Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate

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Abstract
Acid (0.2 M sodium acetate, pH 4.0) extracts of granules recovered from disrupted human polymorphonuclear granulocytes (PMN) exhibited in vitro antimicrobial activity against Salmonella typhimurium. To minimize proteolytic destruction or modification of antimicrobial proteins derived from these granules, the PMN were pretreated with the serine protease inhibitor diisopropyl fluorophosphate. Fractionation of such extracts by carboxymethyl Sephadex G-75 chromatography resulted in the recovery of at least 2 antimicrobial, cationic proteins. These proteins differed substantially in antimicrobial activity, amino acid composition and MW (MW 37,000 and 57,000). These 2 proteins exhibited diminished activity against a polymyxin B-resistant (PBr) mutant of S. typhimurium compared with their activity against the isogenic parental polymyxin B-sensitive (PBs) strain. Expression of the relevant mutation (prmA) in the PBr mutant decreases the electronegativity of lipid A, owing to increased 4-amino-4-deoxy-L-arabinosylation at the 4'' phosphate residue. Evidently, at least 2 different cationic proteins account for the antimicrobial capacity of extracts from human PMN granules. The availability of anionic charges in the outer membrane of S. typhimurium due to free lipid A phosphates apparently dictates phenotypic levels of resistance to both of the cationic proteins extracted from human PMN granules.