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The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal
S P Koo1, M R Yeaman, C C Nast
1Department of Medicine, St. John's Cardiovascular Research Center, Los Angeles County-Harbor UCLA Medical Center, Torrance, California 90509, USA. KOO@AFP76.HUMC.EDU
Abstract:
Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural 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 observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts 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 voltage 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 as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.
Insights
Thrombin-induced platelet microbicidal protein (tPMP-1) directly targets and damages the bacterial cytoplasmic membrane, leading to Staphylococcus aureus death. This membrane disruption is dependent on bacterial growth phase and voltage, indicating a direct mechanism of action for tPMP-1.
Area of Science:
- Antimicrobial Peptides
- Bacterial Pathogenesis
- Membrane Biophysics
Background:
- Thrombin-induced platelet microbicidal protein (tPMP-1) is a cationic peptide with broad-spectrum antimicrobial activity.
- tPMP-1 exhibits efficacy against Staphylococcus aureus, a significant human pathogen.
- Previous studies suggested tPMP-1 disrupts the bacterial cytoplasmic membrane, but the directness of this interaction was unclear.
Purpose of the Study:
- To investigate whether the staphylococcal cytoplasmic membrane is a direct or indirect target of tPMP-1.
- To determine the influence of bacterial growth phase and cell wall on tPMP-1 activity.
- To characterize the biophysical mechanism of tPMP-1-induced membrane damage.
Main Methods:
- In vitro activity of tPMP-1 against protoplasts from logarithmic (LOG) and stationary (STAT) phase Staphylococcus aureus strains (susceptible 19S and resistant 19R).
- Monitoring protoplast lysis via optical density and ultrastructural changes using transmission electron microscopy (TEM).
- Electrophysiological studies using anionic planar lipid bilayers to assess tPMP-1 interaction under varying voltages.
Main Results:
- tPMP-1 caused significant lysis and membrane disruption in LOG-phase protoplasts of susceptible S. aureus (19S), but not stationary-phase protoplasts.
- tPMP-1 activity was independent of the bacterial cell wall, as evidenced by protoplast lysis.
- tPMP-1 permeabilized lipid bilayers in a voltage-dependent manner, increasing conductance at -90 mV but not -30 mV.
Conclusions:
- tPMP-1 directly targets the staphylococcal cytoplasmic membrane as a primary mechanism of action.
- The antimicrobial activity of tPMP-1 is influenced by the bacterial growth phase.
- tPMP-1 induces bacterial death, at least partly, by voltage-dependent membrane permeabilization.