Fig. 1.

TaiP binds directly to ATG16L1 through its C-terminal domain to promote C. trachomatis infection. (A) HeLa cells were cotransfected with Flag-CT622 and the indicated GFP-tagged construct for 24 h, lysed, and IP was performed with anti-Flag coupled beads. Proteins were separated on SDS/PAGE, transferred on a poly(vinylidene difluoride) (PVDF) membrane, and probed with the indicated antibody ([IB]: immunoblot). An aliquot of each cell lysate was loaded on a separate gel to visualize the expression of Flag-CT622 and of each of the GFP-tagged proteins (input, Left). (B) Same as in A, using full-length CT622 or constructs corresponding to the N-terminal (CT622^Nterm, amino acids 1–345) or C-terminal (CT622^Cterm, amino acids 346–647) domains. (C) HeLa cells were infected for 35 h with Ctr^{Δct622+CT622-Flag} bacteria, lysed, and IP was performed with anti-Flag coupled beads. Proteins were separated on SDS/PAGE, transferred on a PVDF membrane and probed with anti-Flag and anti-ATG16L1 antibodies. (D) Recombinant ATG16L1 (100 nM) was incubated with recombinant GST-CT622 or GST-CT622^Nterm (100 nM) for 60 min at 4 °C before performing GST-PD using glutathione beads. PD fractions were analyzed by Western blot as in A. GST-CT622^Nterm used here as a negative control shows the level of nonspecific ATG16L1 binding to the beads. ATG16L1 was pulled-down together with TaiP, demonstrating that the interaction is direct. The experiment shown is representative of three independent experiments. (E) Quantification of the effect of KO atg16l1 on inclusion size. WT or atg16l1 KO cells seeded on coverslips were infected with Ctr^WT, Ctr^ΔtaiP, or Ctr^{ΔtaiP+TaiP-Flag} for 20 h at multiplicity of infection (MOI) = 0.2. After fixation, infected cells were permeabilized, and the inclusion membrane was stained with antibodies against the inclusion protein Cap1. The area of inclusion was measured using imageJ software. The dot plot shows the median ± SD of three independent experiments (n > 50 in total) and displays the P values of the Student’s t tests. The Right shows the absence of ATG16L1 in atg16l1 KO whole cell lysates probed by Western blot with anti-ATG16L1 antibodies. ACTIN IB serves as a loading control. (F) Quantification of the effect of TaiP expression on inclusion size. Cells were transfected with constructs for Flag-CymR or Flag-TaiP for 24 h before being infected with Ctr or Ct^ΔtaiP. The cells were fixed 20-h postinfection, permeabilized, and the inclusion membrane was stained with antibodies against the inclusion protein Cap1. The area of inclusion was measured using imageJ software. The dot plot displays the median ± SD of three independent experiments (n > 50 in total) and displays the P values of the Student’s t test.