Inhibition of the Protein Phosphatase CppA Alters Development of Chlamydia trachomatis.

Chlamydiae are obligate intracellular Gram-negative bacterial pathogens that undergo an essential, but poorly understood, biphasic developmental cycle transitioning between the infectious elementary body and the replicative reticulate body. Ser/Thr/Tyr phosphorylation has been increasingly recognized for its role in regulating bacterial physiology. Chlamydia spp. encode two Hanks'-type kinases in addition to a type 2C protein phosphatase (PP2C; CppA) and appears capable of global protein phosphorylation. While these findings substantiate the importance of protein phosphorylation in Chlamydia, the physiological impact of protein phosphorylation remains enigmatic. In this study, we investigated the in vivo role of CppA by using recombinant protein point mutants and small-molecule inhibitors. Recombinant CppA (rCppA) amino acid point mutants based upon missense mutations identified in growth-deficient Chlamydia trachomatis strains exhibited reduced, but not a complete loss of, phosphatase activity toward p-nitrophenyl phosphate (pNPP) and phosphopeptides. To more directly explore the importance of CppA in chlamydial development, we implemented a chemical "knockout" approach using derivatives of 5,5'-methylenedisalicylic acid (MDSA). Several MDSA derivatives significantly reduced CppA activity in vitro and the growth of C. trachomatis L2, C. trachomatis D, and Chlamydia muridarum in a cell culture infection model. The inhibition of C. trachomatis L2 growth was more pronounced when treated at earlier infection time points, and the removal of the inhibitors after 12 h postinfection did not rescue progeny production. Our findings revealed that altered CppA activity reduces chlamydial growth and that CppA function is likely crucial for early differentiation events. Collectively, our findings further support the importance of the protein phosphorylation network in chlamydial development.IMPORTANCEChlamydia is a significant cause of disease in humans, including sexually transmitted infections, the ocular infection trachoma, and pneumonia. Despite the critical roles of protein phosphatases in bacterial physiology, their function in pathogenesis is less clear. Our findings demonstrate that CppA, a broad-specificity type 2C protein phosphatase (PP2C), is critical for chlamydial development and further substantiate reversible phosphorylation as a key regulatory mechanism in Chlamydia Additionally, our work highlights the potential of CppA to serve as a novel target for future therapeutic strategies and supports the feasibility of designing more potent PP2C phosphatase inhibitors for Chlamydia and other pathogenic bacteria.

CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity.

UNLABELLED: Protein phosphorylation has become increasingly recognized for its role in regulating bacterial physiology and virulence. Chlamydia spp. encode two validated Hanks'-type Ser/Thr protein kinases, which typically function with cognate protein phosphatases and appear capable of global protein phosphorylation. Consequently, we sought to identify a Ser/Thr protein phosphatase partner for the chlamydial kinases. CTL0511 from Chlamydia trachomatis L2 434/Bu, which has homologs in all sequenced Chlamydia spp., is a predicted type 2C Ser/Thr protein phosphatase (PP2C). Recombinant maltose-binding protein (MBP)-tagged CTL0511 (rCTL0511) hydrolyzed p-nitrophenyl phosphate (pNPP), a generic phosphatase substrate, in a MnCl2-dependent manner at physiological pH. Assays using phosphopeptide substrates revealed that rCTL0511 can dephosphorylate phosphorylated serine (P-Ser), P-Thr, and P-Tyr residues using either MnCl2 or MgCl2, indicating that metal usage can alter substrate preference. Phosphatase activity was unaffected by PP1, PP2A, and PP3 phosphatase inhibitors, while mutation of conserved PP2C residues significantly inhibited activity. Finally, phosphatase activity was detected in elementary body (EB) and reticulate body (RB) lysates, supporting a role for protein dephosphorylation in chlamydial development. These findings support that CTL0511 is a metal-dependent protein phosphatase with broad substrate specificity, substantiating a reversible phosphorylation network in C. trachomatis IMPORTANCE: Chlamydia spp. are obligate intracellular bacterial pathogens responsible for a variety of diseases in humans and economically important animal species. Our work demonstrates that Chlamydia spp. produce a PP2C capable of dephosphorylating P-Thr, P-Ser, and P-Tyr and that Chlamydia trachomatis EBs and RBs possess phosphatase activity. In conjunction with the chlamydial Hanks'-type kinases Pkn1 and PknD, validation of CTL0511 fulfills the enzymatic requirements for a reversible phosphoprotein network. As protein phosphorylation regulates important cellular processes, including metabolism, differentiation, and virulence, in other bacterial pathogens, these results set the stage for elucidating the role of global protein phosphorylation in chlamydial physiology and virulence.

The Impact of Protein Phosphorylation on Chlamydial Physiology.

Chlamydia are Gram negative bacterial pathogens responsible for disease in humans and economically important domesticated animals. As obligate intracellular bacteria, they must gain entry into a host cell where they propagate within a parasitophorous organelle that serves as an interactive interface between the bacterium and the host. Nutrient acquisition, growth, and evasion of host defense mechanisms occur from this location. In addition to these cellular and bacterial dynamics, Chlamydia differentiate between two morphologically distinct forms, the elementary body and reticulate body, that are optimized for either extracellular or intracellular survival, respectively. The mechanisms regulating and mediating these diverse physiological events remain largely unknown. Reversible phosphorylation, including classical two-component signaling systems, partner switching mechanisms, and the more recently appreciated bacterial Ser/Thr/Tyr kinases and phosphatases, has gained increasing attention for its role in regulating important physiological processes in bacteria including metabolism, development, and virulence. Phosphorylation modulates these events via rapid and reversible modification of protein substrates leading to changes in enzyme activity, protein oligomerization, cell signaling, and protein localization. The characterization of several conserved chlamydial protein kinases and phosphatases along with phosphoproteome analysis suggest that Chlamydia are capable of global and growth stage-specific protein phosphorylation. This mini review will highlight the current knowledge of protein phosphorylation in Chlamydia and its potential role in chlamydial physiology and, consequently, virulence. Comparisons with other minimal genome intracellular bacterial pathogens also will be addressed with the aim of illustrating the importance of this understudied regulatory mechanism on pathogenesis and the principle questions that remain unanswered.