Supplementation with sphingosine stabilizes CpoS-deficient inclusions.

(A–B) Supplementation of growth media with sphingoid bases protected cells from cpoS mutant-induced death and restored bacterial growth. HeLa cells were infected with the indicated strains (4 IFU/cell), and parallelly treated with 5 µM of the indicated metabolites (dhSph, dihydrosphingosine; Sph, sphingosine; C6-dhCer, C6-dihydroceramide; C6-Cer, C6-ceramide; DMSO, solvent only) or left untreated (control). Nuclei count (A) at 25.5 hpi is displayed normalized to the uninfected untreated control, while inclusion count (B) is displayed normalized to the CTL2-infected untreated control (mean ± SD, n = 3, one-way ANOVA with Dunnett’s post-hoc test; for each infection condition, indicated are significant differences compared to the DMSO control). (C–D) Stabilizing effect of sphingosine on CpoS-deficient inclusions. GFP10-expressing HeLa cells were infected with the indicated strains (5 IFU/cell) and parallelly treated with 5 µM sphingosine (Sph) or left untreated (control). Cells were fixed, stained (HCS, cells; Slc1, bacteria), and imaged at 24 hpi. (C) 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 strain, indicated are significant differences compared to the untreated control). (D) Representative images (scale = 100 µm). The data underlying this figure can be found in S11 Data .