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. 2020 Jul 13;16(7):e1008220.
doi: 10.1371/journal.ppat.1008220. eCollection 2020 Jul.

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

Alexander Kehl  1   2 , Vera Göser  1 , Tatjana Reuter  1 , Viktoria Liss  1 , Maximilian Franke  1 , Christopher John  1 , Christian P Richter  2 , Jörg Deiwick  1 , Michael Hensel  1   3
Affiliations

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

Alexander Kehl et al. PLoS Pathog. .

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.

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Conflict of interest statement

No authors have competing interests

Figures

Fig 1
Fig 1. RNAi screen setup and validation.
A) Intracellular phenotypes of STM under screening conditions. HeLa LAMP1-GFP cells were infected with mCherry-labelled STM WT or ssaV strains and imaged live 8 h p.i. by SDCM. Images display the presence of STM in LAMP1-positive SCV (blue arrowhead), the induction of SIF formation by STM WT (white arrowhead), and the lack of SIF formation by the STM ssaV strain. Scale bar, 10 μm. B) Controls for siRNA-mediated knockdown. HeLa LAMP1-GFP cells were reverse transfected with scrambled AllStars siRNA or PLK1 siRNA for 72 h and then imaged. Scale bar, 20 μm. C) Validation of SKIP siRNA knockdown. HeLa LAMP1-GFP cells were reverse transfected with AllStars or SKIP siRNA for 72 h. Then, RT-PCR targeting SKIP was performed. Depicted is the mean with standard deviation of three biological replicates (n = 3) each performed in triplicates. Statistical analysis was performed using Student’s t-test and indicated as: ***, p < 0.001. D) SKIP knockdown as a control for the inhibition of SIF formation. HeLa LAMP1-GFP cells were first reverse transfected with AllStars or SKIP siRNA for 72 h. Then, cells were infected with mCherry-labelled STM WT (MOI = 15) and imaged live by SDCM 1–7 h p.i in hourly intervals. Blue arrowheads indicate SIF-forming or non-SIF-forming single bacteria or microcolonies, white arrowheads indicate SIF. Scale bar, 10 μm.
Fig 2
Fig 2. Basic workflow of the trafficome RNAi screen.
24 96-well plates with clear optical bottoms per biological replicate (with 3 biological replicates in total, n = 3) were automatically spotted with siRNAs. HeLa LAMP1-GFP cells were seeded for reverse transfection with each plate also containing negative, positive, and phenotype-specific controls. After 72 h of incubation, infection with STM WT (and ssaV as control, MOI = 15) was performed, followed by LCI for the acquisition of eight positions per well with single bacteria-focused Z-planes with hourly intervals of imaging from 1–7 h p.i. on an SDCM system. Subsequent phenotypic scoring was performed using the SifScreen utility. The MATLAB-based data input mask allows the entry of well- and position-specific information on general cell behavior and Salmonella/SMM phenotypes and the generation of a results report.
Fig 3
Fig 3. Interaction network of selected trafficome hits.
The interaction of selected high-ranking hits (scoring cutoff of ≥8, see also Table 1) was visualized using the STRING database (confidence view). Borders delineate clusters related to the cytoskeleton (i, iii), SNAREs (ii), or COPI and clathrin-coated vesicles (iv).
Fig 4
Fig 4. Influence of host factor silencing on SIF formation.
HeLa LAMP1-GFP cells were not transfected (mock), or reverse transfected with siAllStars or the indicated siRNA, infected with STM WT or SPI2-deficient ssaV expressing mCherry as indicated, and SIF-positive infected cells were counted. Depicted are means with standard deviation for three biological replicates (n = 3). Statistical analysis was performed against siAllstars + WT with Student’s t-test and indicated as: n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 5
Fig 5. RAB proteins identified by the trafficome screen colocalize with SIF and SCV.
A) Direct interaction network of RAB1B as visualized by STRING. B) and C) HeLa cells either stably transfected with LAMP1-GFP (green) or transiently transfected with LAMP1-mCherry (red) were co-transfected with plasmids encoding various RAB GTPases (RAB7A, RAB1A, RAB1B, RAB3A, RAB8A, RAB8B, RAB9A, RAB11A) fused to GFP (green) or mRuby2 (red) and then infected with STM WT expressing mCherry or GFP. Living cells were imaged from 6–9 h p.i. by CLSM and images are shown as maximum intensity projections (MIP). Insets magnify structures of interest and white arrowheads indicate colocalization with SIF. Scale bars, 10 μm (overviews), 1 μm (details).
Fig 6
Fig 6. SNARE proteins identified by the trafficome screen colocalize with SIF and SCV.
A) Direct interaction network of STX7 as visualized by STRING. B) and C) HeLa cells either stably transfected with LAMP1-GFP (green), or transiently transfected with LAMP1-mCherry (red) were co-transfected with plasmids encoding various SNAREs (STX7, VTIB, STX8, VAMP7, VAMP8, VAMP2, VAMP3, VAMP4) fused to GFP (green) or mRuby2 (red). Infection and imaging were performed as for Fig 5. Insets magnify structures of interest and white and blue arrowheads indicate colocalization with SIF and SCV, respectively. Scale bars, 10 μm (overviews), 1 μm (details).
Fig 7
Fig 7. CLTA identified by the trafficome screen colocalizes with SIF and SCV.
A) Direct interaction network of CLTA as visualized by STRING. B) HeLa cells stably transfected with LAMP1-GFP (green) were co-transfected with a plasmid encoding CLTA fused to mRuby2 (red). Infection and imaging were performed as for Fig 5. Insets magnify structures of interest and white and blue arrowheads indicate colocalization with SIF and SCV, respectively. Scale bars, 10 μm (overviews), 1 μm (details).
Fig 8
Fig 8. Newly identified interactions of intracellular STM with host factors.
Depicted are central eukaryotic endomembrane organelles possibly playing a role in the newly identified interplays of host factors with SIF. Magnifications show the interactions of clathrin (i), late secretory and/or recycling-related RAB3A, RAB8A/B, and VAMP2/3/4 (ii), late endo-/lysosomal VTI1B, STX8, and VAMP8 (iii), and early secretory RAB1A/B (iv) with other host factors added as discussed in the text. Solid lines represent interactions identified here or otherwise known, dashed lines represent putative interactions. COP, coat protein complex; EE, early endosome; ER, endoplasmic reticulum; LE, late endosome; Lys, lysosome; RE, recycling endosome; SCV, Salmonella-containing vacuole; SIF, Salmonella-induced filaments; STM, S. Typhimurium; SV, secretory vesicle.
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Grants and funding

This work was supported by BMBF grant 0315834D ‘Medizinische Infektionsgenomik’, and the DFG by priority programme SPP 1580 grant HE 1964/18-2 to M.H, the Z project of SFB944. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.