A substantial reduction in both the number and size of inclusions was seen with chlamydiae harvested from HeLa cells exposed to compound D7

(bottom panels). Similar results were obtained with undiluted chlamydial lysates and with lysates harvested at 84 hpi (data not shown). Discussion Chlamydiae are obligate intracellular pathogens that have a unique biphasic developmental cycle. We have previously shown that C. pneumoniae contains three Ser/Thr protein kinases and that one of these, PknD, is a membrane-associated Integrin inhibitor kinase that phosphorylates CdsD, a structural protein of the type III secretion system [45]. In the selleck present study we have identified a selective inhibitor of PknD and show that this compound blocks phosphorylation of CdsD in vitro, retards the intracellular growth rate and decreases Smoothened Agonist cell line the number of infectious C. pneumoniae produced following infection of HeLa cells. To elucidate the role of PknD in the chlamydial developmental cycle, we screened a small library of known eukaryotic kinase inhibitors in an attempt to identify

a PknD inhibitor. In this study we show that compound D7 is a potent inhibitor of C. pneumoniae PknD activity in vitro. PknD autophosphorylation and subsequent phosphorylation of the substrate CdsD were completely inhibited by compound D7. When added to C. pneumoniae-infected HeLa cells, the 3′ pyridyl oxindole compound retarded chlamydial replication. The restriction of the developmental cycle was not due to the induction of chlamydial persistence as seen with interferon-γ or iron deprivation [34, 38]

since PB were not detected in inclusions when viewed by electron microscopy. Compound D7 also decreased the number of infectious C. pneumoniae upon passage suggesting that the compound interferes with an essential step in C. pneumoniae development. The mechanism of chlamydial growth retardation by compound D7 is unknown but an involvement of host cell JAK3 is unlikely because the expression of JAK3 is restricted to the hematopoietic cell lineage [49–51] and HeLa cells do not express JAK3. The absence of JAK3 in Chlamydia-infected HeLa cells is supported by a recent study that failed to detect the induction or expression of the JAK3 substrate, STAT5, in C. trachomatis-infected HeLa cells [52]. In addition, other potent JAK3 inhibitors (compounds D4, D5 and D6) did not selleck kinase inhibitor interfere with C. pneumoniae growth in HeLa cells. Therefore the mechanism of C. pneumoniae growth retardation in HeLa cells is unlikely due to an effect of compound D7 on JAK3 activity. Our data also rule out an effect of compound D7 on the MEK/ERK signaling pathway required for chlamydial infection and intracellular growth. Activation of the MEK/ERK pathway has been shown to be essential for chlamydial invasion of HeLa cells [43], and sustained activation of Raf-MEK-ERK-cPLA2 is also required for acquisition of glycerophospholipids and growth by C. pneumoniae [48].