Immunochemical and biological characterization of three capsular polysaccharides from a single Bacteroides fragilis strain.

Although Bacteroides fragilis accounts for only 0.5% of the normal human colonic flora, it is the anaerobic species most frequently isolated from intra-abdominal and other infections with an intestinal source. The capsular polysaccharides of B. fragilis are part of a complex of surface polysaccharides and are the organism's most important virulence factors in the formation of intra-abdominal abscesses. Two capsular polysaccharides from strain NCTC 9343, PS A1 and PS B1, have been characterized structurally. Their most striking feature is a zwitterionic charge motif consisting of both positively and negatively charged substituent groups on each repeating unit. This zwitterionic motif is essential for abscess formation. In this study, we sought to elucidate structural features of the capsular polysaccharide complex of a commonly studied B. fragilis strain, 638R, that is distinct from strain 9343. We sought a more general picture of the species to establish basic structure-activity and structure-biosynthesis relationships among abscess-inducing polysaccharides. Strain 638R was found to have a capsular polysaccharide complex from which three distinct carbohydrates could be isolated by a complex purification procedure. Compositional and immunochemical studies demonstrated a zwitterionic charge motif common to all of the capsular polysaccharides that correlated with their ability to induce experimental intra-abdominal abscesses. Of interest is the range of net charges of the isolated polysaccharides-from positive (PS C2) to balanced (PS A2) to negative (PS 3). Relationships among structural components of the zwitterionic polysaccharides and their molecular biosynthesis loci were identified.

Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis.

Zwitterionic capsular polysaccharides from pathogenic bacteria have peculiar immunological properties. They are capable of eliciting T-cell proliferation and modulating the course of abscess formation. To understand the molecular basis of this characteristic immune response, we are conducting detailed structure-function studies on these polysaccharides. We have identified, purified, and characterized an abscess-modulating polysaccharide, PS A2, from the clinical strain Bacteroides fragilis 638R. Here, we report the elucidation of both the chemical and three-dimensional structures of PS A2 by NMR spectroscopy, chemical methods, gas chromatography-mass spectrometry, and restrained molecular dynamics calculations. PS A2 consists of a pentasaccharide repeating unit containing mannoheptose, N-acetylmannosamine, 3-acetamido-3,6-dideoxyglucose, 2-amino-4-acetamido-2,4,6-trideoxygalactose, fucose, and 3-hydroxybutanoic acid. PS A2 is zwitterionic and carries one cationic free amine and one anionic carboxylate in each repeating unit. It forms an extended right-handed helix with two repeating units per turn and a pitch of 20 A. Positive and negative charges are exposed on the outer surface of the polymer in a regularly spaced pattern, which renders them easily accessible to other molecules. The helix is characterized by repeated large grooves whose lateral boundaries are occupied by the charges. The three-dimensional structure of PS A2 explicitly suggests mechanisms of interaction between zwitterionic polysaccharides and proteins.

Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.

Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced. Ex vivo expanded hEPCs may thus have utility as a "supply-side" strategy for therapeutic neovascularization.

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity.

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.