Genome-wide identification of modulators of Chlamydia trachomatis parasitophorous vacuole stability highlights an important role for sphingolipid supply
Fig 3
CpoS deficiency causes potentially cytotoxic alterations in sphingolipid metabolite levels.
(A) Infection with the cpoS mutant enhanced the dihydroceramide (dhCer) to ceramide (Cer) ratio in infected cells. Cell extracts from HeLa cells infected with the indicated strains (10 IFU/cell) were prepared at 14 hpi, and the indicated lipids were quantified by LC–MS/MS. Shown are selected metabolite levels (expressed as “fmol/pmol total sphingolipids”) or ratios (mean ± SD, n = 3, one-way ANOVA with Tukey’s post-hoc test; if not specified otherwise, indicated are significant differences compared to uninfected cells). Full data are presented in S5 Fig. (B) Mass spectrometric analysis confirming SKI-II to increase the dhCer to Cer ratio. Cell extracts from uninfected and CTL2-infected (5 IFU/cell) HeLa cells, treated with SKI-II (6.25 µM) at 0 hpi, were prepared at 14 hpi, and the indicated lipids were quantified by LC–MS/MS and displayed as ratio (mean ± SD, n = 3, two-way ANOVA with Sidak’s post-hoc test; indicated are significant differences compared to the DMSO control). (C) SKI-II induced death in cells infected with CTL2. HeLa cells were treated with SKI-II (6.25 µM) or solvent only (DMSO) and were parallelly infected with the indicated strains (5 IFU/cell). Resorufin fluorescence at 25.5 hpi is displayed normalized to a CTL2-infected untreated control (mean ± SD, n = 4, two-way ANOVA with Sidak’s post-hoc test; for each strain, indicated are significant differences compared to the DMSO control). (D–E) SKI-II induced death specifically in infected but not uninfected cells. HeLa cells were treated with the indicated concentrations of SKI-II or with solvent only (DMSO) and were parallelly infected with CTL2 (5 IFU/cell) or left uninfected. (D) Resorufin fluorescence at 34 hpi is displayed normalized to the uninfected untreated control (mean ± SD, n = 3, two-way ANOVA with Sidak’s post-hoc test; if not specified otherwise, indicated are for each infection condition significant differences compared to the DMSO control). (E) Representative images of DNA (Hoechst) staining (scale = 80 µm). The data underlying this figure can be found in S7 Data.