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.
Highlights
Secretory phospholipases A2 are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA2 enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis
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/acyl carrier protein synthetase (Aas) inactivation perturbs both the outer and inner membranes by bypassing bacterial membrane maintenance mechanisms to trigger specific interfacial activation of sPLA2
We recently showed that OMand cell wall-depleted spheroplasts generated from WT E. coli BL21(DE3) cells were resistant to added venom sPLA2, whereas the corresponding spheroplasts from mutant (⌬lplT or ⌬aas) bacteria were sensitive [23]
Summary
Functionally related genes are often co-transcribed in one bicistronic operon. Consistent with the TLC assays, both ⌬lplT and ⌬aas strains showed progressive PL degradation, with nearly 30% of radiolabeled bacterial fatty acids recovered in the extracellular medium after 2 h of incubation, whereas there was little detectable fatty acid released from the WT bacteria (Fig. 5a) These findings indicate an unexpected effect of LplT/Aas on the resistance of E. coli to psPLA2 attack. Lipid A released from the extracted LPS of both WT and mutant strains and analyzed by mass spectrometry revealed only hexaacylated lipid A (Fig. S9), indicating that OM PagP is not activated in the absence of LplT/Aas. The extent of PL degradation by the combined action of AF ϩ psPLA2 in the mutant strains suggests strongly that PL breakdown and accumulation of LPL under bactericidal conditions extend to the IM where essential energy-dependent and -generating machinery reside. These findings suggest that the function of LplT and Aas contributes to the stability of the IM, with vulnerability to psPLA2 a sensitive marker of IM instability (see “Discussion”)
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