Alternatively, PknD may be involved in a signaling pathway indirectly
related to replication and that when inhibited only slows the rate of replication. It is also possible that PknD is an essential enzyme required for replication, but is only partially inhibited in cell culture by the concentration of compound D7 used in our growth experiments. Indeed, it is known that chlamydial isolates can be heterogeneous in nature and therefore a subpopulation of Chlamydia may have been partially resistant to the this website effects of compound D7. Nonetheless, C. pneumoniae grown in the presence of compound D7 and subsequently passaged onto fresh HeLa cell monolayers failed to propagate and develop inclusions suggesting PknD may also be involved in the production of infectious bacteria. Inhibition of PknD could manifest as multiple biological effects if there is more than one PknD substrate, or if the affected biological events are linked. SB202190 More work is needed to elucidate the role of PknD and the exact
mechanism by which compound D7 inhibits the growth and development of C. pneumoniae. These experiments, however, will be difficult to conduct in the absence of a genetic transformation system for chlamydiae. Conclusion We have identified a novel inhibitor of C. pneumoniae growth and development, and its biological effects may be mediated via inhibition of PknD. It is tempting to speculate that PknD plays an essential role in the developmental cycle of C. pneumoniae, which may include mafosfamide a role in replication and/or in the production of infectious progeny, but this hypothesis
cannot be directly tested in the absence of a PknD knockout. The approach of using novel chemicals in cell culture to inhibit other Ser/Thr protein kinases of chlamydiae viz. Pkn1 or Pkn5 may prove fruitful in elucidating their roles in chlamydial development. Methods Reagents and Cell Lines Minimal essential medium (MEM) (Invitrogen, Burlington) containing Earle’s salts and L-glutamine was supplemented with 10% fetal bovine serum. The Calbiochem InhibitorSelect Protein Kinase Inhibitor Library I containing 80 receptor tyrosine kinase inhibitors and atypical kinase inhibitors was from EMD (San Diego). MP Biomedicals (Santa Ana) supplied radiolabelled ATP ([γ-32P]-ATP) for the in vitro kinase assays. HeLa 229 cells were obtained from ATCC (Manassas). CHIR98014 Chlamydophila pneumoniae CWL029 and Chlamydia trachomatis serovar D were obtained from ATCC (cat. #VR1310 and #VR885, respectively). E. coli Rosetta pLysS and BL21(DE3) pLysS were from Novagen (EMD). Epidermal growth factor (EGF) and the MEK inhibitor U0126 were from Sigma (Oakville). U0126 was resuspended in DMSO immediately prior to addition to cell culture in the MEK/ERK activation experiment. Protein Expression and Purification GST-PknD KD and His-FHA-2 were prepared as described [45]. Key parameters for preparing active kinase domain included cooling the E. coli cultures to 20°C prior to induction, inducing with 0.
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