We have recently purified to near homogeneity two very similar proteins, one from human and one from rabbit polymorphonuclear leukocytes, that are potently bactericidal toward a range of gram-negative bacterial species. The immediate objectives of this proposal are to further analyze the actions of these proteins on bacterial viability and envelope structure and function.
Our specific aims are: I. To better define the biological function of these proteins by further examining their intracellular distribution, production during cellular differentiation, antibacterial spectrum, and role in the killing of gram-negative bacteria by intact leukocytes. II. To examine further the interaction of these proteins with the gram-negative bacterial envelope in relation to bactericidal action and effects on envelope structure and function. III. To relate binding to bacteria, bactericidal and membrane-active properties to protein structure, by analyzing fragments that may be formed by proteolysis during 1) interaction with bacteria, 2) purification, 3) brief exposure to added proteases.
These aims have their origin in our previous work and their pursuit will rely heavily on well-tested methods (most of which are already employed in this laboratory) including the use of: assays for measuring the interaction of PMN with bacteria; bacterial mutants; purification of proteins; immunological techniques employing specific antisera; fine-structural analysis; radioisotopic labeling of proteins; myeloid cell lines in culture. Our long-term objectives are to enhance our understanding of leukocyte function and host-defense against infection. These objectives should be met by the proposed studies on these two well-defined proteins, which are the most potent bactericidal mammalian proteins, specific for gram-negative bacteria, yet described. In addition, the planned scrutiny of the structure-function relationships in these proteins may provide clues for the design of new antibiotics by identifying smaller functional peptides involved in bactericidal action.
