The Salmonella virulence protein SifA is a G protein antagonist

Abstract

Salmonella's success at proliferating intracellularly and causing disease depends on the translocation of a major virulence protein, SifA, into the host cell. SifA recruits membranes enriched in lysosome associated membrane protein 1 (LAMP1) and is needed for growth of Salmonella induced filaments (Sifs) and the Salmonella containing vacuole (SCV). It directly binds a host protein called SKIP (SifA and kinesin interacting protein) which is critical for membrane stability and motor dynamics at the SCV. SifA also contains a WxxxE motif, predictive of G protein mimicry in bacterial effectors, but whether and how it mimics the action of a host G protein is not known. We show that SKIP's pleckstrin homology domain, which directly binds SifA, also binds to the late endosomal GTPase Rab9. Knockdown studies suggest that both SKIP and Rab9 function to maintain peripheral LAMP1 distribution in cells. The Rab9:SKIP interaction is GTP-dependent and is inhibited by SifA binding to the SKIP pleckstrin homology domain, suggesting that SifA may be a Rab9 antagonist. SifA:SKIP binding is significantly tighter than Rab9:SKIP binding and may thus allow SifA to bring SKIP to the SCV via SKIP's Rab9-binding site. Rab9 can measurably reverse SifA-dependent LAMP1 recruitment and the perinuclear location of the SCV in cells. Importantly, binding to SKIP requires SifA residues W197 and E201 of the conserved WxxxE signature sequence, leading to the speculation that bacterial G protein mimicry may result in G protein antagonism.GTPase ͉ LAMP1 ͉ Rab9 ͉ SKIP ͉ lysosome

Rab Proteins and the Compartmentalization of the Endosomal System

“…Rab9 interaction with a Golgi-tethering factor, GCC185, promotes interaction with Rab6, Arl1, and the CLASP microtubule-anchoring protein and is suggested to integrate dynein-mediated delivery with docking of endosome-derived vesicles to the Golgi (Hayes et al 2009). Additional insights into the regulation of Rab7 and Rab9 endosome motility-cooperativity with Arflike (Arl) proteins and kinesin-interacting proteins-have in part been gained through analyses of how bacterial pathogens usurp RabGTPase effectors during the establishment of an intracellular niche (Jackson et al 2008;Garg et al 2011;Mrakovic et al 2012;Stein et al 2012b). What emerges from the composite work is that Rab GTPases interact in a nucleotide-dependent manner with microtubule motor complexes and thereby directly control cytoskeletal translocation.…”

Mechanisms and functions of lysosome positioning

“…Cholesterol accumulation in Niemann-Pick C disease also alters Rab9A activity and its association with late endosomes (Ganley and Pfeffer, 2006), suggesting that Rab9A might also be implicated in cholesterol-mediated regulation of late endosome positioning. Rab9A interacts with the PH domain of SKIP (Jackson et al, 2008) and of the Rab36 GAP RUTBC2 (also known as SGSM1) (Nottingham et al, 2012;Zhang et al, 2014), but it is unclear whether these interactions account for the role of Rab9A in lysosome positioning. Overexpression of Rab34 and Rab36 causes lysosome clustering (Wang and Hong, 2002;Chen and Yu, 2013).…”

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

“…Interestingly, SKIP also interacts, through its PH domain, with the late endosome GTPase Rab9. This interaction is important to maintain peripheral LAMP1 distribution in cells and is inhibited by SifA, suggesting that bacterial G protein mimicry may result in G protein antagonism [218].…”