Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides

Abstract

Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms – to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition towards a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and S. sciuri membrane lipids extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains of S. aureus is unlikely to be the sole factor responsible for the observed increased antibiotic resistance.

Growth-Environment Dependent Modulation of Staphylococcus aureus Branched-Chain to Straight-Chain Fatty Acid Ratio and Incorporation of Unsaturated Fatty Acids

“…Zones of inhibition were markedly lower in both strains on serum agar for rifampicin, chloramphenicol, and tetracycline. However, membrane permeability studies clearly need to be done in a more simplified system such as lipid vesicles of defined fatty acid composition [58] to simplify the interpretation of any differences observed. Cells grown in serum had higher hemolytic activity, and MH broth and serum- grown cells had lower autolytic activities than cells grown in the other media (unpublished observations).…”

Antimicrobial Peptides Targeting Gram-Positive Bacteria

“…To some extent, phosphatidylethanolamine (PE) is also present in Gram-positive cytoplasmic membranes. Moreover, phospholipids of Gram-positive species are characterized by a high content of branched-fatty acids, which were shown to influence the activity of AMPs [ 91 ]. The main physicochemical properties of these major bacterial phospholipids are summarized in Table 3 .…”

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

“…Further, other dicationic styryl probes such as di-4-ANEPPDHQ have been observed to flip-flop in model vesicles using SHG (67).The fact that we observe signatures of FM 4-64 flip-flop in S. aureus but not E. faecalis may also be explained by the differences in their membrane fluidity and phosopholipid head group charge. As discussed above, the membranes of S. aureus have more phospholipids with branched acyl chains than E. faecalis, and this has previously been shown to increase the membrane fluidity (51,52). Previous theoretical and experimental studies have both shown that increasing the fluidity of the different membranes being investigated was directly correlated with an increase in the flip-flop rate observed.…”

“…These differences in temperature response for the two bacterial species may be attributed to differences in the fatty acid tails of the phospholipids which are integral to establishing the degree of membrane fluidity. More fluidic membranes are typically composed of shorter acyl chains (50), along with chains that are branched or unsaturated (51,52). S. aureus is known to have a greater percentage of branched acyl chain structures in its lipidome in comparison to E. faecalis (4,48,49,53,54).…”