Figure 4.

Glycolysis and OxPhos are both required for bacterial proliferation in epithelial cells, glycolysis being also required for the initiation of infection. A2EN cells were infected with CTDm and treated with indicated concentrations of GNE-140 (A), oligomycin (B), or phenformin (C) for 48 h. Cells were lysed and bacterial IFU were determined by reinfecting fresh HeLa cells as described in the Experimental procedures. D, A2EN cells were infected with CTDm and treated with indicated concentrations of oligomycin or phenformin for 48 h. Cells were lysed for DNA extraction and samples were analyzed by qPCR. Bacterial load was determined by quantifying the bacterial glgA gene and normalizing it to cell number using the 36B4 gene. A2EN (E and G) or primary cells (F) were infected for 24 h in the presence of the indicated concentration of drug before being fixed and analyzed by flow cytometry to determine the percentage of infected cells. A2EN cells were infected at MOI 20 with CTDm and treated or not with GNE-140 (H) or oligomycin (I). Cells were fixed 6 hpi, then permeabilized before immunostaining with anti-Cap1 followed with Alexa488-conjugated secondary antibodies. DNA is stained with Hoechst. J and K, HeLa cells were infected with CTDm and treated 12 hpi with indicated concentrations of GNE-140 (J) or oligomycin (K) before IFU were determined 48 hpi, as described in the Experimental procedures. The results of 3 independent experiments and the mean are displayed. For each panel, the results of 3 or 4 independent experiments and the mean are displayed. Significance is defined as: ns (not significant) p > 0.05; (∗) = p ≤ 0.05; (∗∗) = p ≤ 0.01; (∗∗∗) = p ≤ 0.001; (∗∗∗∗) = p ≤ 0.0001. CTD, C. trachomatis serovar D; hpi, hours postinfection; IFU, infection forming unit; OxPhos, oxidative phosphorylation; qPCR, quantitative PCR.