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

Area of Science:

• Bacteriology
• Molecular Biology
• Biochemistry

Background:

• Protein phosphorylation regulates bacterial physiology and virulence.
• Chlamydia spp. possess Ser/Thr protein kinases, suggesting a need for phosphatases.
• CTL0511 is a predicted type 2C Ser/Thr protein phosphatase (PP2C) in Chlamydia trachomatis with homologs across Chlamydia species.

Purpose of the Study:

• Identify a Ser/Thr protein phosphatase partner for chlamydial kinases.
• Characterize the enzymatic activity and substrate specificity of CTL0511.
• Investigate the role of protein dephosphorylation in Chlamydia development.

Main Methods:

• Recombinant maltose-binding protein (MBP)-tagged CTL0511 (rCTL0511) was expressed and purified.
• Enzymatic assays were performed using p-nitrophenyl phosphate (pNPP) and phosphopeptide substrates.
• The effect of metal ions (MnCl2, MgCl2) and phosphatase inhibitors (PP1, PP2A, PP3) was assessed.
• Site-directed mutagenesis was used to investigate the role of conserved PP2C residues.
• Phosphatase activity was measured in elementary body (EB) and reticulate body (RB) lysates.

Main Results:

• Recombinant CTL0511 hydrolyzed pNPP in a MnCl2-dependent manner.
• rCTL0511 dephosphorylated P-Ser, P-Thr, and P-Tyr residues, with metal ion usage altering substrate preference.
• Phosphatase activity was independent of PP1, PP2A, and PP3 inhibitors.
• Mutation of conserved PP2C residues significantly reduced phosphatase activity.
• Phosphatase activity was detected in both EB and RB lysates of Chlamydia trachomatis.

Conclusions:

• CTL0511 is a metal-dependent protein phosphatase with broad substrate specificity.
• The findings validate CTL0511 as a functional PP2C in Chlamydia trachomatis.
• These results establish the enzymatic basis for a reversible phosphoprotein network in Chlamydia, impacting physiology and virulence.