Shigella flexneri causes bacterial dysentery by invading colonic epithelial cells, rapidly multiplying within their cytoplasm, and then spreading intercellularly to neighboring cells. Invasion and intracellular spread of this pathogen can be assessed by infecting epithelial cell monolayers and observing the formation of plaques as the bacteria replicate and spread to adjacent cells. Intercellular spread requires actin-based motility, which is catalyzed by the polar-localized bacterial surface protein IcsA. Although the roles of specific membrane proteins, including IcsA, in the pathogenesis of S. flexneri have been examined, there have been few studies on the roles of specific membrane lipids. We found that the presence and distribution of specific phospholipids, particularly cardiolipin, influences intracellular growth and cell-to-cell spread of the bacteria.
S. flexneri has three cardiolipoin synthase genes, clsA, clsB, and clsC. We determined that ClsA is the primary cardiolipin synthase of S. flexneri, and a clsA mutant has little or no detectable cardiolipin during exponential growth. Cardiolipin synthesis increases during stationary phase, and a minor cardiolipin synthase, ClsC, contributes to the increased cardiolipin synthesis. To determine whether cardiolipin synthesis was required for S. flexneri invasion or intracellular replication, we constructed mutations in the cardiolipin synthase genes and tested their invasion and plaque formation in epithelial cell monolayers. While mutations in clsB and clsC had no detectable effects on virulence, the clsA mutant was highly attenuated. The clsA mutant was invasive but failed to spread to adjacent cells, and no plaques were formed. Analysis of clsA bacteria in the epithelial cell cytoplasm showed that they initially replicated and were motile, but within 8 hours after invasion, the cells were defective in replication, were no longer motile, and were unable to spread.
Cardiolipin is found in both the inner and outer membranes and, in E. coli, is restricted to the poles of the cell. Export of cardiolipin to the outer membrane requires the anionic phospholipid transporter PbgA. We constructed a pbgA mutant and confirmed that it had normal levels of cardiolipin in the inner membrane but lacked cardiolipin in the outer membrane. The pbgA mutant was unable to produce plaques in monolayers, indicating that cardiolipin in the outer membrane is required for S. flexneri virulence. Unlike the wild-type strain, the pbgA mutant failed to localize IcsA to the pole of the cells and lacked intracellular motility. Normal levels of IcsA were found in the outer membrane of the pbgA mutant, but the protein was not restricted to the pole of the cells. IcsA was found to bind cardiolipin in vitro, indicating that cardiolipin at the cell pole facilitates IcsA localization. These data suggest that cardiolipin in the outer membrane binds IcsA, resulting in concentration of the protein at the pole of the bacterial cells. This allows actin polymerization at the pole, resulting in motility of the cells in the host cell cytoplasm.