1.6.1.1 HEp-2 cell adherence
In 1979, Cravioto et aL, showed that the majority o f EPEC outbreak strains could adhere to HEp-2 human epithelial cells in tissue culture in the presence of D-mannose. Attachment of these outbreak strains to HEp-2 cells was later found to be encoded on a large 55-60 MDa plasmid (Baldini et al., 1983). The name EPEC adherence factor (EAF) was given to this property and through volunteer studies the EAF adhesion system was shown to be important in the ability o f EPEC to cause diarrhoea. EPEC adhere to HEp-2 cells in two distinct patterns, termed localised adherence (LA) and diffuse adherence (DA), (Scaletsky et aL, 1984; Nataro et a l, 1985a). EPEC showing LA, adhere in discrete microcolonies to localised areas of the HEp-2 cell surface, whereas those showing DA adhere to the entire cell surface. LA and DA are encoded for by different plasmids and use of a 1 kilobase (Kb) EAF DNA probe derived from a 55 MDa EAF plasmid, pMAR2 from strain E2348, showed that the LA phenotype was EAF plasmid encoded (Nataro et aL, 1985a). Nataro et al. (1985b) subdivided the EPEC category into two classes on the basis of hybridisation with the 1 Kb EAF probe. Class I EPEC (EAF+ ) exhibit LA to HEp-2 cells, whereas class II EPEC (E A F)
exhibit either DA or no adherence at all to HEp-2 cells. Both class I and class II EPEC are associated with diarrhoeal disease. An ELISA using antiserum to EAF plasmid- associated factors has subsequently been developed for the detection of classic EPEC displaying LA (Albert et al., 1991). In diarrhoeagenic E. coli strains exhibiting the DA phenotype at least two different adhesins have so far been identified. Bilge et aL (1989) have characterised a region of chromosomal DNA coding for a fimbrial adhesin. More recently the cloning and expression of a 6 Kb DNA fragment from a 100 Kb (2 MDa) plasmid harboured by EPEC clinical isolate 2787 which confers the DA phenotype to recipient E. coli K12 strains, as well as the identification of a 100 KDa protein (AIDA-I- adhesin involved in diffuse adherence) as the DA-mediating adhesin has been described (Benz & Schmidt, 1989, 1992). Serologically related proteins o f similar size were detected by Western blotting in other DA+ diarrhoea-associated EPEC strains (Benz & Schmidt, 1992).
HEp-2 cell adherence was originally suggested to be a good model of in vivo EPEC adherence (Baldini et al., 1983). However, in the original description of HEp-2 cell adherence by Cravioto et aL (1979), 29% of 17 non-EPEC, non-ETEC strains that were possible causes of five diarrhoeal outbreaks were adherent to HEp-2 cells. Mathewson
et aL (1985) reported that nontoxigenic, HEp-2 cell adherent strains of E. coli displaying
LA or DA and which did not belong to the recognised EPEC serogroups were an important cause of travellers' diarrhoea. They proposed, therefore that HEp-2 cell adherence may be a virulence factor in E. coli strains independent of serogroup, and for this reason Mathewson proposed that HEp-2 cell-adherent E. coli strains be referred to as enteroadherent E. coli (EAEC), in order to avoid reference to and confusion with strains belonging to EPEC serogroups. More recently evidence suggests that EAEC are identifiable by a particular pattern of adherence to Hep-2 cells that is clearly distinguishable from both LA and DA (Levine, 1987).
1.6.1.2 Attaching and effacing activity
In vivo studies have shown that EPEC strains adhere avidly to the mucosa of the small
intestine with local destruction of microvilli and pedestal formation (Moon etal., 1983). The characteristic attaching and effacing (A /E ) lesion produced is thought to be responsible for the diarrhoea associated with EPEC (Robins-Browne, 1987) and strains producing this type of lesion have been referred to as attaching and effacing E. coli (AEEC).
Levine et al. (1985) showed that a 94 KDa outer membrane protein was detected in wild type EPEC strains but not in EAF plasmid-cured derivatives of these strains, suggesting that the 94 KDa membrane protein of EPEC may be encoded for by the EAF plasmid. However, an isogenic EPEC strain cured of the pMAR2 plasmid continued to demonstrate A /E adherence to HEp-2 cells (Knutton et aL, 1987a) and enterocytes in organ culture (Knutton et al., 1987b). Others have also indicated that adherence of EPEC to HEp-2 cells does not depend on the expression of fimbrial adhesins (Scotland
et aL, 1983b). Knutton et al. (1987a, b) provided evidence that the LA to HEp-2 cells
and the A /E adherence are two genetically distinct phenomena. They proposed a two stage model for EPEC adherence with a fimbrial mediated initiation step (plasmid encoded and responsible for HEp-2 LA) and a subsequent phase of A /E adherence (encoded for by genes on the chromosome). It was evident that the second stage can occur in the absence of the first stage, but the presence of plasmid-encoded adhesins appears to greatly enhance the ability o f EPEC to colonise the mucosa (Tzipori et a t, 1989). In addition to enabling initial colonisation, the EAF adherence plasmid may provide tissue specificity for human epithelial cells (Jerse et aL, 1991). Studies using
TnphoA mutagenesis indicated that the 94 KDa outer membrane protein was encoded
by a chromosomal locus (eae - E. coli A /E ). Furthermore the eae gene has been shown to be necessary for A /E activity on human tissue culture cells (Jerse et aL, 1990) and to share significant homology to invasin, a known virulence factor of Yersinia
pseudotuberculosis (Jerse & Kaper, 1991). Expression of the eae gene was found to be
positively regulated by the EAF plasmid (Jerse & Kaper, 1991). This may explain the earlier observations that in vivo (Knutton et aL, 1987b) and in vitro (Tzipori et al., 1989), increased numbers of A /E lesions are produced by EPEC strains that possess the EAF plasmid.