CRITICAL ROLE OF LIPID-COMPOSITION IN MEMBRANE PERMEABILIZATION BY RABBIT NEUTROPHIL DEFENSINS

We have examined the interactions of the six known rabbit neutrophil d efensin antimicrobial peptides with large unilamellar vesicles (LW) ma de from various lipid mixtures based an the lipid composition of Esche richia coli membranes. We find that the permeabilization of LUV made f rom E. coli whole lipid extracts differs dramatically from that of sin gle-component LUV made from palmitoyl-oleoyl-phosphatidylglcerol (POPG ). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mix ture of the six defensins cause fast nonpreferential leakage of high m olecular weight dextrans as well as the low molecular weight fluoropho re/ quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xy lene-bis-pyridium bromide (DPX) from E. coli whole lipid LUV through l arge, transient membrane lesions. In contrast, release of ANTS/DPX for m POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selstad, M. E., and White, S. H. (1996) Bioche mistty 35, 11888-11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. Coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LW, Examination of She sequences of the six d efensins suggests that the inactivity of NP-4 and NP-5 may be due to t heir lower net positive charge and/or the substitution of a Thr for th e Arg or Lys that follows the fourth Cys residue. We found the presenc e of three major lipid components of E. coli whole lipid to be essenti al for creation of the large lesions observed in LUV: phosphatidyletha nolamine, phosphatidylglycerol, and cardiolipin, Cardiolipin appears t o play a key role because no leakage can be induced when only phosphat idylglycerol, and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabil ization of cell membranes by rabbit defensins.