Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies

Abstract

Summary. The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule-and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membraneassociated epitopes in the large capsule-and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were speciesspecific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Multiple inverted DNA repeats of Bacteroides fragilis that control polysaccharide antigenic variation are similar to the hin region inverted repeats of Salmonella typhimurium

“…Each of these mAbs reacts with antigenically variable epitopes associated with both the LC and non-capsulate/EDL phases. After PAGE and immunoblotting, these mAbs label HMMPS with associated ladder patterns characteristic of heteropolymeric PS with repeating units (Lutton et al, 1991). QUBF7 reacts with PS C (Coyne et al, 2000) which lacks an invertible region upstream of the biosynthesis locus.…”

“…Immunoblotting of these antigenically variable PSs after PAGE reveals patterns characteristic of heteropolymeric PSs with repeating subunits. Ladders with two different sizes of steps have been observed (Lutton et al, 1991), one with a ladder pattern suggesting that it may be similar to the O-antigen of smooth Gramnegative bacteria identified by some workers (Poxton & Brown, 1986;Delahooke et al, 1995), but not others (Lindberg et al, 1990;Comstock et al, 1999).…”

“…Images were viewed with a Leitz Ortholux fluorescence microscope, a Nikon DMX1200 digital camera and Lucia GF software. PAGE and immunoblotting were carried out as described previously (Lutton et al, 1991).…”

“…Expression of the different capsular types is heritable; populations can be enriched by subculture from different interfaces of Percoll step density gradients (Patrick & Reid, 1983). In addition, antigenic variation of individual capsular types can be demonstrated using mAb labelling; variable proportions of bacterial cells within these populations label with individual mAbs (Lutton et al, 1991). The LC and EDL phases have shared epitopes; however, the SC is antigenically different (Reid et al, 1985;Lutton et al, 1991).…”

“…In addition, antigenic variation of individual capsular types can be demonstrated using mAb labelling; variable proportions of bacterial cells within these populations label with individual mAbs (Lutton et al, 1991). The LC and EDL phases have shared epitopes; however, the SC is antigenically different (Reid et al, 1985;Lutton et al, 1991). mAb labelling indicates that there are potentially six different high-molecular-mass polysaccharides (HMMPS) associated with both the EDL and LC.…”