Abstract
The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-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 of 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 (soluble N-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.
Highlights
The food-borne, facultative intracellular pathogen Salmonella enterica serovar Typhimurium (STM) is the etiological agent of gastroenteritis in humans or systemic infections in mice [1]
The facultative intracellular pathogen Salmonella enterica serovar Typhimurium induces the reorganization of the endosomal system of mammalian host cells
To identify such factors for the formation and dynamics of endosomal compartments in Salmonella-infected cells, we performed a live cell imaging-based RNA inference (RNAi) screen to investigate the role of 496 mammalian proteins involved in cellular logistics
Summary
The food-borne, facultative intracellular pathogen Salmonella enterica serovar Typhimurium (STM) is the etiological agent of gastroenteritis in humans or systemic infections in mice [1]. An early step in disease is the active invasion of epithelial cells This process is dependent on the translocation of effector proteins by STM into the host cell through a type 3 secretion system (T3SS) encoded by genes in Salmonella pathogenicity island 1 (SPI1) [2, 3]. After invasion STM, similar to many other intracellular pathogens, establish a replicative niche in host cells, termed Salmonella-containing vacuole (SCV). This process is dependent on the function of a distinct T3SS, encoded by genes in SPI2 [4, 5] and translocating another set of effectors [6]. Other canonical organelle markers such as the mannose-6-phosphate receptor are excluded [14]
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