The purpose of this project is to define pathogenic mechanisms employed by group B Streptococcus (GBS) to abrogate a specific host defense system, the classical complement pathway. Incubation of a soluble type Ia or III GBS capsular polysaccharide in human serum leads to Cl activation and depletion of C4. The mechanism by which the type Ia polysaccharide activates Cl will be defined by use of purified Clq, Clr and Cls and purified polysaccharides. Clq, Clr and Cls will be purified from human plasma by euglobulin precipitation and further purified by gel filtration. The Cl subcomponents will be identified by SDS-PAGE and Western Blot analysis. Polysaccharide activation of Cl will be measured by Cl esterolytic activity and by cleavage of C4, the natural substrate of Cl. Each subcomponent will be incubated with the polysaccharide to identify the site of Cl activation. Next the site on the polysaccharide that activates Cl will be identified by use of native and chemically degraded capsular polysaccharides. These degraded polysaccharides will be used in the Cl activation assays as just described. The relationship of the site on the GBS capsular polysaccharide that activates Cl and the site on the polysaccharide that promotes GBS virulence will be examined in a neonatal rat model. Rats will be injected with native or degraded capsular polysaccharides and challenged with type Ia GBS. Changes in virulence will be assessed by the number of CFU in CSF and serum and by mortality. After identification of the mechanism of fluid phase complement activation by soluble GBS polysaccharides, the mechanism by which the classical pathway is activated by whole type Ia organisms will be investigated. These studies will employ a classical complement pathway-dependent radiolabeled bacterial uptake assay. H3 type Ia will be incubated with purified Cl subcomponents. The classical pathway will be completed by use of a Cl depleted reagent and association with polymorphonuclear leukocytes (PMN's) measured. The site of complement activation on the bacterial surface will be examined by chemically altering the organisms' surface structures and assaying the organisms for uptake by PMN's. These studies are directed at identification of mechanisms of GBS pathogenesis. Advances in this area will lead to better understanding of GBS disease, in general, and potentially to better methods of prevention and treatment in the future.
| Levy, N J; Kasper, D L (1986) Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway. J Immunol 136:4157-62 |
