Genome-wide identification of modulators of Chlamydia trachomatis parasitophorous vacuole stability highlights an important role for sphingolipid supply
Fig 5
Novel reporters for inclusion damage revealed instability of CpoS-deficient inclusions.
(A) Composition of the three GFP11-tagged constructs. (B) The principle of detecting inclusion damage using the split-GFP approach. (C) Fluorescence microscopic detection of inclusion damage during infection with CTL2-cpoS::cat. HeLa cells were transfected with a plasmid driving GFP1–10 expression, infected with the indicated strains (5 IFU/cell), and then fixed, stained (DNA (Hoechst) staining), and imaged at 26 hpi (scale = 20 µm). (D) Confocal fluorescence microscopic images displaying different grades of inclusion damage. GFP1–10-expressing HeLa cells were infected with the indicated strain (10 IFU/cell) and then fixed, stained (DNA (Hoechst) staining), and imaged at 24 hpi (scale = 10 µm). (E) FIB-SEM analysis validating inclusion damage at the ultrastructural level. GFP1–10-expressing HeLa cells were infected with the indicated strain and fixed at 24 hpi. A cell containing green-fluorescent bacteria was identified by fluorescence microscopy (left, scale = 10 µm, asterisks highlight inclusions) and subjected to FIB-SEM analysis (right, scale = 1 µm, one selected slice of the volume shown in S1 Movie, green arrows highlight bacteria in the host cell cytosol). (F) Quantitative analysis of inclusion damage in cells infected with the cpoS mutant. GFP10-expressing HeLa cells were infected with the indicated strains (10 IFU/cell) and then 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 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 CTL2/pOmpA-GFP11int). (G) Complementation of the cpoS mutant restored inclusion integrity. GFP10-expressing HeLa cells were co-infected with the indicated strains (5 IFU/cell of each strain) and then fixed, stained, and imaged at 24 hpi. The percentage of infected cells, surviving cells, and infected cells containing cytosolic bacteria was determined by manual image analysis (mean ± SD, n = 4, at least 200 cells per condition and replicate counted, one-way ANOVA with Tukey’s post-hoc test; if not indicated otherwise, indicated are significant differences compared to CTL2/pOmpA-GFP11int + CTL2). Note that due to the fusogenic nature of inclusions, co-infection results in mixed inclusions containing both of the co-infecting strains. The data underlying this figure can be found in S9 Data.