Abstract
To invade epithelial cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) induces macropinocytosis through the action of virulence proteins delivered across the host cell membrane via a type III secretion system. We show that after docking at the plasma membrane S. Typhimurium triggers rapid recruitment of cytosolic SNX18, a SH3-PX-BAR domain sorting nexin protein, to the bacteria-induced membrane ruffles and to the nascent Salmonella-containing vacuole. SNX18 recruitment required the inositol-phosphatase activity of the Salmonella effector SopB and an intact phosphoinositide-binding site within the PX domain of SNX18, but occurred independently of Rho-GTPases Rac1 and Cdc42 activation. SNX18 promotes formation of the SCV from the plasma membrane by acting as a scaffold to recruit Dynamin-2 and N-WASP in a process dependent on the SH3 domain of SNX18. Quantification of bacteria uptake revealed that overexpression of SNX18 increased bacteria internalization, whereas a decrease was detected in cells overexpressing the phosphoinositide-binding mutant R303Q, the ΔSH3 mutant, and in cells where endogenous levels of SNX18 were knocked-down. This study identifies SNX18 as a novel target of SopB and suggests a mechanism where S. Typhimurium engages host factors via local manipulation of phosphoinositide composition at the site of invasion to orchestrate the internalization process.
Highlights
To gain entry into non-phagocytic epithelial cells, various viral and bacterial human pathogens co-opt the endocytic machinery by modulation of phosphoinositide metabolism (PizarroCerda and Cossart, 2004) or actin cytoskeleton (Haglund and Welch, 2011)
Given that the phosphoinositide binding of Sorting nexin 18 (SNX18) is essential for membrane recruitment, and this recruitment is dependent on the PtdIns-phosphatase activity of SopB, we investigated whether SNX18 is recruited to the plasma membrane subdomains in response to SopB-mediated manipulation of phosphoinositide composition, since it has been previously reported that SopB is essential for local increase in PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and depletion of PtdIns(4,5)P2 at Salmonella-induced membrane ruffles (Terebiznik et al, 2002; Mason et al, 2007; Mallo et al, 2008)
We identify a function for SNX18 in this process and demonstrate that engagement of SNX18 occurs via the action of the Salmonella Salmonella pathogenicity island 1 (SPI1)-T3SS effector SopB
Summary
To gain entry into non-phagocytic epithelial cells, various viral and bacterial human pathogens co-opt the endocytic machinery by modulation of phosphoinositide metabolism (PizarroCerda and Cossart, 2004) or actin cytoskeleton (Haglund and Welch, 2011). One of the most efficient routes for pathogens to enter the host cells is macropinocytosis, a distinct form of endocytosis characteristic for its non-selective and high turnover uptake of extracellular fluid into macropinosomes (Lim et al, 2008; Kerr et al, 2010).
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