We have shown previously that
Chlamydophila pneumoniaecontains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. Results
in vitrokinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniaeas evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniaegrown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatisserovar D or Salmonella entericasv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. Conclusion
We have identified a 3'-pyridyl oxindole compound that inhibits the
in vitrokinase activity of C. pneumoniaePknD and inhibits the growth and production of infectious C. pneumoniaeprogeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.