Fig 2: TepP is required for C. trachomatis replication in A2EN cells and to recruit CrkL and PI3K to nascent inclusions. (A to D) Phenotypic characterization of a C. trachomatis ?tepP::bla insertional mutant. (A) Immunoblot analysis of TepP expression in HeLa cells infected for 24 h with the indicated strains. Slc1, loading control. (B) A2EN cells were infected with CTL2 or ?tepP::bla bacteria, and the levels of IFIT1 (black) and IFIT2 (gray) transcripts from the strains described in the panel A legend were assessed by quantitative RT-PCR. DNA transfection was used as a positive control for the induction of IFIT genes. (C) Time course of appearance of major tyrosine-phosphorylated (p-Tyr) proteins in A2EN cell infected with CTL2 or ?tepP::bla insertional mutants or left uninfected (Uninf.). MOMP, bacterial major outer membrane protein (arrowhead); Tub, tubulin. (D) The replication potential of ?tepP::bla mutants was assessed in HeLa and A2EN cells by the generation of inclusion-forming units (IFU). (E and F) Subcellular localization of CrkL, PI3K (p110a), and GSK-3ß in Chlamydia-infected cells. A2EN cells were infected with CTL2 or ?tepP::bla bacteria for 4 h and immunostained by anti-MOMP (green), anti-CrkL, anti-p110a, or anti-GSK-3ß (red). Hoechst (blue) was used to detect DNA. Quantification of Chlamydia (MOMP) and p110a, CrkL, and GSK-3ß colocalization was performed on a single-cell basis (n = 20 and 30 cells/replicate). Student’s t test showed that there was a statistically significant difference between CTL2 and ?tepP::bla bacteria, with a P value of <0.001.

Fig 3: TepP is phosphorylated by Src kinases. (A) In vitro phosphorylation reactions were performed by incubating recombinant 6×His-TepP with ATP and cell lysates derived from the indicated cell lines. The degree of TepP phosphorylation was assessed by immunoblotting with anti-pY antibodies after reisolation of TepP on nickel beads. TepP was phosphorylated under all conditions except after incubation with SYF cell lysates and the buffer-only control. (B) TepP-FLAG immunoprecipitations from cell lines infected with CTLM062G1 expressing TepP-FLAG. TepP phosphorylation was assessed after IP with anti-FLAG antibodies and immunoblotting with anti-pY antibodies. Note the decreased levels of phosphorylation in Src, Yes, and Fyn-deficient cells. (C) Mouse embryo fibroblast (MEF), SYF, and SYF-plus-c-Src cell lines were infected for 4 h with C. trachomatis at an MOI of 20, fixed, and stained. Infected cells were immunostained by anti-MOMP (green), anti-CrkL, or anti-p110a (red) antibodies.

Fig 4: TepP forms complexes with PI3K and CrkL in infected cells. (A) Schematic representation of epithelial proteins that associate with TepP during infection. A2EN cells were infected with C. trachomatis expressing TepP-FLAG for 4 h, and cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibodies. Bound proteins were digested with trypsin, and the resulting peptides were identified by mass spectrometry (MS). Sizes of circles correspond to the relative numbers of peptides identified for each protein by LC-MS/MS. (B) Immunoblot validation of TepP-interacting proteins. A2EN cells were infected for 4 h with CTLM062G1 transformed with a TepP-FLAG expression plasmid or vector-only control (Vec.), and cell lysates were subjected to IP with anti-FLAG antibodies. Bound proteins were detected by immunoblotting with specific antibodies. (C and D) Reciprocal co-IP of CrkL (C), PI3K (D), and TepP. A2EN cells were infected with CTLM062G1 strains as described for panel B, and at 4 h postinfection, CrkL and p110a were subjected to IP. Co-IP of proteins was assessed by immunoblot analysis.