Diversity in antistaphylococcal mechanisms among membrane-targeting antimicrobial peptides

Many antimicrobial peptides permeabilize the bacterial cytoplasmic membrane . However, it is unclear how membrane permeabilization and antimicrobial ac tivity are related for distinct peptides. This study investigated the relat ionship between Staphylococcus aureus membrane permeabilization and cell de ath due to the following antistaphylococcal peptides: thrombin-induced plat elet microbicidal protein 1 (tPMP-1), gramicidin D, and protamine. Isogenic S. aureus strains ISP479C and ISP479R (tPMP-1 susceptible and resistant, r espectively), were loaded with the fluorochrome calcein and exposed to a ra nge of concentrations of each peptide. Flow cytometry was then used to moni tor membrane permeabilization by quantifying the release of preloaded calce in. Killing was determined by quantitative culture at time points simultane ous to measurement of membrane permeabilization. Membrane permeabilization and killing caused by tPMP-1 occurred in a time- and concentration-dependen t manner, reflecting the intrinsic tPMP-1 susceptibilities of ISP479C and I SP479R. In comparison, gramicidin D killed both S. aureus strains to equiva lent extents in a concentration-dependent manner between 0.5 to 50 mug/ml, but cell permeabilization only occurred at the higher peptide concentration s (25 and 50 mug/ml). Protamine permeabilized, but did not kill, either str ain at concentrations up to 10 mg/ml. Regression analyses revealed differen t relationships between membrane permeabilization and staphylocidal activit y for the distinct antimicrobial peptides. Taken together, these findings d emonstrate that permeabilization, per se, does not invariably result in sta phylococcal death due to distinct antimicrobial peptides. Thus, although ea ch of these peptides interacts with the S. aureus cytoplasmic membrane, div ersity exists in their mechanisms of action with respect to the relationshi p between membrane permeabilization and staphylocidal activity.