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

Abstract

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 MnCl 2 -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 MnCl 2 or MgCl 2 , 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. IMPORTANCEChlamydia 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. C hlamydia spp. are obligate intracellular bacterial pathogens that are widely distributed in nature and are responsible for significant diseases in humans and a variety of animals (1, 2). The human-specific pathogen Chlamydia trachomatis is the leading cause of preventable blindness and the most common reportable bacterial sexually transmitted infection both in the United States and worldwide (3, 4). C. trachomatis infections of the conjunctiva and the urogenital tract may lead to chronic diseases, including trachoma, pelvic inflammatory disease, and infertility (1). Further understanding of the physiology of these highly significant pathogens is critical for developing therapeutics and vaccines.While Chlamydia spp. differ in host range and virul...

Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

“…Similarly, the Kcat values were calculated to be 0.2089 s −1 and 0.093s −1 for REL606 and MG1655 PphC, respectively. Previously reported kinetic values for known bacterial PP2Cs range from 0.35mM to 5.7mM pNPP for Km and 0.1 to 7.4 s −1 for Kcat (22–24, 26, 27, 31, 40–43), indicating that PphC has relatively low phosphatase activity in vitro as compared to previously characterized bacterial PP2C-like phosphatases.…”

“…Multiple sequence alignment of YegK with known PP2C phosphatases from other Gram-negative bacteria shows presence of these 11 motifs but with low overall sequence homology. However, unlike other bacterial PP2C homologs (22, 23, 25, 28, 30), YegK contains only six of the eight absolutely conserved residues that are involved in metal binding, coordination and catalysis (Fig. 1A).…”

“…To further confirm the bioinformatic identification of PphC as a PP2C-like phosphatase, the aspartic acid residue (D46) in motif II was mutated to asparagine. A similar approach was used in the analysis of the PP2C-like phosphatase Cpp1 from Chlamydia trachomatis (22). The mutant protein (PphC-D46N) was purified as above and phosphatase activity was compared to wild type PphC.…”

“…The substrate specificity of PphC was further examined using commercially available phosphopeptides. Previously characterized PP2Cs have demonstrated preferential specificity to phosphoserine/threonine peptides over phosphotyrosine peptides (22, 23, 30, 40). Phosphatase assays were performed with phosphoserine RRA(pS)VA, phosphothreonine KR(pT)IRR and phosphotyrosine RRLIEDAE(pY)AARG peptide substrates (Fig.…”

The Impact of Protein Phosphorylation on Chlamydial Physiology

“…We recently characterized a protein phosphatase from C. trachomatis , Cpp1, which we hypothesize to partner with Pkn1 and PknD completing a reversible phosphoprotein network (Claywell and Fisher, 2016). All sequenced Chlamydia spp.…”