MbovP0725, a secreted serine/threonine phosphatase, inhibits the host inflammatory response and affects metabolism in Mycoplasma bovis

Abstract

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.

Importance: Mycoplasma 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.

Keywords: HAD phosphatase; MbovP0725; Mycoplasma bovis; anti-inflammatory response; transcriptomics and metabolomics.

Fig 1

MbovP0725 is a secreted phosphatase of the HAD superfamily. (A) Proteins homologous to MbovP0725 were identified by hhpred online server and alignment with sequences using CLUSTALW and ESPript. (B) Purification of MbovP0725. The protein was purified with nickel affinity chromatography and resolved with SDS-PAGE. (C) Confirmation of secreted MbovP0725 in culture supernatant. (D) Phosphatase activity of rMbovP0725 (30 µg) with p-nitrophenyl phosphate (pNPP) as substrate in the presence of 5 mM Mg²⁺ for 0, 0.25, 0.5, 1, 1.5, 2, 4, and 12 h. (E) Phosphatase activity of different concentrations of rMbovP0725. Various amounts (0, 5, 10, 20, 30, and 50 µg) of rMbovP0725 protein were added to the reaction buffer containing pNPP and 5 mM Mg²⁺. (F) Effect of different monovalent and divalent cations (K⁺, Na⁺, Ca²⁺, Mn²⁺, and Mg²⁺) on the phosphatase activity of rMbovP0725. (G) Effect of different concentrations of Mg²⁺ (0.0625, 0.125, 0.25, 0.5, 1, 5, and 10 mM) on the phosphatase activity of rMbovP0725. (H) Effect of different pHs (5, 6, 7, 8, 9, and 10) on the phosphatase activity of rMbovP0725. (I) Effect of different temperatures (4, 16, 30, 37, 42, 55, and 65°C) on the phosphatase activity of rMbovP0725. Without rMbovP0725 or pNPP was used as a negative control (NC). Values represent the mean ± SD.

Fig 2

MbovP0725 is a serine/threonine phosphatase. (A) Comparison of the substrate preferences of MbovP0725. Amounts of released inorganic phosphate were measured by the malachite green assay. The mean values of the three replicates are shown. (B) Schematic representation of the predicted key phosphorylation sites of MbovP0725. (C) Bacterial expression and purification of the site-directed mutagenesis for D15A, T48A, K210A, and D236A of MbovP0725. (D) Phosphatase activity of MbovP0725 and its mutant protein with pNPP as substrate. Values represent the mean ± SD.

Fig 3

MbovP0725 mutant strain T8.66 elicited a stronger inflammatory response of BoMac cells and less adhesion to epithelial cells compared to the wild-type strain and its complement strain CT8.66. (A) Visualization of MbovP0725 expression in HB0801, T8.66, and CT8.66 using western blotting assay. (B) Growth curves of HB0801, T8.66, and CT8.66 strains. Growth of M. bovis at each time point was determined with a CFU plating assay. (C) Microscopic view of the colony morphology of HB0801, T8.66, and CT8.66 strains. (D–F) MbovP0725 disruption could enhance the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in response to BoMac infected with different strains, assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (G, H) T8.66 decreased its adhesion ability compared to WT strain and CT8.66. The binding to EBL and MAC-T cells was determined by counting the number of CFU associated with cell monolayers following incubation with 10⁸ CFU. The data are the mean Mycoplasma titers from three independent assays. Two-way ANOVA was used to determine the statistical significance of differences between the treatments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 indicate statistically significant differences.

Fig 4

Transcriptome analysis of the DEGs in T8.66 compared with WT HB0801. (A) The volcano map of DEGs between T8.66 and HB0801. The x-axis is the log2 fold change, and the y-axis is the −log10 (P-value). The horizontal line is the threshold of P-value =0.05. Red dots indicate gene upregulation, and green dots indicate gene downregulation. Gray dots denote genes with no significant change. (B) Bubble chart of GO enrichment analysis of DEGs. P-values range from 0 to 1. The size of the dot denotes the number of DEGs. The gene ratio is the number of the DEGs to the number of annotated genes. (C) Bubble chart of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The y-axis is the different KEGG pathways, and the x-axis is the gene ratio, which represents the ratio of the number of DEGs to the total number of annotated genes in this pathway. The size of the dot correlates with the number of DEGs annotated in the pathway. (D) Validation of RNA-seq results by qRT-PCR.

Fig 5

The metabolomic analysis of MbovP0725 activity in M. bovis. (A and B) PCA score chart for all samples in pos (A) and neg (B) modes. The abscissa is PC1, and the PCA score graph shows 95% confidence intervals. Each dot represents a sample, and different groups are labeled with different colors. Six replicates were included for both HB0801 and T8.66. (C and D) Bubble plots for metabolic pathway enrichment analysis in positive (C) and negative (D) modes. The x-axis enrichment factor (RichFactor) is the number of differential metabolites annotated to the pathway divided by all identified metabolites annotated to the pathway. The larger the value, the greater the proportion of differential metabolites annotated to the pathway. The dot size represents the number of DAMs annotated to this pathway. (E) Volcano map of DAMs in both positive and negative modes. The log2 of fold change plotted against the −log10 of the P-value. The two vertical dotted lines in the figure are the two fold change threshold, and the horizontal dotted line is the P-value at a 0.05 threshold. Red and green dots represent up- and downregulated DAMs, respectively, and gray dots represent non-significant metabolites. A variable with a VIP score close to or greater than 1 was considered important in the model. VIP, variable importance in projection. (F) Bubble chart of KEGG pathways involving significantly enriched DAMs in both pos and neg modes.

Fig 6

Heatmap including 20 metabolites from MbovP0725 mutant metabolomic analysis with higher VIP scores. Samples and genotype are represented in columns. High-intensity measurements compared to average intensity are green, and low-intensity measurements are represented by white.

Fig 7

MbovP0725 inhibited pro-inflammatory cytokine production. (A and B) IL-1β, IL-6, and TNF-α mRNA levels of BoMac and MAC-T cells treated with rMbovP0725 protein at 1, 4, 10, and 20 µg/mL and LPS at 1 µg/mL were measured by qRT-PCR. Two-way ANOVA was used to determine the statistical significance of differences between the treatments. Samples without LPS or MbovP0725 treatment were used as controls. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 indicate statistically significant differences.

Fig 8

Intracellular MbovP0725 expression inhibited pro-inflammatory cytokine gene production. Recombinant eukaryotic vector pEGFP-MbovP0725 transfection decreased cytokine mRNA expression including IL-1α, IL-6, and TNF-α in (A) BoMac and (B) MAC-T cells. EGFP-vector mean cells transfected with empty vector. NC indicates PBS-treated cells. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 indicate statistically significant differences.

Fig 9

MbovP0725 suppresses pro-inflammatory response depending on its phosphatase activity. Wild-type rMbovP0725 protein was able to reduce the mRNA expression response to 1 µg/mL LPS treatment, whereas its mutant D15A constructs exhibited reduced ability to suppress the inflammatory response similar to the blank group with LPS treatment alone. NC means no LPS stimulation. Data are the means of three independent assays. Standard deviations are indicated by error bars. In the figure, P-values are indicated by asterisks: ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Fig 10

MbovP0725, but not its mutant protein D15A, attenuated the activation of eukaryotic MAPK pathways induced by LPS. (A and B) Western blot analysis of activation and phosphorylation of MAPK P38 and ERK1/2 was achieved by incubating MAC-T cells with different doses of rMbovP0725 (A) or its mutant protein D15A (B) after stimulation with LPS for 2 h. (C and D) Densitometry qualification of phosphorylated P38 and ERK1/2 was normalized by total P38 and ERK (^#P < 0.001 vs NC group; ^***P < 0.001 vs LPS group). (E and F) The gray value of phosphorylated P38 and ERK1/2 levels was conducted by normalizing to total P38 and ERK. (G) Pull-down assays showed P38 and ERK as interactors of MbovP0725. The 293T cells transfected with pCAGGS-HA-MbovP0725, its point mutant protein D15A, and HA-empty vector, respectively, following the whole cell lysates were immunoprecipitated with anti-HA tagged antibody and immunoblotted with P38 and ERK antibody. IB, immunoblotting.