Fig 1.
A CRISPR screen identified host factors contributing to C. trachomatis L2-induced cytotoxicity.
(A) CRISPR screening procedure. The screen was conducted in two independent replicates (R1 + R2). (B) Venn diagram illustrating the selection of screening hits. (C) STRING interaction network displaying interactions among and between “general infection hits” (lilac nodes) and “mutant-specific hits” (green nodes), refined by manual grouping and annotations. Mixed groups were deemed to represent biological processes of general importance for infection (lilac group labels), while groups containing only “mutant-specific hits” were considered to have specific importance during infection with the CpoS-deficient mutant (green group labels). The data underlying this figure can be found in S4 Data.
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.
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.
Fig 4.
The cpoS mutant displays an increased dependence on host de novo ceramide synthesis.
(A–B) SKI-II treatment eradicated infected cells from cultures infected with CTL2. HeLa cells were treated with the indicated concentrations of SKI-II or with solvent only (DMSO), and parallelly infected with a GFP-expressing derivative of CTL2 (5 IFU/cell). Bacterial inclusions were detected microscopically at 34 hpi. (A) Inclusion numbers displayed relative to CTL2 inclusion count in the untreated control (mean ± SD, n = 2–3, one-way ANOVA with Dunnett’s post-hoc test; indicated are significant differences compared to the DMSO control). (B) Representative images showing inclusions (GFP) and DNA (Hoechst) staining (scale = 40 µm). (C–D) MYR and LCS, but not GW4869, reduced inclusion count after infection with the cpoS mutant. HeLa cells were treated with the indicated inhibitors (LCS, 125 µM; MYR, 1 µM; GW4869, 12.5 µM) or solvent only (DMSO), and parallelly infected with GFP-expressing derivatives of the indicated strains (1 IFU/cell). Bacterial inclusions were detected microscopically at 26.5 hpi. (C) Data displayed as percentage relative to inclusion count and average inclusion area detected for CTL2 in the absence of inhibitors (mean ± SD, n = 4, one-way ANOVA with Dunnett’s post-hoc test; for each strain, indicated are significant differences compared to the DMSO control). (D) Representative images showing inclusions (GFP) and DNA (Hoechst) staining (scale = 40 µm). (E) Differential susceptibility of cpoS mutant and wild-type bacteria to SPT inhibition. HeLa cells were treated with the indicated inhibitors and parallelly infected with GFP-expressing derivatives of the indicated strains (1 IFU/cell). GFP fluorescence measured at 25.5 hpi is displayed relative to fluorescence detected for the respective strain in the absence of inhibitors (mean ± SD, n = 3–4, 2-way ANOVA with Sidak’s post-hoc test; for each concentration, indicated are significant differences between the two strains). (F–G) Depletion of SPTLC1 or deficiency in KDSR partially restored inclusion count after infection with the cpoS mutant but increased the occurrence of inclusion-free cells. The indicated HeLa cells lines were infected with the indicated strains (5 IFU/cell). (F) Inclusion numbers detected at 24 hpi displayed normalized to the number of inclusions detected for CTL2 in the parental cells (mean ± SD, n = 3, two-way ANOVA with Dunnett’s post-hoc test; for each strain, indicated are significant differences compared to the parental cells). (G) Representative images showing inclusions (detected by immunofluorescence staining of the bacterial protein Slc1) and DNA (Hoechst) staining (scale = 40 µm). The data underlying this figure can be found in S8 Data.
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.
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.
Fig 7.
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.
Fig 8.
Model describing how a disruption of both sphingolipid supply routes may destabilize inclusions in a potentially therapeutically beneficial manner.
C. trachomatis can acquire sphingolipids in two ways, by recruiting the host ceramide transfer protein CERT to acquire ceramides (Cer) then converted to sphingomyelin (SM) at the inclusion [48], and by hijacking sphingolipid-laden membrane vesicles and directing them to the inclusion [47]. When CpoS is absent, the vesicular transport of sphingomyelin to the inclusion is compromised [11], leading to inclusion damage at mid stages of infection, followed by premature host cell death. We hypothesize that under conditions of impaired ceramide de novo synthesis, the CERT-mediated transport is compromised, while the bacteria may still be able utilize the already available lipid pool through CpoS-mediated modulation of host vesicular transport, leading to more minor inclusion damage that manifests at late stages of infection. A combination of ceramide synthesis disruption and CpoS deficiency, which should compromise both transport routes, results in early inclusion destabilization and clearance of infection while keeping the host cells alive. Hence, an effective therapeutic targeting of inclusion stability may have to aim for blocking both transport routes simultaneously.