Coli surface antigen 6 of enterotoxigenic Escherichia coli. Detection and mucosal binding

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children and travellers in developing countries. The bacteria colonise the intestinal mucosa by means of colonisation factors (CFs) and produce diarrhoegenic heat-labile and/or heat-stable enterotoxins. Hitherto, 21 CFs have been identified. One of these is the afimbrial coli surface antigen 6 (CS6) which is found on a large proportion of ETEC strains in epidemiological studies. The aim of this thesis was to further characterise CS6, i.e. by developing genotypic and phenotypic diagnostic assays, by investigating the binding of CS6 to intestinal mucosa and by studying CS6-specific immune responses in humans.A gene probe, specific for a structural subunit of CS6, and enzyme-linked immunosorbent assays (ELISA) based on polyclonal CS6-specific sera were compared with regard to their capability to identify CS6-positive bacteria. Both methods identified CS6 with high sensitivity and specificity; however, some strains were found to be genotypically positive but phenotypically negative. In an attempt to improve the immunological detection of CS6 even further, CS6-specific monoclonal antibodies were produced and employed in ELISAs and dot blot assays and concordant results were obtained. When using the different CS6-specific antibodies in immunoelectron microscopy surface labelling of CS6-expressing bacteria was seen but no fimbrial or fibrillar structure could be observed.Caco-2 cells and human enterocytes were found to express both similar and unique binding structures for ETEC CFs. Bacteria expressing CS6 did not bind to any of these cells. However, they bound to carbohydrate structures on rabbit ileal and human duodenal enterocytes but only if the enterocytes were isolated using a new approach, i.e., isolated in the absence of EDTA in an attempt to preserve surface-associated structures. CS6, and other CFs, were shown to bind to carbohydrate moieties on both enterocyte and goblet cell derived components of intestinal mucosa, possibly mucus constituents.CS6-specific IgA antibodies were demonstrated, by means of immunoblotting, in faecal extracts and blood of Bangladeshi patients convalescing from natural ETEC disease with CS6-expressing bacteria. Bangladeshi and Swedish volunteers also responded with CS6-specific antibodies in faeces and serum following vaccination with an oral ETEC vaccine containing formalin-inactivated CS6-expressing ETEC bacteria.