SummaryMembrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

SYNOPSIS Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are well documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the free fatty acid docosahexaenoic acid (DA), but not oleic acid (OA), is decreased and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may alone, or in conjunction with Ly, exert antibacterial action against P. aeruginosa. We found that DA and Ly synergistically inhibit the metabolic activity of P. aeruginosa, in contrast to OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of Ly. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA leading to bacterial cell death. Our study elucidates a molecular basis for the synergistic action of free fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.

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The effects of a mammalian cyclic antimicrobial peptide, rhesus theta defensin 1 (RTD-1) and its open chain analogue (oRTD-1), on the phase behaviour and structure of model membrane systems (dipalmitoyl phosphatidylcholine, DPPC and dipalmitoyl phosphatidylglycerol, DPPG) were studied. The increased selectivity of RTD-1 for anionic DPPG over zwitterionic DPPC was shown by differential scanning calorimetry. RTD-1, at a molar peptide-lipid ratio of 1:100, induced considerable changes in the phase behaviour of DPPG, but not of DPPC. The main transition temperature, T m , was unchanged, but additional phase transitions appeared above T m. oRTD-1 induced similar effects. However, the effects were not observable below a peptide:lipid molar ratio of 1:50, which correlates with the weaker biological activity of oRTD-1. Small-and wide-angle X-ray scattering revealed for DPPG the appearance of additional structural features induced by RTD-1 above T m , which were interpreted as correlated lamellar structures , with increased order of the fatty acyl side chains of the lipid. It is proposed that after initial electrostatic interaction of the cationic rim of the peptide with the anionic DPPG headgroups, leading to stabilized lipid-peptide clusters, the hydrophobic face of the peptide assists in its interaction with the fatty acyl side chains eventually leading to membrane disruption.