THE CYTOPLASMIC MEMBRANE IS A PRIMARY TARGET FOR THE STAPHYLOCIDAL ACTION OF THROMBIN-INDUCED PLATELET MICROBICIDAL PROTEIN

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, ca tionic peptide released from rabbit platelets exposed to thrombin in v itro. tPMP-1 is microbicidal against a broad spectrum of bloodstream p athogens, including Staphylococcus aureus. Preliminary evidence sugges ts that tPMP-1 targets and disrupts the staphylococcal cytoplasmic mem brane. However, it is not clear if the cytoplasmic membrane is a direc t or indirect target of tPMP-1. Therefore, we assessed the in vitro ac tivity of tPMP-1 versus protoplasts prepared from logarithmic-phase (L OG) or stationary-phase (STAT) cells of the genetically related S. aur eus Strains 19S and 19R (tPMP-1 susceptible and resistant, respectivel y). Protoplasts exposed to tPMP-1 (2 mu g/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and u ltrastructural alterations (by transmission electron microscopy [TEM]) . Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption ob served by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the st aphylococcal cell wall. In contrast to 19S, neither LOG nor STAT proto plasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in v oltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP- 1 appears to directly target the staphylococcal cytoplasmic membrane a s a primary event in its mechanism of action. Specifically, tPMP-1 lik ely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.