Fig. 3.

TaiP blocks the TMEM59/ATG16L1 complex to promote C. trachomatis inclusion expansion. (A) Cells transfected with the indicated siRNA were infected with Ct^ΔtaiP the following day at MOI = 0.2. Cells were fixed 20 h later, permeabilized, and the inclusion membrane was stained with anti-Cap1 antibodies. Inclusion areas were measured using imageJ software. The box and whiskers plots represent the median area and 90th percentile of intracellular inclusions (n > 50 cells from three independent experiments). Statistical analysis was performed using a one-way ANOVA test with a Dunnett’s multiple comparison test to small interfering control (siCtrl). (B) Cells transfected with the indicated plasmids were infected or not 12 h later with Ct^WT, Ct^ΔtaiP, or Ctr^{ΔtaiP+TaiP-Flag} bacteria at MOI = 1. Cells were lysed 30 h later, and IP was performed with anti-HA coupled beads. Proteins were separated on SDS/PAGE, transferred on a PVDF membrane, and probed with the indicated antibodies. (C) Lysates from cells expressing HA-TMEM59 or GFP-ATG16L1 were mixed in the presence of 100 pmol of GST-TaiP or GST-TaiP^Nterm (for negative control) for 90 min at 4 °C in a final volume of 1.5 mL. TMEM59 was IP with anti-HA coupled beads and the levels of GFP-ATG16L1 in the IP fraction was analyzed by Western blot. IBs of the input fractions show the expression of each individual protein and represents 1.5% of the total reaction and comassie staining shows purified GST-TaiP and GST-TaiP^Nterm. The histogram on the Right displays the mean ± SD of three independent experiments and the P value of the Student’s t test. Addition of GST-TaiP decreased the amount of GFP-ATG16L1 that co-IP with HA-TMEM59 by about 50%. (D) HeLa cells were cotransfected with Flag-TaiP, Flag-TaiP^D480A, and GFP-ATG16L1 constructs for 24 h, lysed, and IP was performed with anti-Flag antibody. Proteins were separated on SDS/PAGE, transferred on a PVDF membrane, and probed with the indicated antibody. An aliquot of each cell lysate was loaded on separate gels to visualize the expression of the tagged proteins (input). Quantification on the Right shows the decrease in the amount of ATG16L1 that co-immunoprecipitates with Flag-TaiP^D480A compared to Flag-TaiP. Statistical analysis was performed using Student’s unpaired t test, n = 4. (E) Quantification of the average inclusion size in cells expressing Flag-CymR, Flag-TaiP, and Flag-TaiP^D480A. Cells were transfected for 24 h before they were infected for 20 h with Ctr^ΔtaiP at MOI = 0.2. After fixation and permeabilization, inclusions were stained using antibodies against Cap1. Inclusion areas were measured using imageJ software. The dot plot shows the median ± SD of three independent experiments (n > 50 cells in each experiment) and displays the P values of the Student’s t test.