Figure 5.

PI15 silencing and over-expression inhibits CPAF activation. (A) Cells transfected with control (siControl) or PI15 (siPI15) siRNAs were infected with Chlamydia and CPAF maturation was accessed by immunoblotting. Total lysates were prepared from both siPI15 as well as siControl transfected cells after 24 h of Chlamydia infection. (B) Effect of silencing of PI15 on CPAF maturation was accessed in HeLa cells with lentivirus-mediated stable knock down of PI15. Lysates of cells were harvested 24 h post infection when PI15.f cannot be detected. Cells with empty vector control were tested in parallel. kDa, molecular weight of protein markers represented as kilodaltons. (C) Progeny formation of Chlamydia is affected by suppression of PI15 expression. HeLa cells with normal PI15 expression and with shRNA-mediated knock down PI15 were infected with wild type Chlamydia (Wt Ctr) or the RST17 (CPAF-) mutant. Cells were lysed 36 h post infection and equal amounts of cell lysates were used to infect fresh cells. Chlamydial DNA was quantified 24 h post infection by qPCR and was normalized to the total cell count. Data represent ± SEM from 3 independent experiments. (D) Transient over-expression of PI15 inhibits CPAF maturation. 293T cells were transiently transfected with Flag-tagged PI15 for 48 h and were then infected with C. trachomatis for different time intervals. Total lysates were prepared and processed for immunoblotting. CK8, cytokeratin-8; −, Empty vector; +, PI15-flag. Quantities of chlamydial proteins (cHtrA full length form, cHsp60 and CPAF) were determined by normalization to actin. (E) Similar experiments were carried out in T-REx-293 cells carrying a Doxycycline-inducible gene of PI15-flag under 4TO vector backbone. Repressor protein is expressed under the 6TR vector backbone. Cells carrying 6TR alone were used as control. T-REx-293 cells were induced for 48 h with Doxycycline and then washed thoroughly with PBS before Chlamydia infection. hpi, hours post infection; −, Empty vector; +, PI15-flag. (F, G) Quantification of chlamydial growth in presence of PI15. Secondary infection assays were carried out from the above experiment, to determine effects of stable PI15 overexpression on chlamydial development. Lysed infected cells were used to infect fresh cells and the number of chlamydial inclusions was counted and presented as bar diagram (F). Chlamydial DNA was quantified 24 h post infection by qPCR and was normalized to the total cell count (G). Data represent ± SEM from 3 independent experiments.