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Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

Figure 1

GBF1 function is required for SM acquisition but not for C. trachomatis replication.

(A) HeLa cells were infected with C. trachomatis L2 for 24 hrs and then fixed and stained with antibodies to GBF1 (red) and BIG1 (green). Bacteria and host DNA were detected using DAPI (blue). The cis and trans polarity of the Golgi was maintained in C. trachomatis L2-infected cells. N, host nucleus. *, inclusion. Scale bar = 5 µm. (B) HeLa cells were transfected with Arf1-GFP for 18 hrs, infected with C. trachomatis L2 for 24 hrs in the absence or presence of 10 µM BFA, and then fixed and stained with antibodies to GBF1 (red). Enlargements of boxed regions are shown to the right. Images represent a single z slice from confocal images. The exposure time for each filter set for all images was identical. Arf1-GFP localized to the region between two closely apposed inclusions (white arrow) and to a thin rim around the inclusion (red arrow) whereas GBF1 was excluded from these regions. *, inclusion. Scale bar = 5 µm. (C) Western blot analysis of siRNA-treated samples. GAPDH was used as a loading control. (D) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then labeled with BODIPY FL-Ceramide to visualize SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (E) HeLa cells were infected with C. trachomatis for 24 hrs, treated with 10 µM BFA or GCA during the last 3 hrs of infection, and then labeled with BODIPY FL-Ceramide to analyze SM acquisition by the inclusion. The exposure time for all images was identical. Dashed red lines demarcate the inclusions. Scale bar = 5 µm. (F) HeLa cells were depleted of GBF1, BIG1, and/or BIG2 for 3 days, infected with C. trachomatis L2 for 24 hrs, and then analyzed for progeny formation as described in Materials and Methods. Values (mean ± standard error) are shown as percentage of control siRNA-treated samples. No significant decrease in progeny formation was observed. IFU, inclusion forming units.

Figure 1

doi: https://doi.org/10.1371/journal.ppat.1002198.g001