Interactions of Enteropathogenic Escherichia coli with Pediatric and Adult Intestinal Biopsy Specimens during Early Adherence

Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea almost exclusively in young children. The basis for this age discrimination has never been determined, but it may be related to host cell receptors. During infection, EPEC strains express type IV bundle-forming pili composed of repeating subunits of the protein called bundlin. The very first interaction between EPEC and in vitro-cultured epithelial cells is mediated by the binding of α-bundlin to a carbohydrate receptor that contains, at a minimum, the N-acetyllactosamine (LacNAc) glycan sequence. However, bundlins expressed from the β-bundlin allele do not bind LacNAc glycan sequences. Herein, we investigated whether EPEC strains use α-bundlin to mediate early adherence to human intestinal biopsy specimens cultured in vitro by assessing the ability of isogenic EPEC mutants expressing either the α₁- or β₁-bundlin allele or a bundlin-deficient EPEC strain to bind to these specimens. Furthermore, we directly compared the abilities of a wild-type EPEC strain to bind to the epithelial surfaces of both human adult and pediatric biopsy specimens. Our results demonstrate that β-bundlin does not act as an adhesin during early EPEC adherence to adult duodenal biopsy specimens. The results also indicate that EPEC binds equally well to adult and pediatric biopsy specimens in an early adherence assay. This result is supported by the finding that the early adherence of EPEC to both adult and pediatric biopsy specimens was inhibited by LacNAc neoglycoconjugates, suggesting that organisms expressing α-bundlin-type bundle-forming pili initially bind to related glycan receptors in both age groups.Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea in young children, an illness associated with the EPEC-mediated disruption of the small-intestinal epithelium (21, 22, 24). While EPEC strains cause clinical disease predominantly in children under the age of 2 years, disease can also be elicited in adult volunteers given a very high infectious dose of bacteria (1, 7, 9, 11). The basis for this age discrimination by EPEC has never been determined but may be related to either differences in susceptibility to EPEC colonization between adults and infants or to acquired protective immunity from repeated infections in adults.EPEC pathogenesis is dependent on a two-stage mechanism of adherence of the bacteria to host enterocytes. In the first step, EPEC cells bind to the host cell via their type IV bundle-forming pili (BFP) in a process known as localized adherence (LA). Following LA, EPEC strains inject effector proteins and their own receptor, a protein called the translocated intimin receptor (Tir), into the host cell via a type III secretion system. Tir is inserted into the host cell plasma membrane, where it acts as the receptor for the EPEC adhesin intimin and recruits cytoskeletal components, the net effect of which is the effacement of the microvilli and the formation of actin-rich pedestals at the site of EPEC adherence. This is known as the attaching-and-effacing process.BFP are homopolymers of a protein called bundlin. EPEC strains express one of two bundlin types, α or β, based on sequence homology (5). There are currently three α-bundlin alleles and seven β-bundlin alleles that have been described among diverse EPEC strains (4, 5). The α-bundlin proteins are N-acetyllactosamine (LacNAc) lectins, whereas the β-bundlins are not, since they lack the carbohydrate binding domain found in the α-bundlins (13, 16). The divergence between bundlins may be the result of selective immunological pressure, since the β-bundlins are less immunogenic in rabbits and mice than are the α-bundlins (8). It appears that α₁-bundlin is the only adhesin mediating early (45-min) LA of EPEC strain E2348/69 to HEp-2 cells, since replacing the α₁-bundlin allele in this strain with β-bundlin alleles 1 to 3 abolishes early adherence despite the expression of BFP (16). The early LA of this strain, and of all the other α-bundlin allele-expressing EPEC strains tested to date, can also be inhibited by LacNAc conjugated to bovine serum albumin (BSA) or gold nanoparticles (LacNAc-Au) (14-16). We previously reported (14) that LacNAc-based neoglycoconjugates inhibit α₁-bundlin-expressing EPEC LA as a result of their ability to (i) competitively inhibit the binding of the organisms to host cell glycan receptor sequences and (ii) induce BFP retraction, resulting in the dispersal of EPEC autoaggregates. Those strains that harbor the native β-bundlin allele are either nonadherent after a 45-min incubation with HEp-2 cells or not inhibited by LacNAc neoglycoconjugates (16). The inhibition of the α-bundlin allele-carrying strains is also lost when the bacteria are incubated on HEp-2 tissue culture cells for extended periods of time (3 h) (16). These data are consistent with evidence presented previously by Cleary and colleagues (6) that demonstrates a requirement for BFP during EPEC adherence to Caco-2 cells during the first 60 min of infection. After the first 60 min, other adhesins such as intimin and EspA then contribute to EPEC adherence.Recently, significant differences were observed in the mechanism by which EPEC induces attaching and effacing lesions in tissue culture cells compared to ex vivo human intestinal biopsy specimens (3, 20). As such, we sought to determine if the roles that we have demonstrated for bundlin and LacNAc in E2348/69 early LA to intestinal cells grown in tissue culture also apply to early EPEC adherence to human intestinal explants. Of particular interest was whether the β-bundlins, represented in these studies by the β₁-bundlin allele, had completely lost the ability to act as adhesins as a result of selective pressure or whether tissue culture cells simply lack the appropriate receptor for β-bundlin. We also wished to determine if the predilection of EPEC for causing disease in the pediatric population might be related to BFP-mediated early adherence.