We will determine the mechanism by which polymorponuclear (PMN) granulocytes kill engulfed gonococci by nonoxidative means. We have shown that lysosomal cathepsin G is a candidate for such intraphagosomal killing of gonococci. Gonococci that survive within PMNs may do so by resisting the antibacterial action of cathepsin G. To evaluate how gonococci are killed by or otherwise resist cathepsin G we will isolate mutants that have decreased susceptibility to cathepsin G. These mutants may resemble those gonococci that persist in PMNs. The mutants will be analyzed for changes in outer membrane proteins and lipopolysaccharide since these components may serve as binding sites for cathepsin G. Changes in membrane lipids and peptidoglycan will also be evaluated since their composition may influence insertion of cathepsin G and postbinding antimicrobial events. We will define the saturable and specific binding characteristics of cathepsin G, the outer membrane sites for binding, and whether cathepsin G interacts with peptidoglycan or the transpeptidases involved in the terminal stages of peptidoglycan synthesis. Binding of cathepsin G to outer membrane proteins and LPS will be evaluated by the use of liposomes containing defined proteins or LPS. Monoclonal antibodies to cell surface antigens will be used to test for their ability to inhibit specific binding of cathepsin G. Differences in surface accessibility of antigens that bind cathepsin G amongst isogenic strains differing in susceptibility to cathepsin G will be evaluated by electron microscopy using gold-conjugated antibodies. We will evaluate the capacity of cathepsin G to kill gonococci by depolarizing its membrane or by damaging the outer membrane such as to inhibit cell division. The capacity of cathepsin G to damage the cell envelope and thereby release pharmacologically active peptidoglycan and lipopolysaccharide will be tested since intraphagosomal killing of gonococci may have pathophysiologic consequences.
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