Antimicrobial action of rabbit leukocyte CAP18(106-137)

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C terminal and (CAP18(106-137)) has been found to be the most active, The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry, Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 mu M) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential {bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(4)(3)]}, or membrane integrity (propidium iodide [PI]) before flow cytometric analysis, Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC(4)(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media, In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake, In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 mu M) inhibited the uptake of both dyes, These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 mu M fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation, The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.