Mycoplasma bovis is a global concern for the cattle industry owing to its high rates of infection and resulting morbidity, but its pathogenesis remains poorly understood. Metabolic pathways and characteristics of M. bovis clinical strain were elucidated by comparing the differential expression of metabolites between M. bovis clinical strain NX114 and M. bovis international reference strain PG45. Metabolites of M. bovis in the logarithmic stage were analyzed based on the non-targeted metabolomic technology of ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). We found 596 metabolites with variable expression, of which, 190 had substantial differences. Differential metabolite analysis of M. bovis NX114 showed organic acids and their derivatives, nucleosides, and nucleotide analogs as important components. We found O-Phospho-L-serine (SEP) as a potential signature metabolite and indicator of pathogenicity. The difference in nucleic acid metabolites reflects the difference in growth phenotypes between both strains of M. bovis. According to KEGG enrichment analysis, the ABC transporter synthesis route had the most differential metabolites of the first 15 differential enrichment pathways. This study reflects the species-specific differences between two strains of M. bovis and further enriches our understanding of its metabolism, paving the way for further research into its pathogenesis.

Mycoplasma synoviae infection rates in chickens are increasing worldwide. Genomic studies have considerably improved our understanding of M. synoviae biology and virulence. However, approximately 20% of the predicted proteins have unknown functions. In particular, the M. synoviae ATCC 25204 genome has 663 encoding DNA sequences, among which 155 are considered encoding hypothetical proteins (HPs). Several of these genes may encode unknown virulence factors. This study aims to reannotate all 155 proteins in M. synoviae ATCC 25204 to predict new potential virulence factors using currently available databases and bioinformatics tools. Finally, 125 proteins were reannotated, including enzymes (39%), lipoproteins (10%), DNA-binding proteins (6%), phase-variable hemagglutinin (19%), and other protein types (26%). Among 155 proteins, 28 proteins associated with virulence were detected, five of which were reannotated. Furthermore, HP expression was compared before and after the M. synoviae infection of cells to identify potential virulence-related proteins. The expression of 14 HP genes was upregulated, including that of five virulence-related genes. Our study improved the functional annotation of M. synoviae ATCC 25204 from 76% to 95% and enabled the discovery of potential virulence factors in the genome. Moreover, 14 proteins that may be involved in M. synoviae infection were identified, providing candidate proteins and facilitating the exploration of the infection mechanism of M. synoviae.

Background: Mycoplasma synoviae infection in chickens is associated with development of synovitis and arthritis, leading to a decline in poultry production and significant economic repercussions. However, the mechanism of this infection remains imperfectly known. Results: Prokaryotic transcriptome sequencing was used to identify differentially expressed genes (DEGs) in M. synoviae when exposed to mixed cultures of chicken macrophages and chicken embryo fibroblast cells, as well as M. synoviae cultured in vitro . Of 416 DEGs, 262 are upregulated and 122 are downregulated. Ten DEGs were randomly selected and subjected to verification through quantitative reverse transcriptase PCR. The upregulated DEGs exhibited significant enrichment in various Gene Ontology (GO) terms, notably transmembrane transport, transporter activity, and membrane protein complex. Additionally, these DEGs were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including ABC transporters, quorum sensing, aminoacyl-tRNA biosynthesis, DNA replication, and the bacterial secretion system. The 5’ untranslated region, transcription starting point and noncoding small RNA (sRNA) were also revealed, thirty-one upregulated genes were predicted to interact with sRNA NZ_CP011096_predRNA9. Conclusion: These findings revealed the expression of M. synoviae transcripts when exposed, which beneficial for the treatment of M. synoviae’s target proteins study, will also help to enhance understanding of mechanisms underlying M. synoviae infection.

Mycoplasma synoviae (M. synoviae) is an avian pathogen responsible for respiratory disease and synovitis. The VlhA (variable lipoprotein hemagglutinin) family of surface adhesins plays a critical role in host cell attachment, yet the specific residues and structural determinants governing this interaction remain incompletely understood. In this study, we selected the C-terminal lipid-associated membrane hemagglutinin (LAM HA) domain within the VlhA family as the bait protein, based on its conserved C-terminal region. Yeast two-hybrid screening identified 18 LAM HA-interacting host proteins, and molecular docking pinpointed five residues (S83, R85, Y88, N124, K192) as the most frequent interaction hotspots. To assess their collective functional relevance, we constructed a combined deletion mutant lacking these five candidate residues. At the optimal pH of 6.0–6.5, wild-type LAM HA showed the highest titer (1:2), significantly exceeding that at pH 7.0–7.5 (no activity) and pH 5.0–5.5 (1:1) (p < 0.05), whereas the deletion mutant displayed a reduced titer (from 2 to 1; p = 0.05). Secondary structure analysis at the same pH revealed decreased α-helix content (from 7.90% to 7.57%), along with increased β-sheet (from 38.92% to 38.83%) and random coil (from 10.83% to 10.44%). Molecular dynamics simulations revealed that the deletion mutant exhibited elevated RMSF (from 2.83 ± 1.88 Å to 4.18 ± 2.65 Å) and Rg (from 40.48 ± 1.31 Å to 54.16 ± 4.47 Å) at pH 6.0–6.5, while RMSD showed a slight decrease (11.17 ± 1.33 Å vs. 12.14 ± 0.21 Å), collectively indicating compromised structural stability of the mutant. Collectively, these findings suggest that the LAM HA domain contributes to hemagglutination activity through pH-sensitive conformational stability and specific residue clusters—a property that may be relevant to M. synoviae colonization in the acidic microenvironments of the respiratory tract and synovial fluid during infection.

Mycoplasma synoviae is an avian pathogen that causes respiratory disease and synovitis, and its hemagglutinin plays a critical role in host cell adhesion. However, the key residues and structural mechanisms underlying hemagglutination remain unclear. In this study, domain analysis of the hemagglutinin family of Mycoplasma synoviae revealed that it contains long-chain and short-chain types, among which LAM HA (VY93_RS01465) was selected as the bait protein due to its complete C-terminal conserved domain. Through yeast two-hybrid screening, 18 host proteins interacting with LAM HA were identified. Furthermore, five key amino acid residues S83, R85, Y88, N124, and K192 were found to mediate hemagglutination activity. Deletion of these residues reduced the hemagglutination titer of LAM HA under acidic conditions. Secondary structure analysis showed that the deletion mutation decreased the α-helix content while increasing the proportions of β-sheet and random coil. Molecular dynamics simulations revealed that the mutant exhibited generally higher root mean square deviation and root mean square fluctuation values than the wild-type under different pH conditions, with a marked decrease in structural stability particularly at pH 5.0 and 6.0. These findings indicate that LAM HA, as a critical adhesin, exerts its hemagglutination function dependent on specific key residues and pH-sensitive conformational stability. IMPORTANCE: Mycoplasma synoviae ( M. synoviae ) causes significant economic losses to the poultry industry worldwide. Lipid-related membrane protein hemagglutinin (LAM HA) is a surface adhesin essential for host cell attachment, but its precise amino acid residues and structural features have not been defined. In this study, five key residues (S83, R85, Y88, N124, and K192) were identified as critical for LAM HA-mediated hemagglutination activity. Deletion of these residues altered the secondary structure composition, reduced conformational stability under acidic pH conditions, and decreased hemagglutination activity. These findings reveal a previously unknown structure-function relationship of M. synoviae LAM HA, demonstrating that its hemagglutination activity depends on specific residues and pH-sensitive structural integrity. This provides new insights into the molecular mechanisms of M. synoviae adhesion and offers potential targets for the development of novel intervention strategies against avian mycoplasmosis.