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

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.

A host E3 ubiquitin ligase regulatesSalmonellavirulence by targeting an SPI-2 effector involved in SIF biogenesis

“…Additional proteins potentially modified by members of the SseK have been identified in cells ectopically expressing the effectors or in cells infected with S. Typhimurium strains expressing specific effectors (Meng et al, 2020; Newson et al, 2019). These results are consistent with observations made in an earlier RNAi screen which revealed that the canonical mammalian late endo-/lysosomal vesicle fusion machinery (SNARE and Rab GTPase) is involved in SIF biogenesis(Kehl et al, 2020). Our finding that multiple coiled-coil containing-domain proteins are Arg-GlcNAcylated in infected cells suggests that SseKs may act coordinately to target endomembrane components to facilitate S. Typhimurium replication.…”

Predicting host dependency factors of pathogens in Drosophila melanogaster using machine learning

“…We were interested how this compares to infected human cells. Kehl et al [65] performed a focused screen knocking down genes of the trafficome in Salmonella enterica infected HeLA cells [65] . Indeed, when comparing our gold standard and our list of predHDF, we found a good overlap, specifically in the list of predHDF (n = 22, 10.6% of predHDF, compared to n = 21 genes, 2.5% of the gold standard, GS-1-out-of-12-HDF).…”

Mycoplasma bovis inhibits autophagy in bovine mammary epithelial cells via a PTEN/PI3K-Akt-mTOR-dependent pathway

“…Furthermore, Akt also has an important role in mediating infection of epithelial cells and macrophages by Salmonella ( Reggio et al, 2020 ). Several hypotheses have been proposed to explain the mechanism by which Akt promotes bacterial intracellular proliferation or survival, including the idea that by accelerating the recycling of the RAB14 small G protein, Akt delays phagosome-lysosome fusion and thus may enhance bacterial intracellular survival ( Kehl et al, 2020 ). Therefore, we inferred that M. bovis inhibited bMECs autophagy and promoted intracellular survival of bMECs through activation of the PI3K-Akt-mTOR signaling pathway.…”