Genome-wide identification of modulators of Chlamydia trachomatis parasitophorous vacuole stability highlights an important role for sphingolipid supply
Fig 6
Early destabilization of inclusions clears infection while keeping host cells alive.
(A–B) Destabilizing effect of SPT inhibition on inclusions. GFP10-expressing HeLa cells were infected with the indicated strains (5 IFU/cell) and parallelly treated with MYR (10 µM) or LCS (125 µM) or left untreated (control). Cells were fixed, stained, and imaged at the indicated time points. (A) The percentage of infected cells, surviving cells, and infected cells containing cytosolic bacteria was determined by manual image analysis (mean ± SD, n = 3, at least 100 cells per condition and replicate counted, 2-way ANOVA with Sidak’s post-hoc test; for each time point, indicated are significant differences compared to the untreated control). (B) Representative images of whole cell (HCS) staining (scale = 100 µm). (C) Inclusion-destabilizing effects of SPTLC1 depletion or KDSR deficiency. The indicated cell lines were transfected with a GFP1–10-expressing plasmid and then infected with the indicated strains. Cells were fixed, stained, and imaged at the indicated time points. The percentage of infected cells, surviving cells, and infected cells containing cytosolic bacteria was determined by manual image analysis (mean ± SD, n = 3, at least 200 cells per condition and replicate counted, one-way ANOVA with Dunnett’s post-hoc test; for each time point (and strain), indicated are significant differences compared to the parental cells). (D–E) Treatment with SKI-II destabilizes CTL2 inclusions. GFP10-expressing HeLa cells were infected with the indicated strains (5 IFU/cell) and parallelly treated with SKI-II (10 µM) or solvent-only (DMSO). Cells were fixed at 24 hpi or 30 hpi, stained (Hoechst, DNA; Slc1, bacteria), and imaged. (D) The percentage of infected cells displaying inclusion damage (ruptured inclusions or early damage indicated by individual cytosolic bacteria) was determined by manual image analysis (mean ± SD, n = 3, at least 200 cells per condition and replicate counted, unpaired t test). (E) Representative images displaying cytosolic bacteria and inclusion rupture (scale = 10 µm). The data underlying this figure can be found in S10 Data.