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A serine/threonine phosphatase encoded by MG_207 of Mycoplasma genitalium is critical for its virulence.

ABSTRACT: Bacterial signal transduction systems like two component system (TCS) and Serine/Threonine kinase (STK) and Serine/Threonine phosphatase (STP) play important roles in the virulence and pathogenesis of bacterial pathogens. Mycoplasma genitalium, a mollicute that causes the urogenital diseases urethritis and cervicitis in men and women, respectively, is a pathogen which lacks TCS but possesses STK/STP. In this study, we investigated the biochemical and virulence properties of an STP protein encoded by the gene MG_207 of this species.We overexpressed MG207 in Escherichia coli overexpression system as a recombinant His10MG207 protein and purified it with affinity chromatography. This recombinant protein readily hydrolyzed the substrate p-nitrophenyl phosphate (pNPP) in a dose-dependent manner. Additional studies using synthetic peptides as substrates revealed that the recombinant protein was able to hydrolyze the threonine phosphate. Further, a transposon insertion mutant strain of M. genitalium (TIM207) that lacks the protein MG207 showed differentially phosphorylated proteins when compared to the wild type G37 strain. Mass spectrometry revealed that some of the key proteins differentially phosphorylated in TIM207 strain were putative cytoskeletal protein encoded by the gene MG_328 and pyruvate dehydrogenase E1 ? chain encoded by the gene MG_274. In addition, TIM207 was noticed to be less cytotoxic to HeLa cells and this correlated with the production of less hydrogen peroxide by this strain. This strain was also less efficient in inducing the differentiation of THP-1 cell line as compared to wild type M. genitalium.The results of the study suggest that MG207 is an important signaling protein of M. genitalium and its presence may be crucial for the virulence of this species.

SUBMITTER: Martinez MA

PROVIDER: S-EPMC3639085 | biostudies-other | 2013 Feb

REPOSITORIES: biostudies-other

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Project description:Mycoplasma species are able to produce and release secreted proteins, such as toxins, adhesins, and virulence-related enzymes, involved in bacteria adhesion, invasion, and immune evasion between the pathogen and host. Here, we investigated a novel secreted protein, MbovP0725, from Mycoplasma bovis encoding a putative haloacid dehalogenase (HAD) hydrolase function of a key serine/threonine phosphatase depending on Mg2+ for the dephosphorylation of its substrate pNPP, and it was most active at pH 8 to 9 and temperatures around 40°C. A transposon insertion mutant strain of M. bovis HB0801 that lacked the protein MbovP0725 induced a stronger inflammatory response but with a partial reduction of adhesion ability. Using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction analysis, we found that the mutant was upregulated by the mRNA expression of genes from the glycolysis pathway, while downregulated by the genes enriched in ABC transporters and acetate kinase-phosphate acetyltransferase pathway. Untargeted metabolomics showed that the disruption of the Mbov_0725 gene caused the accumulation of 9-hydroxyoctadecadienoic acids and the consumption of cytidine 5'-monophosphate, uridine monophosphate, and adenosine monophosphate. Both the exogenous and endogenous MbvoP0725 protein created by purification and transfection inhibited lipopolysaccharide (LPS)-induced IL-1β, IL-6, and TNF-α mRNA production and could also attenuate the activation of MAPK-associated pathways after LPS treatment. A pull-down assay identified MAPK p38 and ERK as potential substrates for MbovP0725. These findings define metabolism- and virulence-related roles for a HAD family phosphatase and reveal its ability to inhibit the host pro-inflammatory response.ImportanceMycoplasma bovis (M. bovis) infection is characterized by chronic pneumonia, otitis, arthritis, and mastitis, among others, and tends to involve the suppression of the immune response via multiple strategies to avoid host cell immune clearance. This study found that MbovP0725, a haloacid dehalogenase (HAD) family phosphatase secreted by M. bovis, had the ability to inhibit the host pro-inflammatory response induced by lipopolysaccharide. Transcriptomic and metabolomic analyses were used to identify MbovP0725 as an important phosphatase involved in glycolysis and nucleotide metabolism. The M. bovis transposon mutant strain T8.66 lacking MbovP0725 induced a higher inflammatory response and exhibited weaker adhesion to host cells. Additionally, T8.66 attenuated the phosphorylation of MAPK P38 and ERK and interacted with the two targets. These results suggested that MbovP0725 had the virulence- and metabolism-related role of a HAD family phosphatase, performing an anti-inflammatory response during M. bovis infection.

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A serine/threonine phosphatase encoded by MG_207 of Mycoplasma genitalium is critical for its virulence.

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