Doctoral Dissertations

Fall 1997

Dissertation

Microbiology

Degree Name

Doctor of Philosophy

Thomas Pistole

Abstract

Macrophages have been shown to bind pathogenic microorganisms in the absence of exogenous opsonins, but the precise mechanisms are poorly understood. Microbial recognition by macrophages under these conditions becomes particularly important in individuals who have decreased amounts of opsonins such as immunocompromised individuals and neonates. This study was undertaken to investigate the mechanisms of adherence between group B Streptococcus and macrophages in opsonin-limiting conditions. Using several adherence assays; visual binding, enzyme-linked immunosorbent, and flow cytometry, GBS was able to adhere to U937 cells, a human macrophage-like cell line, in a dose-dependent manner without exogenous opsonins.

The $\beta\sb2$ integrins, a heterodimeric family of leukocyte receptors, have been implicated in adherence of GBS to macrophages in the absence of opsonins. GBS bound to CD18-deficient bovine polymorphonuclear leukocytes (PMNs) almost as well as bovine PMNs with normal CD18 surface expression, suggesting that CD18 is not essential for adherence and that additional receptors are involved in adhesion. In soluble binding assays, three macrophage proteins (60, 55 and 20 kDa) were found to bind to GBS. The results were confirmed when the experiments were performed using Listeria monocytogenes. In competition assays, the organisms competed for attachment to U937 cells. In addition, Listeria bound to the same U937 proteins as did GBS. These results support the roles of proteins in the interaction of GBS and Listeria to macrophages.

Because adherence involves participation from the host and the bacteria the present study was expanded to investigate the role of GBS envelope proteins in the adherence to macrophages. The GBS adhesin for macrophages was identified using a soluble binding assay. A 21 kDa protein from GBS was shown to bind to U937 cells. Antisera to the 21 kDa protein was able to inhibit adhesion of GBS to U937 cells as measured using flow cytometry. This antisera was able to cross-react with a number of GBS strains and additional Gram positive bacteria, suggesting, this may be a conserved adhesin.

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