effacement in vitro and in animal models. To assess the role of the EHEC eae gene in intimate attachment, we constructed an eae deletion/insertion mutation in wild-type EHEC O157:H7 strain 86-24 by using linear electroporation of a recombinant allele. The mutant obtained was deficient in inducing f-actin accumulation in HEp-2 cells and was incapable of attaching intimately to colonic epithelial cells in a newborn piglet model of infection. Intimate attachment in vivo was restored when the EHEC eae gene or the eaeA gene of EPEC was introduced into the mutant on a plasmid. These results indicate that the eae gene is necessary for intimate attachment of EHEC in vivo. In addition, the complementation achieved by the EPEC locus indicates that the eae gene of EHEC and the eaeA gene of EPEC are functionally homologous.

Enteropathogenic Escherichia coli.

Abstract Enteropathogenic Escherichia coli (EPEC) strains are a leading cause of diarrhea among infants in developing countries. These bacteria spread via fecal–oral transmission and colonize the small intestine. Typical EPEC differs from atypical strains in that they possess the bfp operon and generate a type IV-B class of fimbriae known as the bundle-forming pilus (BFP). EPEC uses BFP to attach to intestinal epithelium. Upon attachment, EPEC translocates effector proteins into the host cell via a type 3 secretion system, which is encoded on a pathogenicity island known as the locus of enterocyte effacement. Translocated effectors modulate the host response in a number of ways, including subversion of immune responses and hijacking of the cytoskeletal system. By inducing actin rearrangement, EPEC causes the formation of actin pedestals onto which it sits, and leads to the effacement of microvilli, giving this bacterium a characteristic attaching and effacing phenotype. EPEC infections are typically self-limiting, with rehydration therapy constituting primary treatment, but they can be protracted and lethal. Breastfeeding provides a great protective effect, validating the use of a vaccine to induce a humoral response in mother or child as a potential means of preventative action. This chapter provides a detailed discussion on EPEC pathogenesis and host cell responses and summarizes the current detection and treatment options available.

HeLa cell adherence, actin aggregation, and invasion by nonenteropathogenic Escherichia coli possessing the eae gene

Enteropathogenic Escherichia coli (EPEC) produce diarrhea in humans by a mechanism that involves close adherence to epithelial cells in the intestine and colon. Close adherence is associated with effacement of microvilli and condensation of actin beneath the bacteria, a process termed attaching/effacing adherence. Attaching/effacing adherence of EPEC occurs in vitro in tissue culture, simplifying the study of the molecular genetics of this process. An EPEC gene (eae) necessary for attaching/effacing adherence was recently characterized. Enterohemorrhagic E. coli and the rabbit-specific RDEC-1 strain adhere in a like fashion in vivo and hybridize with eae. However, these strains adhere poorly to tissue culture cells, complicating the in vitro study of attaching/effacing adherence. In order to develop an in vitro model for the study of attaching/effacing activity of non-EPEC bacteria, a plasmid encoding the F1845 adhesin of an E. coli strain (C1845) isolated from a patient with diarrhea was transformed into RDEC-1 and enterohemorrhagic E. coli. The transformed strains adhered in a diffuse pattern to HeLa cells, and they aggregated HeLa cell actin at points of adherence in the fluorescein-isothiocyanate-labeled phalloidin assay. They also invaded HeLa cells in a gentamicin invasion assay, although not to the extent seen with EPEC. The construction of adherent non-EPEC strains facilitates the molecular study of the attaching/effacing properties and invasiveness of these strains in tissue culture models.

Occurrence of 'attaching and effacing' lesions in the small intestine of calves experimentally infected with bovine isolates of verocytotoxic E coli.

Nine calves (five colostrum-fed, four colostrum-deprived) were challenged with two field strains of Escherichia coli which produced either verocytotoxin 1 (VT1) or verocytotoxin 2 (VT2). Although three colostrum-fed calves had blood and mucus in their faeces, no diarrhoea was observed. Three of the four colostrum-deprived calves had diarrhoea and in two of them severe lesions were detected in the small intestine. Focal changes were detected in the colon of three calves. E coli were associated with the lesions in the small and large intestine and were shown by transmission electron microscopy to be intimately attached to the enterocyte surface with effacement of microvilli (attaching and effacing lesions). This is the first report of E coli which produce VT2 being associated with disease in calves.

Molecular evolution of pathogenic Escherichia coli.

Over the past four decades, Escherichia coli bearing certain of the 164 known somatic O antigens of the genus have been shown to be associated with intestinal infections. These strains may be termed enterovirulent E. coli (EVEC). Only a small percentage of the ^9000 0:H serotypes possible are known to be enteric pathogens. Detailed studies of the pathogenic mechanisms involved permit the separation of EVEC into four groups [1]: (7) enterotoxigenic E. coli (ETEC) that adhere to the mucosa of the small bowel and produce heat-labile (LT) toxin, heat-stable (ST) toxin, or both; (2) enteroinvasive E. coli (EIEC) that produce neither LT nor ST but, like Shigella, penetrate and multiply within the epithelial cells lining the colon; and (5) enteropathogenic E. coli (EPEC) that adhere to the mucosa of the small intestine and produce a characteristic effacement of microvilli. The latter are not invasive and do not produce LT or ST. The fourth group has been termed enterohemorrhagic E. coli (EHEC); this group causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) and are of the serotype 0157:H7. In this issue of the Journal, Whittam et al. [2] present genetic evidence of a clonal descent of E. coli 0157:H7, the strain associated with hemorrhagic colitis and HUS. This report suggests that 0157:H7 is a newly evolved strain originating from a single ancestor. Until recently, 0157:H7 was a rare serotype that was seldom isolated from humans and animals. When isolated during outbreaks of hemorrhagic colitis, these strains have been shown to be neither invasive nor enterotoxigenic. This serotype also was not a recognized enteropathogenic serotype. However, 0157:H7 strains produce high levels of Shigalike toxin (SLT), which is related to Shiga toxin of Shigella dysenteriae type 1 [3]. These toxins also have

Pathogenicity of attaching effacing enteropathogenic Escherichia coli isolated from diarrheic suckling and weanling rabbits for newborn rabbits.

The pathogenicity of six strains of Escherichia coli originating from different commercial rabbitries was tested in neonatal rabbits. Two strains isolated from healthy weaned rabbits (O7:H6 and O9:H?) did not induce any clinical sign or lesion. Two strains (O109:H2) isolated from diarrheic suckling rabbits caused yellow diarrhea 36 to 60 h after inoculation and high mortality between 60 and 72 h after infection. At 12 h after infection, light and electron microscopy showed attachment to epithelial cells and effacement of microvilli from proximal small intestine to colon. Bacteria were often present in the apical cytoplasm of epithelial cells. The two strains isolated from diarrheic weanling rabbits (O109:H2 and O15:H-) did not induce any clinical sign. Attachment to epithelial cells and effacement of microvilli was observed 48 h after inoculation in distal small intestine, cecum, and colon. These data are further evidence for the existence of two groups of attaching effacing enteropathogenic E. coli in rabbits, showing different preferences for age group and intestinal compartment.

Pancreatic acinar ultrastructure in human acute pancreatitis.

Ultrastructural alterations in pancreatic acini from six patients operated for acute necrotizing pancreatitis are described. One of the patients suffered from biliary tract disease, the rest had excessive alcohol intake as the presumed aetiology. Areas of the pancreatic parenchyma showing oedematous inflammation in light microscopy were studied in the electron microscope. Findings in acinar cells included changes in zymogen granules and an increased autophagocytosis in addition to unspecific organelle alterations. Zymogen granules showed increase in size and number, loss or variation of electron-density and peripheral dissolution. Increased autophagic activity was indicated by several autophagic vacuoles and residual bodies. Acinar lumina were dilated showing effacement of microvilli and invaginations in the luminal plasma membrane of the acinar cells. In acinar lumina and in the interstitium fibrillar material was observed, with an increasing frequency in those areas showing severe cellular disintegration. These findings suggest: 1) an increased activity of zymogen granules, 2) an increased autophagocytosis, and 3) penetration of acinar luminal contents into the interstitium.