Role of cardiolipin in Shigella flexneri pathogensis
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Shigella flexneri causes bacterial dysentery in humans by invading colonic epithelial cells, and then replicating and moving within the host cell cytoplasm to spread intercellularly to neighboring epithelial cells. Analysis of S. flexneri phospholipids revealed that cardiolipin, a large anionic phospholipid that localizes to the inner leaflet at the poles of Gram-negative bacterial membrane, makes up approximately 7% of S. flexneri’s total phospholipids and is found in equal proportions between the inner and outer membrane. To characterize the mechanisms S. flexneri uses to synthesize and transport cardiolipin, a series of mutants were construed that disrupted one of the three putative cardiolipin synthases, clsA, clsB, and clsC, or transporter, pbgA. It was determined that ClsA is the major cardiolipin synthase of S. flexneri and the level of phosphatidylglycerol increases in both the inner and outer membrane in the absence of cardiolipin. In addition, PbgA transports cardiolipin and phosphatidylglycerol to the S. flexneri outer membrane, but has no effect on the inner membrane phospholipid composition. Both the clsA and pbgA mutants were unable to form wild type plaques in cultured epithelial cells, and time-lapse microscopy revealed that the clsA mutant was less motile intracellularly and unable to divide during intracellular growth, while the pbgA mutant grew normally but was non-motile. This suggests inner membrane cardiolipin is required for cell division and outer membrane anionic phospholipids are required for motility. Unipolar localization of S. flexneri virulence protein IcsA, a member of the auto-transporter protein family, provides the mechanism for intracellular mobility through the polymerization of host actin. Analysis of IcsA localization on the surface of the clsA and pbgA mutants revealed reduced and pinpoint (respectively) levels of polar localized IcsA; however, both mutants displayed wild type levels of outer membrane IcsA, and surface exposed IcsA passenger domain. Protein-lipid co-sedimentation assays revealed a direct interaction of the polar targeting region in the passenger domain of IcsA with cardiolipin, and to a lesser extent with phosphatidylglycerol. We propose a novel model for IcsA polar localization directed by outer membrane anionic phospholipids, where outer membrane cardiolipin directs IcsA insertion and subsequent auto-transport at the bacterial pole.