PLATELET MICROBICIDAL PROTEINS AND NEUTROPHIL DEFENSIN DISRUPT THE STAPHYLOCOCCUS-AUREUS CYTOPLASMIC MEMBRANE BY DISTINCT MECHANISMS OF ACTION

Platelet microbicidal proteins (PMPs) are hypothesized to exert microb icidal effects via cytoplasmic membrane disruption, Transmission elect ron microscopy demonstrated a temporal association between PMP exposur e, damage of the Staphylococcus aureus cytoplasmic membrane ultrastruc ture, and subsequent cell death, To investigate the mechanisms of acti on of PMPs leading to membrane damage, we used flow cytometry to compa re the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] o r PMP-2) with human neutrophil defensin-l (hNP-1) on transmembrane pot ential (Delta psi), membrane permeabilization, and killing of S. aureu s. Related strains 6850 (Delta psi -150 mV) and JB-1 (Delta psi -100 m V; a respiration-deficient menadione auxotroph of 6850) were used to a ssess the influence of Delta psi on peptide microbicidal effects, Prop idium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC(5)) was used to monit or membrane depolarization (Delta psi), and quantitative culture or ac ridine orange accumulation was used to measure viability, PMP-2 rapidl y depolarized and permeabilized strain 6850, with the extent of permea bilization inversely related to pH, tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related t o pH, Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850, Growth in menadi one reconstituted Delta psi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP -1, Reconstitution of Delta psi also enhanced permeabilization and kil ling of JB-1 due to tPMP-1 or PMP-2, Both PMP-2 and tPMP-1 caused sign ificant reductions in viability of strain 6850, In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both s trains 6850 and JB-1 equally, and in a manner directly related to pH, Collectively, these data indicate that membrane dysfunction and cell d eath due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanism s, These findings may also reveal new insights into the microbicidal a ctivities versus mammalian cell toxicities of antimicrobial peptides.