Monoclonal antibodies to detect capsular diversity among Bacteroides fragilis isolates

Abstract

The capsular polysaccharide complex (CPC) of Bacteroides fragilis is composed of two distinct polysaccharides, designated PS A and PS B, and is a major virulence factor of this microorganism. In order to investigate the antigenic diversity of the CPCs of B. fragilis strains, we generated and characterized 10 monoclonal antibodies (MAbs) directed to the CPCs of three reference strains. The specificities of the MAbs were determined by enzyme-linked immunosorbent assay and dot-immunobinding assay. At least one MAb was specific for each PS A and PS B of the three strains. The MAbs were used to detect capsular antigens on the surface of 231 B. fragilis isolates from different geographical areas by a whole-cell dot-immunobinding assay. Over half of the strains, regardless of the country of origin, reacted with at least one MAb. Clinical extraintestinal infection isolates were significantly more reactive than fecal isolates, suggesting an association between capsular composition and the propensity to cause clinical infections. The patterns of reactivity of the isolates with the 10 MAbs were very different and sometimes extremely complex and indicated a sharing of epitopes among different capsular polysaccharides. The reactive strains could be grouped according to 32 different patterns; some patterns were relatively common, while others were rarer and were shown by only one or two strains. These results show that B. fragilis capsular polysaccharides are antigenically extremely diverse. This complexity and the large number of nonreactive strains indicate that a typing system based on B. fragilis capsular antigens will be difficult to establish.

Analysis of a Capsular Polysaccharide Biosynthesis Locus ofBacteroides fragilis

“…To locate genes involved in capsular polysaccharide biosynthesis in strain 9343, transposon mutants were generated with B. fragilis 638R. MAb 4D5 (638R capsule specific [33]) was used to screen the mutant bank. The DNA at the junction of the transposon insertion of mutant 2-42 (4D5 negative) was homologous to rmlA, a gene known to be present in various bacterial polysaccharide biosynthesis loci.…”

Vaginal carriage of enterotoxigenic Bacteroides fragilis in pregnant women

“…Studies describing the molecular subtyping of B. fragilis are sparse: the most recent review on the subject, dating back to 1992, proclaims that besides serological assays, exploration of the newer molecular biology-based techniques is mandatory (44). The recent availability of various monoclonal antibodies recognizing capsular antigens shows interisolate degrees of variability too extensive to be helpful in typing studies (38). More recently, the first DNA typing studies were presented.…”

Bacteroides fragilis NCTC9343 Produces at Least Three Distinct Capsular Polysaccharides: Cloning, Characterization, and Reassignment of Polysaccharide B and C Biosynthesis Loci

“…E. coli DH5␣ containing pNJR6⍀4 and the conjugal helper plasmid R751 was mated with B. fragilis US52540 overnight aerobically on BHIS plates, plated onto BHIS containing erythromycin and gentamicin, and incubated anaerobically. The resulting colonies were screened by colony immunoblot with MAb G9 (11).…”