Fig 1.
Chlamydia triggers host cell ferroptosis during the late stage of its developmental cycle.
(A) Host cell death induced by Chlamydia trachomatis serovar D at various multiplicities of infection (MOIs) was assessed over a time course of 0–72 hours post-infection using a propidium iodide (PI) staining assay. Scale bars represent 200 μm. (B) The number of PI-positive cells per 200× field was quantified for subsequent statistical analysis. Statistical significance was determined using a two-way ANOVA with Bonferroni’s multiple comparisons. Individual data points represent the number of PI-positive cells per 200× field, with the line indicating the mean of positive cells across all 200× fields. Data were derived from three independent experiments, with five 200× fields analyzed per experiment (n=3). (C) The release of lactate dehydrogenase (LDH) was measured in both mock- and Chlamydia trachomatis serovar D-infected HeLa-229 cells over a time course of 0–72 hours post-infection. Statistical analysis was performed using a two-way ANOVA with Bonferroni’s multiple comparisons. Individual data points represent the mean, and the bars represent the standard deviation (SD) of the mean (n=3). (D) The level of lipid peroxidation in Chlamydia trachomatis serovar D-infected cells at different MOIs throughout the infection was evaluated using C11-BODIPY staining, followed by flow cytometric analysis. (E) Statistical analysis of the data from (D) was conducted using a two-way ANOVA with Bonferroni’s multiple comparisons. Data are presented as mean ± SD (n=3). (F) The release of LDH and the level of lipid ROS in Chlamydia trachomatis serovar L1 (CT-L1) (MOI 3)-infected HeLa-229 cells were measured and compared to mock-infected HeLa-229 cells (n=3). (G) The release of LDH and the level of lipid ROS in Chlamydia muridarum (CM) (MOI 2)-infected McCoy cells were measured and compared to mock-infected McCoy cells at 48 h.p.i. The Student’s t-test was used for statistical analysis of (F) and (G). Data are presented as the mean ± SD (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Fig 2.
Suppression of host cell ferroptosis impairs Chlamydia progeny release in-vitro.
(A) A schematic diagram illustrates a series of reported pharmaceuticals that block ferroptosis by inhibiting lipid ROS accumulation. (B) The release of LDH and the levels of lipid ROS in Chlamydia trachomatis serovar D (MOI 10)-infected HeLa-229 cells were assessed following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. Statistical analysis was conducted using a one-way ANOVA with Bonferroni’s multiple comparisons (n=3). (C) Immunoblot analysis of chlamydial MOMP from cell supernatant and GAPDH from cell lysate were conducted following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. (D) The copy number of the chlamydial cryptic plasmid in the cell supernatant of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) for 72 hours. Statistical analysis was conducted using a one-way ANOVA with Bonferroni’s multiple comparisons (n=3). (E) The copy number of ompA in the total culture (cell supernatant and monolayer) of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined following treatment with ferrostatin-1 (10 μM) and liproxstatin-1 (1 μM) over a time course. Statistical analysis was conducted using a two-way ANOVA test (n=3). (F, G) The release of LDH, lipid ROS levels, and the copy number of the chlamydial cryptic plasmid of cell supernatant in Chlamydia trachomatis serovars L1 (MOI 3)- (F) and A (MOI 3)- (G) infected cells were measured following treatment with liproxstatin-1 (1 μM) for 72 hours. The Student’s t-test was used for statistical analysis of (F) and (G) (n=3). (H) The release of LDH and the level of lipid ROS in Chlamydia muridarum (CM) (MOI 2)-infected McCoy cells were measured following treatment with trolox (3.2 mM) for 48 hours. The Student’s t-test was used for statistical analysis (n=3). (I) Immunoblot analysis of chlamydial HSP60 in the cell supernatant and β-actin in the cell lysate was performed following treatment with trolox (3.2 mM) for 48 hours. (J) The copy number of the Nigg II plasmid in the cell supernatant and total culture (cell supernatant and monolayer) of CM (MOI 2)-infected McCoy cells was quantified over a time-course following treatment with trolox (3.2 mM). The Student’s t-test was used for statistical analysis of the cell supernatant data (left panel), while a two-way ANOVA was applied to the data from the total culture (cell supernatant and monolayer) over the time course (right panel). Data are presented as the mean ± SD (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Fig 3.
Ferroptosis inhibitor vitamin E reduces Chlamydia muridarum burden and pathology in the mouse genital tract.
(A) Gross pathology images of whole genital tracts from Chlamydia muridarum-infected C57BL/6 mice, fed natural ingredient diets containing low (7 mg/kg) or high (120 mg/kg) vitamin E, were collected at 56-day post-infection (d.p.i). White arrow indicates hydrosalpinx (n = 8 per group) (B) Incidence of hydrosalpinx at 56 d.p.i. (***, P < 0.001, by Fisher’s exact test; n = 8 per group). (C) Severity of hydrosalpinx at 56 d.p.i. Both individual bilateral scores (dots) and medians (bars) are shown. Statistical analysis was performed using the Mann-Whitney test (n = 8 per group). (D) Lower genital tract shedding course of Chlamydia muridarum following intravaginal inoculation with 2 × 105 IFU. Shedding was quantified from cervicovaginal swabs collected on the indicated days post-infection. Points represent the IFU of infectious EBs collected from each mouse, and the lines represent the means of 8 mice per group. Statistical analysis was performed using a two-way ANOVA. The P value is indicated in the panel. The table (bottom) shows the mean IFU and the ratio between the two groups on the indicated days (n = 8 per group). (E) Micrographs of H&E-stained oviducts from infected mice at 56 d.p.i. For each infected mouse, both a broad view of tissue sections (10×; scale bar, 250 μm) and an amplified view (40×; scale bar, 50 μm) are shown. The rectangular frame with an arrow indicates foci of chronic inflammatory cell infiltration (n = 8 per group). (F) Bilateral chronic inflammatory cell infiltrate scores were determined from H&E-stained tissue sections. Statistical analysis was performed using the Mann-Whitney test (n = 8 per group). ns, not significant. (G) Diameter of uterine horns was measured from H&E-stained tissue sections. Statistical analysis was performed using the Mann-Whitney test (n = 8 per group). ns, not significant.
Fig 4.
Chlamydia trachomatis triggers host cell ferroptosis through SLC7A11 downregulation and consequent glutathione depletion.
(A) Immunoblot analysis of multiple ferroptosis-associated proteins and chlamydial HSP60 was performed in cells infected with various MOIs of Chlamydia trachomatis serovar D (CT-D) at 72 h.p.i., compared to mock-infected cells. (B) Immunoblot analysis of SLC7A11, GPx4, GAPDH, and chlamydial HSP60 was conducted in cells infected with Chlamydia trachomatis serovar L1 (CT-L1) (MOI 3) and Chlamydia muridarum (CM) (MOI 2), compared to mock-infected cells. (C) A schematic representation of the SLC7A11-GSH-GPx4 pathway in the regulation of ferroptosis. (D) Intracellular glutathione (GSH) levels were measured in CT-D (MOI 5)-infected cells at 72 h.p.i by flow cytometry, compared to mock-infected cells. The fraction of cells with high intracellular GSH was calculated. The Student’s t-test was used for statistical analysis. Data are presented as the mean ± SD (n=3). **, P < 0.01.
Fig 5.
CPAF mediates chlamydial-induced ferroptosis through proteolytic degradation of SLC7A11 in host cells.
(A) The mRNA expression of SLC7A11 and GPx4 in Chlamydia trachomatis serovar D (MOI 10)-infected cells was measured by SYBR Green qPCR at 72 h.p.i., compared to mock-infected cells. Statistical analysis was performed using Student’s t-test. Data are presented as the mean ± SD (n=3). (B) Immunoblot analysis of SLC7A11, GPx4, and cHSP60 in CT-D (MOI 10) infected cells was performed at 72 h.p.i., following a 6-hour pre-treatment with lactacystin (10 μM) or MG132 (10 μM). (C) The copy number of ompA in the total culture (cell supernatant and monolayer) of Chlamydia trachomatis serovar D (MOI 10)-infected cells was determined before and after treatment with lactacystin (10 μM). Statistical analysis was performed using a two-way ANOVA test (n=3). (D) The release of LDH from cells infected with the CPAF-deficient strain (L2-17/mCherry) (MOI 1) or the CPAF-supplemented strain (L2-17/CPAF) (MOI 1) was measured at 72 h.p.i. Statistical analysis was performed using a one-way ANOVA test with Bonferroni’s multiple comparisons. Data are presented as the mean ± SD (n=3). (E) Immunoblot analysis of SLC7A11, TfR1, GPx4, GAPDH, and CPAF was performed across three groups: mock infection, CPAF-deficient strain (L2-17/mCherry) (MOI 1) infection, and CPAF-supplemented strain (L2-17/CPAF) (MOI 1) infection at 72 h.p.i. (F) Immunoblot analysis of SLC7A11, vimentin, CPAF, and GAPDH in Chlamydia trachomatis serovar D (MOI 3) -infected cells was performed using the hot SDS loading buffer lysis protocol over the course of infection. (G), (H) The release of LDH (G) and the level of lipid ROS (H) in HeLa-229 cells following the induction of CPAF expression by DMSO or doxycycline treatment were quantified. Statistical analysis was performed using Student’s t-test. Data are presented as the mean ± SD (n=3). (I) The expression of CPAF and the degradation of SLC7A11 and GPx4 were confirmed in HeLa-229 cells after a 24-hour treatment with doxycycline, as determined by western blotting. (J) Intracellular glutathione (GSH) levels were determined in HeLa-229 cells following the induction of CPAF with doxycycline treatment. Statistical analysis was performed using Student’s t-test. Data are presented as the mean ± SD (n=3). (K) Immunoblot analysis was performed to examine the degradation of SLC7A11 in cell lysates following a 30-minute incubation with recombinant wild-type CPAF (rCPAF) or the H105A mutant. An activated CPAFc fragment was observed in the wild-type CPAF group, but not in the CPAF (H105A) group. The degradation of recombinant wild-type CPAF was blocked by lactacystin. **, P< 0.01; ***, P<0.001; ns, not significant.
Fig 6.
Chlamydial protease-like activity factor targets SLC7A11 for degradation to induce ferroptosis and facilitate progeny releases.
A schematic diagram illustrates the key findings of this study, showing how Chlamydial protease-like activity factor directly degrades the host protein SLC7A11, leading to GSH depletion, lipid ROS accumulation of lipid ROS, and ultimately triggering host cell ferroptosis, which facilitates Chlamydia progeny release.