A trafficome-wide RNAi screen reveals deployment of early and late secretory host proteins and the entire late endo-/lysosomal vesicle fusion machinery by intracellularSalmonella

Abstract

The intracellular lifestyle ofSalmonella entericais characterized by the formation of a replication-permissive membrane-bound niche, theSalmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellularSalmonellaestablish a unique network of variousSalmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, theSalmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (solubleN-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, which is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, providing a potential link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed us to characterize completely new host factor interactions, and to strengthen observations from several previous studies. Author summary The facultative intracellular pathogenSalmonella entericaserovar Typhimurium induces the reorganization of the endosomal system of mammalian host cells. This activity is dependent on translocated effector proteins of the pathogen. The host cell factors required for endosomal remodeling are only partially known. To identify such factors for the formation and dynamics of endosomal compartments inSalmonella-infected cells, we performed a live cell imaging-based RNAi screen to investigate the role of 496 mammalian proteins involved in cellular logistics. We identified that endosomal remodeling by intracellularSalmonellais dependent on host factors in the following functional classes: i) the late endo-/lysosomal SNARE (solubleN-ethylmaleimide-sensitive factor attachment protein receptor) complex, ii) the early secretory pathway, represented by regulator GTPases RAB1A and RAB1B, iii) the late secretory pathway and/or recycling endosomes represented by GTPases RAB3A, RAB8A, RAB8B, and the SNAREs VAMP2, VAMP3, and VAMP4, and iv) clathrin-coated structures. The identification of these new host factors provides further evidence for the complex manipulation of host cell transport functions by intracellularSalmonellaand should enable detailed follow-up studies on the mechanisms involved.