title: Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action.
abstract: 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.
authors.affiliation: Division of Infectious Diseases, St. John's Cardiovascular Research Center, LAC-Harbor UCLA Medical Center, Torrance, California 90509, USA. yeaman@afp76.humc.edu
authors.forename: M R
authors.initials: MR
authors.lastname: Yeaman
authors.forename: A S
authors.initials: AS
authors.lastname: Bayer
authors.forename: S P
authors.initials: SP
authors.lastname: Koo
authors.forename: W
authors.initials: W
authors.lastname: Foss
authors.forename: P M
authors.initials: PM
authors.lastname: Sullam
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001798:Blood Proteins / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D018925:Chemokines; D023082:Defensins; D005434:Flow Cytometry; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D009504:Neutrophils / Q000378:metabolism; D011817:Rabbits; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000648:ultrastructure*; D023084:alpha-Defensins; D001620:beta-Thromboglobulin
chemical_list: D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D018925:Chemokines; D023082:Defensins; C061615:PPBP protein, human; D023084:alpha-Defensins; D001620:beta-Thromboglobulin; C406413:human neutrophil peptide 1; C077150:platelet microbicidal protein, Oryctolagus cuniculus