Action of Daptomycin on Membranes

Daptomycin is a clinically important 13 amino acid lipopeptide antibiotic. Its N-terminus is acylated with n-decanol and its C-terminal 10 amino acids form a ring. It is structurally and size-wise different from other well-known membrane-active antimicrobials. Daptomycin is known to disrupt the cytoplasmic membrane function of Gram-positive bacteria by causing leakage of potassium (and potentially other) ions, leading to the loss of membrane potential and cell death. The critical factor affecting the function of daptomycin is its interaction with negatively charged lipids such as PG in a calcium (Ca++) dependent manner. Based on previous research on cell membranes, daptomycin has been assumed to insert and aggregate in the membrane, and then to alter the membrane curvature. However these details have not been demonstrated by biophysical studies. In our aspirated GUV experiments, we found that with a DOPG-containing GUV and a sufficient concentration of Ca++, daptomycin can extract lipid molecules, and form lipid-peptide aggregations. The lipid-peptide aggregates did not occur if cardiolipin replaced PG, or if other divalent ions, such as Mg++, Ba++ replaced Ca++. Similarly, daptomycin did not bind to a GUV when cardiolipin substituted for DOPG or in the absence of Ca++. Daptomycin with Ca++ did bind to a pure DOPC GUV, but had no other effects. Furthermore, with the presence of daptomycin, DOPG and Ca++, we found Ca++ permeates into the GUV, while a content dye, Texas red dextran, did not leak out. This result suggests that daptomycin and Ca++ do not form pores on the membrane of DOPG-contained GUV, but cause leakage of ions. Finally, daptomycin with DOPG and Ca++ produces a negative exciton CD couplet centered at the 225 nm absorption peak of Trptophan1 and Kynurenine13, whereas in all other conditions, the exciton CD couplet is positive.