Genome-wide identification of modulators of Chlamydia trachomatis parasitophorous vacuole stability highlights an important role for sphingolipid supply
Fig 2
Deficiencies in ceramide synthesis provide specific protection against the cpoS mutant.
(A) Illustration of pathways in ceramide synthesis and metabolism. Enzymes whose deficiency protected cells from cpoS mutant-induced toxicity in the screen are highlighted in bold green, pharmacologic inhibitors used in this study in bold gray. Asterisks mark enzymes selected for genetic validation via individual CRISPR/Cas9-mediated gene knockout or knockdown. The names of the inhibitors are given in the main text. Enzymes: ACER/ASAH, ceramidase; CERK, ceramide kinase; CERS, ceramide synthase; CPP, ceramide-1-phosphate phosphatase; DEGS, dihydroceramide desaturase; GBA, glucosylceramidase; KDSR, 3-ketodihydrosphingosine reductase; LPP, lipid phosphate phosphatase; SGMS, sphingomyelin synthase; SGPP, sphingosine-1-phosphate phosphatase; SMPD, sphingomyelinase; SPHK, sphingosine kinase; SPL, sphingosine phosphate lyase; SPT, serine palmitoyl transferase; UGCG, UDP-glucose ceramide glycosyltransferase. (B–D) Selected inhibitors targeting sphingolipid metabolism protected cells against cpoS mutant-induced death. HeLa cells were treated with the indicated inhibitors (LCS, 125 µM; MYR, 1 µM; GW4869, 12.5 µM) or solvent only (DMSO), and were parallelly infected with the indicated strains (5 IFU/cell). Resorufin fluorescence (B) and nuclei count (C) at 25.5 hpi are displayed normalized to a CTL2-infected untreated control (mean ± SD, n = 4, two-way ANOVA with Dunnett’s post-hoc test; for each strain, indicated are significant differences compared to the DMSO control). (D) Representative images of DNA (Hoechst) staining (scale = 80 µm). (E) western blot analysis confirming the depletion (knockdown, KD) of SPTLC1 and absence (knockout, KO) of KDSR in the respective HeLa cell lines. (F–H) Depletion of SPTLC1 or deficiency in KDSR partially protected cells against cpoS mutant-induced death. The indicated HeLa cell lines were infected with the indicated strains (5 IFU/cell). Resorufin fluorescence (F) and nuclei count (G) at 24 hpi are displayed normalized to an uninfected control (mean ± SD, n = 3, one-way ANOVA with Dunnett’s post-hoc test; for each strain, indicated are significant differences compared to the parental (wild-type) cells). (H) Representative images of DNA (Hoechst) staining (scale = 80 µm). The data underlying this figure can be found in S6 Data.