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



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.
Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA.
Ja E
JE
Claywell
Department of Microbiology, Southern Illinois University, Carbondale, Illinois, USA dfisher@siu.edu.
Derek J
DJ
Fisher
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002690:Chlamydia Infections / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D000071636:Protein Phosphatase 2C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D000814:Aniline Compounds; D001426:Bacterial Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; C068798:4-aminophenylphosphate; D000071636:Protein Phosphatase 2C