Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis

Abstract

Bacteroides fragilis, though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses. B. fragilis 9343 expresses at least three capsular polysaccharides-polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50 B. fragilis isolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature of B. fragilis.

FIG. 1

(A) Cosmids and subclones used in sequencing the 9343 PS A locus. B, BamHI; E, EcoRI; P, PstI; B/S, compound site resulting from cloning Sau3AI-digested DNA into the vector BamHI site. (B) Diagrammatic representation of the PCRs showing the positions of the primers and the span of the product. PCRs marked with an asterisk were successful in more than 98% of the strains tested. (C) ORF map of the B. fragilis 9343 PS A locus. The region deleted in mutant strain 9343ΔPSA and the positions of the primers used for EPCR are indicated above the ORFs. Flanking ORFs not contributing to the production of PS A are shaded. Arrows within the ORFs indicate direction of transcription. (D) G+C content of each of the 9343 PS A locus ORFs.

FIG. 2

Western immunoblot of B. fragilis wild-type and mutant strains. Bacterial cultures were grown to an optical density at 600 nm of 0.8, pelleted by centrifugation, and lysed by boiling in 1× loading buffer. Bacteria equivalent to 40 μl of the original culture were added to each well of the 4-to-20% gradient SDS-polyacrylamide gel. After separation, the samples were transferred to polyvinylidene difluoride membranes and probed with PS A-specific MAb CE3 (A), PS B-specific MAb G9 (B), and PS C-specific MAb QUBF7 (C). Lanes (all panels): 1. B. fragilis 9343; 2. 9343ΔPSA; 3. B. fragilis B16; 4. B. fragilis B16 mutant 65.