Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi 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 Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.

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The regulation of glycine betaine accumulation has been investigated in Salmonella typhimurium. The size of the glycine betaine pool in the cells is determined by the external osmotic pressure and is largely independent of the external glycine betaine concentration. Analysis of the activity of the Prop and ProU transport systems suggests that other systems must be active in the regulation of the glycine betaine pool. Addition ofp-chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulphonate (PCMBS) to cells that have accumulated glycine betaine provokes rapid loss of glycine betaine. The route of glycine betaine efflux under the influence of PCMB is independent of either the Prop or ProU transport systems. Rapid loss of the accumulated pool of glycine betaine in the presence of PCMB is specific to glycine betaine and proline; accumulated pools of serine and lysine are not significantly affected by the -SH reagent. A specific glycine betainelproline efflux system is postulated on the basis of these data and its role in the regulation of glycine betaine and proline accumulation is discussed.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic, antimicrobial peptide released from rabbit platelets when stimulated with thrombin. We studied the relationship between staphylococcal transmembrane potential (⌬) and tPMP staphylocidal activity. A genetically related pair of Staphylococcus aureus strains, 6850 and JB1, which differ in ⌬ generation (؊143 and ؊97 mV, respectively) were used. Mutant JB-1 was substantially less susceptible to tPMP than the parental strain, 6850. Menadione supplementation, which normalized the ⌬ of strain JB-1, did not restore JB-1 tPMP susceptibility. These findings suggest that the staphylocidal activities of tPMP require factors other than or in addition to an intact ⌬.