A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication
ABSTRACT 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.
Full-textDOI: · Available from: James B Mahony, Jun 30, 2015
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ABSTRACT: Present-day members of the Chlamydiaceae contain parasitic bacteria that have been co-evolving with their eukaryotic hosts over hundreds of millions of years. Likewise, a type III secretion system encoded within all genomes has been refined to complement the unique obligate intracellular niche colonized so successfully by Chlamydia spp. All this adaptation has occurred in the apparent absence of the horizontal gene transfer responsible for creating the wide range of diversity in other Gram-negative, type III-expressing bacteria. The result is a system that is, in many ways, uniquely chlamydial. A critical mass of information has been amassed that sheds significant light on how the chlamydial secretion system functions and contributes to an obligate intracellular lifestyle. Although the overall mechanism is certainly similar to homologous systems, an image has emerged where the chlamydial secretion system is essential for both survival and virulence. Numerous apparent differences, some subtle and some profound, differentiate chlamydial type III secretion from others. Herein, we provide a comprehensive review of the current state of knowledge regarding the Chlamydia type III secretion mechanism. We focus on the aspects that are distinctly chlamydial and comment on how this important system influences chlamydial pathogenesis. Gaining a grasp on this fascinating system has been challenging in the absence of a tractable genetic system. However, the surface of this tough nut has been scored and the future promises to be fruitful and revealing.Frontiers in Microbiology 01/2010; 1:114. DOI:10.3389/fmicb.2010.00114 · 3.94 Impact Factor
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ABSTRACT: Chlamydiae are obligate intracellular pathogens that likely require type III secretion (T3S) to invade cells and replicate intracellularly within a cytoplasmic vacuole called an inclusion body. Chlamydia pneumoniae possess a YscL ortholog, CdsL, that has been shown to interact with the T3S ATPase (CdsN). In this report we demonstrate that CdsL down-regulates CdsN enzymatic activity in a dose-dependent manner. Using Pepscan epitope mapping we identified two separate binding domains to which CdsL binds viz. CdsN(221-229) and CdsN(265-270). We confirmed the binding domains using a pull-down assay and showed that GST-CdsN(221-270), which encompasses these peptides, co-purified with His-CdsL. Next, we used orthology modeling based on the crystal structure of a T3S ATPase ortholog from Escherichia coli, EscN, to map the binding domains on the predicted 3D structure of CdsN. The CdsL binding domains mapped to the catalytic domain of the ATPase, one in the central channel of the ATPase hexamer and one on the outer face. Since peptide mimetics have been used to disrupt essential protein interactions of the chlamydial T3S system and inhibit T3S-mediated invasion of HeLa cells, we hypothesized that if CdsL-CdsN binding is essential for regulating T3S then a CdsN peptide mimetic could be used to potentially block T3S and chlamydial invasion. Treatment of elementary body with a CdsN peptide mimetic inhibited C. pneumoniae invasion into HeLa cells in a dose-dependent fashion. This report represents the first use of Pepscan technology to identify binding domains for specific T3S proteins viz. CdsL on the ATPase, CdsN, and demonstrates that peptide mimetics can be used as anti-virulence factors to block bacterial invasion.Frontiers in Microbiology 02/2011; 2:21. DOI:10.3389/fmicb.2011.00021 · 3.94 Impact Factor
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ABSTRACT: Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.Molecular Biology and Evolution 06/2011; 28(12):3253-70. DOI:10.1093/molbev/msr161 · 14.31 Impact Factor