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The sRNA Regulated Protein DdbA Is Involved in Development and Maintenance of the Chlamydia trachomatis EB Cell Form.

ABSTRACT: The chlamydial small non coding RNA, IhtA, regulates the expression of both HctA and DdbA, the uncharacterized product of the C. trachomatis L2 CTL0322 gene. HctA is a small, highly basic, DNA binding protein that is expressed late in development and mediates the condensation of the genome during RB to EB differentiation. DdbA is conserved throughout the chlamydial lineage, and is predicted to express a small, basic, cytoplasmic protein. As it is common for sRNAs to regulate multiple mRNAs within the same physiological pathway, we hypothesize that DdbA, like HctA, is involved in RB to EB differentiation. Here, we show that DdbA is a DNA binding protein, however unlike HctA, DdbA does not contribute to genome condensation but instead likely has nuclease activity. Using a DdbA temperature sensitive mutant, we show that DdbAts creates inclusions indistinguishable from WT L2 in size and that early RB replication is likewise similar at the nonpermissive temperature. However, the number of DdbAts infectious progeny is dramatically lower than WT L2 overall, although production of EBs is initiated at a similar time. The expression of a late gene reporter construct followed live at 40°C indicates that late gene expression is severely compromised in the DdbAts strain. Viability assays, both in host cells and in axenic media indicate that the DdbAts strain is defective in the maintenance of EB infectivity. Additionally, using Whole Genome Sequencing we demonstrate that chromosome condensation is temporally separated from DNA replication during the RB to EB transition. Although DdbA does not appear to be directly involved in this process, our data suggest it is a DNA binding protein that is important in the production and maintenance of infectivity of the EB, perhaps by contributing to the remodeling of the EB chromosome.

SUBMITTER: Grieshaber NA

PROVIDER: S-EPMC8343073 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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The sRNA Regulated Protein DdbA Is Involved in Development and Maintenance of the Chlamydia trachomatis EB Cell Form.

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