A wide range of chemical structures having antimicrobial activity have been studied in an effort to treat the increasing emergence of bacteria that are resistant to traditional antibiotics. These agents have varying degrees of toxicity against different bacterial species. We demonstrate, using members of a novel class of antimicrobial agents, the oligomers of acyllysine, that one cause for the difference in species selectivity is the ability to induce the clustering of anionic lipids, resulting in their segregation into domains. This phenomenon occurs only in bacterial membranes composed of both anionic and zwitterionic lipids and not with bacteria whose membrane lipids are largely anionic. As a consequence it can be predicted which bacterial species will be most affected by antimicrobial agents that function principally by this mechanism. This finding allows for the design of new antibiotics with selective toxicity against different groups of bacteria.

Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but in Barth syndrome, where this association is compromised, cardiolipin becomes unstable, which causes the accumulation of monolyso-cardiolipin.

Cardiolipin is a mitochondrial phospholipid with a characteristic acyl chain composition that depends on the function of tafazzin, a phospholipid-lysophospholipid transacylase, although the enzyme itself lacks acyl specificity. We incubated isolated tafazzin with various mixtures of phospholipids and lysophospholipids, characterized the lipid phase by 31P-NMR, and measured newly formed molecular species by mass spectrometry. Significant transacylation was observed only in non-bilayer lipid aggregates and the substrate specificity was highly sensitive to the lipid phase. In particular, tetralinoleoyl-cardiolipin, a prototype molecular species, formed only under conditions that favor the inverted hexagonal phase. In isolated mitochondria, <1 percent of lipids participated in transacylations, suggesting that the action of tafazzin is limited to privileged lipid domains. We propose that tafazzin reacts with non-bilayer type lipid domains that occur in curved or hemifused membrane zones, and that acyl specificity is driven by the packing properties of these domains.

The only human cathelicidin, the 37-residue peptide LL-37, exhibits antimicrobial activity against both gram-positive and gram-negative bacteria. We studied the ability of several fragments of LL-37, exhibiting different antimicrobial activities, to interact with membranes whose compositions mimic the cytoplasmic membranes of gram-positive or of gram-negative bacteria. These fragments are as follows: KR-12, the smallest active segment of LL-37, with the sequence KRIVQRIKDFLR, which exhibits antimicrobial activity only against gram-negative bacteria; a slightly smaller peptide, RI-10, missing the two cationic residues at the N and C termini of KR-12, which has been shown not to have any antimicrobial activity; a longer peptide, GF-17, which shows antimicrobial activity against gram-positive as well as gram-negative bacteria; and GF-17D3, with 3 D-amino-acid residues, which is also selective only for gram-negative bacteria. Those fragments with the capacity to cluster anionic lipids away from zwitterionic lipids in a membrane exhibit selective toxicity toward bacteria containing zwitterionic as well as anionic lipids in their cytoplasmic membranes but not toward bacteria with only anionic lipids. This finding allows for the prediction of the bacterial-species selectivity of certain agents and paves the way for designing new antimicrobials targeted specifically toward gram-negative bacteria.