Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites
Fig 7
Inhibition of ERES cargo loading or specificity reduces C. trachomatis developmental growth.
(A) Experimental design used to test the impact of ERES disruption on Chlamydia developmental growth. Colored bars mark the times when FLI-06 or 4PBA was applied to infected cells. All cells were harvested for IFU determination at 48 hpi. The effects of ERES inhibition were determined for (B,D) primary infection, through measuring the diameters of inclusions at 48 hpi, or (C,E) IFU production at 48 hpi. Bars denote the mean (n = 3; SD); white bars correspond to untreated controls; gray bars are inhibitor treated in decreasing concentration, 10 μM, 5 μM, or 1 μM FLI-06 and 5 mM, 2 mM, 1 mM 4PBA. Colored bars on x-axis correspond to the inhibitor application key in A. Significance determined by one-way ANOVA with Dunnett’s multiple comparisons test, comparing to untreated control. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (F) Infected cells were treated from 24–48 hpi with 10 μM FLI-06, 5 mM 4PBA, or 0.5 μg/mL chloramphenicol (CAM). Genomic DNA was extracted, and genome copy number was determined by using quantitative PCR for the GroEL2 gene. Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test, comparing to control. **, p < 0.01; ***, p < 0.001. (G) Relative expression of genes upregulated during ER stress in infected or mock infected cells after treatment with 10 μM FLI-06, 5 mM 4PBA, or 1 μM thapsigargin from 20–24 hpi. Gray bars are infected, black are uninfected. Expression was determined using quantitative PCR. Significance was determined using a two-way ANOVA with Dunnett’s multiple comparisons test. There was no significant difference between infected and uninfected cells, thapsigargin treatment was significantly different from the control. Uninfected treatments were compared to uninfected control, infected treatments compared to infected control. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.