Figure 5.

Recruitment of full-length CCDC146 to the periphery of the C. trachomatis inclusion does not depend on CT288. (A) A C. trachomatis ctl0540 (ct288 in C. trachomatis strain D/UW3) mutant was generated in strain L2/434 by the targeted insertion of a modified group II intron carrying the aadA gene, conferring spectinomycin resistance. (B) HeLa cells were infected for 24 or 44 h (p.i., post-infection) with C. trachomatis L2/434 or with two C. trachomatis ct288:aadA insertional mutant plaque-purified clones (A,B). Whole cell lysates were analyzed by immunoblotting with antibodies against CT288, C. trachomatis Hsp60 (bacterial loading control) and α-tubulin (loading control for host cells). (C) HeLa cells were infected with the indicated strains at a multiplicity of infection of 5 and recoverable inclusion forming units (IFUs) were determined at 24 and 48 h p.i., Data are mean and standard error of the mean of 4 independent experiments. For each time-point, P-values were calculated by a two-tailed unpaired Student's t-test relative to the L2/434 strain; ns, not significant. (D) HeLa cells transfected with plasmids encoding EGFP or EGFP-CCDC146_FL, and infected for 24 h by C. trachomatis L2/434 or ct288:aadA (clone A) were fixed with methanol, immunolabeled with anti-GFP and anti-Hsp60 antibodies, and appropriate fluorophore-conjugated secondary antibodies, and analyzed by immunofluorescence microscopy. Identical observations were made with clone B (data not shown). Scale bars, 5 μm.