Figure 6
A C. trachomatis ct105::aadA insertional mutant is not defective for intracellular growth in tissue culture cells. (A,C) trachomatis ct105::aadA insertional mutant was generated in the strain L2/434 by the targeted insertion of a modified group II intron carrying a spectinomycin-resistance gene. (A) Hela cells were infected by C. trachomatis L2/434 harboring a plasmid encoding CT105-2HA or by an identical plasmid carrying a ct105::aadA-2HA mutant allele. Whole cell extracts were analyzed by immunoblotting with antibodies against HA, C. trachomatis Hsp60 (bacterial loading control) and α-tubulin (HeLa loading control) using SuperSignal West Pico detection kit (Thermo Fisher Scientific) for Hsp60 and α-tubulin, or SuperSignal West Femto detection kit (Thermo Fisher Scientific) for HA. (B) HeLa cells were infected for 24, 30, or 40 h by C. trachomatis L2/434 or ct105::aadA mutant at the multiplicity of infection of 1, and recoverable inclusion forming units (IFUs) were enumerated. Data ± standard error of the mean of 3 independent experiments. For each time-point, P-values were obtained by one-way ANOVA and Dunnett post-test analyses relative to the L2/434 parental strain; ns, not significant (P ≥ 0.05). (C) Hela cells were infected for 24 h by C. trachomatis L2/434, ct105::aadA mutant, or ct105:aadA harboring pCT105-2HA. Cells were fixed with methanol, labeled with goat anti-C. trachomatis FITC-conjugated antibody and imaged by fluorescence microscopy. Scale bars, 10 µm. (D) The inclusion area was measured (from images as those depicted in C) for 50 particles randomly chosen from independent images using Fiji software. P-values were obtained by one-way ANOVA and Dunnett post-test analyses; *statistical significant (P < 0.05).
