Human neutrophil polymorphonuclear granulocytes (PMN) are in the first line of cellular defense against infection. They phagocytize bacteria and kill them with antimicrobial activities that are oxygen dependent or oxygen independent. The granules of PMN are intrinsic to these activities. Our goal is to continue to define the proteins that account for antimicrobial activities of crude granule extracts. Our main emphasis will be on anti-Proteus activity and purified cationic antimicrobial proteins (CAP), CAP57 and CAP37 that kill Salmonella and E. coli. We will: define genetic and (tissue) environmental factors contributing to sensitivity or resistance of microorganisms; determine the target molecules CAP interact with in the outer membrane of sensitive bacteria. We will complete studies to localize CAPs at the cellular and subcellular levels. We will use techniques required to understand bacterial pathogenesis and host resistance. We will use: cell culture and differential and gradient centrifugation; phagocytosis; assays for antimicrobial action; protein purification, including conventional and high performance liquid chromatography and affinity chromatography; ELISA; monoclonal antibodies; construction of isogenic strains by P22 transduction; and biochemical analysis of lipopolysaccharides, and outer membrane proteins of Salmonella typhimurium which differ in their resistance to CAP. Our long term objective is to define the role of CAP in oxygen independent antimicrobial phagocytosis and to develop monoclonal antibodies for rapid screening of PMN in clinical infectious disease. The research we propose is significant in that it will: a) establish the existence of at least three highly effective Cationic Antimicrobial Proteins (CAPs) whose mode of action in human PMN is oxygen independent; b) provide biochemical evidence for specific target sites on susceptible bacteria to the action of CAP; c) implicate a contribution of low O2 tension to the resistance to CAPs of N. gonorrhoeae; d) provide highly suggestive evidence for the existence of a heretofore unsuspected antimicrobial granule.
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