The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack

Abstract

Secretory phospholipases A₂ (sPLA₂s) are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA₂ enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis. However, most Gram-negative bacteria are naturally resistant to sPLA₂ Here we report a novel resistance mechanism to mammalian sPLA₂ in Escherichia coli, mediated by a phospholipid repair system consisting of the lysophospholipid transporter LplT and the acyltransferase Aas in the cytoplasmic membrane. Mutation of the lplT or aas gene abolished bacterial lysophospholipid acylation activity and drastically increased bacterial susceptibility to the combined actions of inflammatory fluid components and sPLA₂, resulting in bulk phospholipid degradation and loss of colony-forming ability. sPLA₂-mediated hydrolysis of the three major bacterial phospholipids exhibited distinctive kinetics and deacylation of cardiolipin to its monoacyl-derivative closely paralleled bacterial death. Characterization of the membrane envelope in lplT- or aas-knockout mutant bacteria revealed reduced membrane packing and disruption of lipid asymmetry with more phosphatidylethanolamine present in the outer leaflet of the outer membrane. Moreover, modest accumulation of lysophospholipids in these mutant bacteria destabilized the inner membrane and rendered outer membrane-depleted spheroplasts much more sensitive to sPLA₂ These findings indicated that LplT/Aas inactivation perturbs both the outer and inner membranes by bypassing bacterial membrane maintenance mechanisms to trigger specific interfacial activation of sPLA₂ We conclude that the LplT/Aas system is important for maintaining the integrity of the membrane envelope in Gram-negative bacteria. Our insights may help inform new therapeutic strategies to enhance host sPLA₂ antimicrobial activity.

Keywords: bacteria; bacterial resistance; lysophospholipid; membrane biogenesis; membrane envelope; membrane lipid; membrane structure; outer membrane asymmetry; phospholipase A; phospholipase A2; phospholipid turnover.