Mycoplasma bovis (M. bovis) is a significant bovine pathogen associated with various diseases, including bovine bronchopneumonia and mastitis resulting in substantial economic losses within the livestock industry. However, the development of effective control measures for M. bovis is hindered by a limited understanding of its virulence factors and pathogenesis. Nucleomodulins are newly identified secreted proteins of bacteria that internalize the host nuclei to regulate host cell gene expression and serve as critical virulence factors. Although recent reports have initiated exploration of mycoplasma nucleomodulins, the efficiency of conventional techniques for identification is very limited. Therefore, this study aimed to establish high-throughput methods to identify novel nucleomodulins of M. bovis. Using a direct biotinylation (DB) approach, a total of 289 proteins were identified including 66 high abundant proteins. In parallel, the use of proximity-based biotinylation (PBB), identified 28 proteins. Finally, seven nucleomodulins were verified to be nuclear by transfecting the bovine macrophage cell line BoMac with the plasmids encoding EGFP-fused proteins and observed with Opera Phenix, including the known nucleomodulin MbovP475 and six novel nucleomodulins. The novel nucleomodulins were four ribosomal proteins (MbovP599, MbovP678, MbovP710, and MbovP712), one transposase (MbovP790), and one conserved hypothetical protein (MbovP513). Among them, one unique nucleomodulin MbovP475 was identified with DB, two unique nucleomodulins (MbovP513 and MbovP710) with PBB, and four nucleomodulins by both. Overall, these findings established a foundation for further research on M. bovis nucleomodulin-host interactions for identification of new virulence factors.

The most important pathogenic Mycoplasma species in bovines are Mycoplasma bovis (M. bovis) and Mycoplasma mycoides subsp. mycoides (Mmm). Mmm causes contagious bovine pleuropneumonia (CBPP), which is a severe respiratory disease widespread in sub-Saharan Africa but eradicated in several countries, including China. M. bovis is an important cause of the bovine respiratory disease complex (BRD), characterized worldwide by pneumonia, arthritis, and mastitis. Secreted proteins of bacteria are generally considered virulence factors because they can act as toxins, adhesins, and virulent enzymes in infection. Therefore, this study performed a comparative proteomic analysis of the secreted proteins of M. bovis and Mmm in order to find some virulence-related factors as well as discover differential diagnostic biomarkers for these bovine mycoplasmas. The secretome was extracted from both species, and liquid chromatography-tandem mass spectrometry was used, which revealed 55 unique secreted proteins of M. bovis, 44 unique secreted proteins of Mmm, and 4 homologous proteins. In the M. bovis secretome, 19 proteins were predicted to be virulence factors, while 4 putative virulence factors were identified in the Mmm secretome. In addition, five unique secreted proteins of Mmm were expressed and purified, and their antigenicity was confirmed by Western blotting assay and indirect ELISA. Among them, Ts1133 and Ts0085 were verified as potential candidates for distinguishing Mmm infection from M. bovis infection.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1β, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Mycoplasma bovis can lead to a decline in milk quality and yield, thereby causing significant economic losses worldwide. Extracellular vesicles (EVs) are crucial for triggering immune cell responses to infection. This study aimed to demonstrate the immunomodulatory effects of EVs released by bovine mammary epithelial cells (MAC-T cells) infected with M. bovis on bovine macrophages (BoMacs). After EVs were extracted from M. bovis-infected MAC-T cells (M. bovis NX2-EVs) as well as from uninfected MAC-T cells (Ctrl-EVs), they were incubated with BoMacs to assess their potential to induce cytokine expression. The results showed that M. bovis NX2-EV-treated BoMacs exhibited significantly increased expression of TNF-α, IL-1β, and IL-6. Additionally, the differentially expressed genes mainly involved the TNF, NF-kappa B and IL-17 signalling pathways, with endocytosis and megalocytosis recognized as the main pathways through which BoMacs can take up EVs. Furthermore, mass spectrometry and RNA-seq were used to determine the protein and miRNA expression profiles of Ctrl-EVs and M. bovis NX2-EVs. Overall, 27 And 86 proteins were significantly downregulated and upregulated, respectively, in M. bovis NX2-EVs compared with those in Ctrl-EVs. Similarly, a total of 9 miRNAs were upregulated, while 2 miRNAs were downregulated in M. bovis NX2-EVs. Finally, JCHAIN, MAPRE1, miR-1307, and miR-149-5p were identified as differentially expressed proteins and miRNAs in M. bovis NX2-EVs, thus highlighting their involvement in cellular immune regulation and related diseases. These results reveal the mechanism of host resistance to M. bovis infection and provide new insights for exploring the pathogenic mechanism of M. bovis.

Mycoplasma bovis (M. bovis) is characterized by a reduced genomic size and limited synthetic capacity, including the inability to synthesize nucleotides de novo, relies on nucleases for nutrient acquisition and survival. A number of nucleases have been implicated in M. bovis pathogenicity, facilitating substrate degradation and contributing to DNA repair mechanisms that enhance bacterial persistence. The present study confirmed that the T5.808 mutant, in which a novel nuclease gene (Mbov_0701) was disrupted by the mini-Tn4001 transposon, exhibits a growth defect when co-cultured with EBL cells. However, the restoration of Mbov_0701 resulted in the resumption of growth in the mutant. The characterization of MbovP701 revealed that it had high activity in hydrolyzing dsDNA with 5′- to 3′- polarity. Furthermore, the substrates of MbovP701 were extended to include linear dsDNA, ssDNA, RNA, and plasmid DNA. The exonuclease activity is dependent on the presence of Mn²⁺ and/or Mg²⁺ ions, with an optimal pH and temperature of 8.3 and 43 °C, respectively. The truncation experiments of rMbovP701 revealed that YqaJ (41–185 aa) is the key functional domain of MbovP701 exonuclease. In conclusion, the present study identified a novel nuclease in M. bovis that plays an essential role in the proliferation of this minimal organism. This finding elucidates the survival strategy and pathogenesis of M. bovis, suggesting a potential therapeutic strategy for the treatment of M. bovis through targeting the inhibition of MbovP701. Moreover, it provides a foundation for future investigations into the interactions between MbovP701 and other nucleases involved in M. bovis biology.