Johnson Dustin L, Stone Chris B, Bulir David C, Coombes Brian K, Mahony James B
MG DeGroote Institute for Infectious Disease Research and the Department of Pathology and Molecular Medicine, McMaster University, Ontario, Canada.
BMC Microbiol. 2009 Oct 14;9:218. doi: 10.1186/1471-2180-9-218.
We have shown previously that Chlamydophila pneumoniae contains 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.
Using an in vitro kinase 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. pneumoniae as 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. pneumoniae grown 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 trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae.
We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.
我们之前已经表明，肺炎衣原体含有一种双特异性丝氨酸/苏氨酸蛋白激酶，该激酶可磷酸化III型分泌装置的结构成分CdsD。为了进一步研究PknD在生长和发育中的作用，我们试图鉴定一种PknD抑制剂，以确定复制是否需要PknD活性。
使用体外激酶测定法，我们筛选了80种已知的真核蛋白激酶抑制剂对PknD的活性，并鉴定出一种3'-吡啶基羟吲哚化合物，该化合物可抑制PknD的自磷酸化以及CdsD的磷酸化。PknD抑制剂显著延缓了肺炎衣原体的生长速度，电子显微镜观察显示存在非常小的包涵体，其中细菌数量减少，这证明了这一点。这些包涵体包含正常的复制形式，包括原体（EB）、中间体（IB）和网状体（RB），但缺乏持续体（PB），这表明持续性的诱导不是衣原体生长减少的原因。在这种PknD抑制剂存在下培养72或84小时的肺炎衣原体盲传未能产生包涵体，表明该化合物阻断了感染性衣原体EB产生的关键步骤。该化合物对HeLa细胞无毒，不阻断衣原体入侵所需的MEK/ERK途径的激活，也不阻断沙眼衣原体D血清型或鼠伤寒沙门氏菌的细胞内复制，这表明该化合物的抑制作用对肺炎衣原体具有特异性。
我们鉴定出一种3'-吡啶基羟吲哚化合物，该化合物可抑制肺炎衣原体PknD的体外激酶活性，并抑制HeLa细胞中感染性肺炎衣原体子代的生长和产生。总之，这些结果表明PknD可能在肺炎衣原体的发育周期中起关键作用。
