The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

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-1ß, 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.

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

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-1ß, 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.

The distribution characteristics of aerosol bacteria in different types of sheepfolds.

With the development of modern sheep raising technology, the increasing density of animals in sheep house leads to the accumulation of microbial aerosols in sheep house. It is an important prerequisite to grasp the characteristics of bacteria in aerosols in sheep house to solve the problems of air pollution and disease prevention and control in sheep house. In this study, the microorganisms present in the air of sheep houses were investigated to gain insights into the structure of bacterial communities and the prevalence of pathogenic bacteria. Samples from six sheep pens in each of three sheep farms, totaling 18, were collected in August 2022 from Ningxia province, China. A high-volume air sampler was utilized for aerosol collection within the sheep housing followed by DNA extraction for 16S rRNA sequencing. Employing high-throughput 16S rRNA sequencing technology, we conducted an in-depth analysis of microbial populations in various sheep pen air samples, enabling us to assess the community composition and diversity. The results revealed a total of 11,207 operational taxonomic units (OTUs) within the bacterial population across the air samples, encompassing 152 phyla, 298 classes, 517 orders, 853 families, 910 genera, and 482 species. Alpha diversity and beta diversity analysis indicated that differences in species diversity, evenness and coverage between different samples. At the bacterial phylum level, the dominant bacterial groups are Firmicutes, Proteobacteria, and Actinobacteria, among which Firmicutes (97.90-98.43%) is the highest. At the bacterial genus level, bacillus, Bacteroides, Fusobacterium, etc. had higher abundance, with Bacillus (85.47-89.87%) being the highest. Through an in-depth analysis of microbial diversity and a meticulous examination of pathogenic bacteria with high abundance in diverse sheep house air samples, the study provided valuable insights into the microbial diversity, abundance, and distinctive features of prevalent pathogenic bacteria in sheep house air. These findings serve as a foundation for guiding effective disease prevention and control strategies within sheep farming environments.

Exploring Mycoplasma ovipneumoniae NXNK2203 infection in sheep: insights from histopathology and whole genome sequencing.

BACKGROUND: Mycoplasma ovipneumoniae (M. ovipneumoniae) is a significant pathogen causing respiratory infections in goats and sheep. This study focuses on investigating vulnerability of Hu sheep to M. ovipneumoniae infection in the context of late spring's cold weather conditions through detailed autopsy of a severely affected Hu sheep and whole genome sequencing of M. ovipneumoniae. RESULTS: The autopsy findings of the deceased sheep revealed severe pulmonary damage with concentrated tracheal and lung lesions. Histopathological analysis showed tissue degeneration, mucus accumulation, alveolar septum thickening, and cellular necrosis. Immunohistochemistry analysis indicated that M. ovipneumoniae was more in the bronchi compared to the trachea. Genome analysis of M. ovipneumoniae identified a 1,014,835 bp with 686 coding sequences, 3 rRNAs, 30 tRNAs, 6 CRISPRs, 11 genomic islands, 4 prophages, 73 virulence factors, and 20 secreted proteins. CONCLUSION: This study investigates the vulnerability of Hu sheep to M. ovipneumoniae infection during late spring's cold weather conditions. Autopsy findings showed severe pulmonary injury in affected sheep, and whole genome sequencing identified genetic elements associated with pathogenicity and virulence factors of M. ovipneumoniae.

Comparative Proteomic Analysis of Secretory Proteins of Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides Investigates Virulence and Discovers Important Diagnostic Biomarkers.

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.

Whole genome sequence of Mycoplasma ovipneumoniae strain NXNK2203 isolated from Hu sheep in Ningxia province, China.

We report whole-genome sequencing of Mycoplasma ovipneumoniae strain NXNK2203 isolated from the lungs of dead Hu sheep in Ningxia province, China. We get single circular chromosomes of 1,014,835 bp after sequencing. The purpose was to better understand the clinical significance of Mycoplasma infections for the health and welfare of ovine species.

Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation.

Mycoplasmas are successful pathogens that cause debilitating diseases in humans and various animal hosts. Despite the exceptionally streamlined genomes, mycoplasmas have evolved specific mechanisms to access essential nutrients from host cells. The paucity of genetic tools to manipulate mycoplasma genomes has impeded studies of the virulence factors of pathogenic species and mechanisms to access nutrients. This review summarizes several strategies for editing of mycoplasma genomes, including homologous recombination, transposons, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and synthetic biology. In addition, the mechanisms and features of different tools are discussed to provide references and future directions for efficient manipulation of mycoplasma genomes.

Novel mycoplasma nucleomodulin MbovP475 decreased cell viability by regulating expression of CRYAB and MCF2L2.

Nucleomodulins are secreted bacterial proteins whose molecular targets are located in host cell nuclei. They are gaining attention as critical virulence factors that either modify the epigenetics of host cells or directly regulate host gene expression. Mycoplasma bovis is a major veterinary pathogen that secretes several potential virulence factors. The aim of this study was to determine whether any of their secreted proteins might function as nucleomodulins. After an initial in silico screening, the nuclear localization of the secreted putative lipoprotein MbovP475 of M. bovis was demonstrated in bovine macrophage cell line (BoMac) experimentally infected with M. bovis. Through combined application of ChIP-seq, Electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis, MbovP475 was determined to bind the promoter regions of the cell cycle central regulatory genes CRYAB and MCF2L2. MbovP475 has similar secondary structures with the transcription activator-like effectors (TALEs). Screening of various mutants affecting the potential DNA binding sites indicated that the residues 242NI243 within MbovP475 loop region of the helix-loop-helix domain were essential to its DNA binding activity, thereby contributing to decrease in BoMac cell viability. In conclusion, this is the first report to confirm M. bovis secretes a conserved TALE-like nucleomodulin that binds the promoters of CRYAB and MCF2L2 genes, and subsequently down-regulates their expression and decreases BoMac cell viability. Therefore, this study offers a new understanding of mycoplasma pathogenesis.

Annexin A2 regulates Mycoplasma bovis adhesion and invasion to embryo bovine lung cells affecting molecular expression essential to inflammatory response.

Mycoplasma bovis (M. bovis) is an important pathogen of the bovine respiratory disease complex, invading lower respiratory tracts and causing severe pneumonia. However, its molecular mechanism largely remains unknown. Host annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein. The current study sought to determine whether ANXA2 could mediate M. bovis adhesion and invasion thereby affecting its induction of inflammatory response. ANXA2 expression was upregulated in M. bovis-infected bovine lung epithelial cells (EBL), and blocking ANXA2 with an anti-ANXA2 antibody reduced M. bovis adhesion to EBL. Compared with uninfected cells, more ANXA2 was translocated from the cytoplasm to the cell surface after M. bovis infection. Furthermore, RNA interference knockdown of ANXA2 expression in EBL cells resulted in a significant decrease in M. bovis invasion and F-actin polymerization. Next, the transcriptomic study of M. bovis-infected EBL cells with and without ANXA2 knockdown were performed. The data exhibited that ANXA2 knockdown EBL cells had 2487 differentially expressed genes (DEGs), with 1175 upregulated and 1312 downregulated compared to control. According to GO and KEGG analyses, 50 genes potentially linked to inflammatory responses, 23 involved in extracellular matrix (ECM) receptor interaction, and 48 associated with PI3K-AKT signal pathways were upregulated, while 38 mRNA binding genes, 16 mRNA 3'-UTR binding genes, and 34 RNA transport genes were downregulated. Furthermore, 19 genes with various change-folds were selected for qPCR verification, and the results agreed with the RNA-seq findings. Above all, the transcription of two chemokines (IL-8 and CXCL5) and a key bovine ß-defensin TAP in IL-17 signaling pathway were significantly increased in ANXA2 knockdown cells. Moreover, ANXA2 knockdown or knockout could increase NF-κB and MAPK phosphorylation activity in response to M. bovis infection. Additionally, ANXA2 knockdown also significantly decreased the CD44 transcripts via exon V3 and V7 skipping after M. bovis infection. We concluded that M. bovis borrowed host ANXA2 to mediate its adhesion and invasion thereby negatively regulating molecular expression essential to IL-17 signal pathway. Furthermore, CD44 V3 and V7 isoforms might contribute to this ANXA2 meditated processes in M. bovis infected EBL cells. These findings revealed a new understanding of pathogenesis for M. bovis infection.

The attenuated Mycoplasma bovis strain promotes apoptosis of bovine macrophages by upregulation of CHOP expression.

Mycoplasma bovis (M. bovis) is one of the major pathogens in the bovine respiratory disease complex, which includes pneumonia, mastitis, and arthritis and causes a great economic loss in the cattle industry. In China, a live-attenuated vaccine strain M. bovis P150 was obtained by a continuous culture of the wild-type strain M. bovis HB0801 (P1) in vitro for 150 passages. Using the infected bovine macrophage cell line BoMac, this work attempted to investigate the mechanism of P150 attenuation and protective immune response. To begin, we show that M. bovis P150 effectively triggered cytotoxicity and apoptosis in BoMac, although with lower intracellular survival than P1. The transcriptomes of BoMac after infection with M. bovis strains P1 and P150 were sequenced, and bioinformatic analysis identified 233 differentially expressed genes (DEGs), with 185 upregulated and 48 downregulated. Further Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses revealed that the majority of the DEGs were linked to CHOP complex, MAP kinase phosphatase activity and were involved in the IL-17 signaling pathway in immune response, MAPK signaling pathway in signal transduction, and p53 signaling pathway in cell growth and death. Among them, the level of C/EBP homologous protein (CHOP) was significantly upregulated in P150-infected BoMac compared to P1-infected cells at different time points, along with its upstream and downstream genes phosphorylated-PERK, phosphorylated-EIF2α, ATF4, and GADD45A increased in the PERK-dependent ER stress response. The role of CHOP in apoptosis was further verified by M. bovis-induced siCHOP knockdown in BoMac cells. The results showed that CHOP knockdown enhanced P150-induced apoptosis and dramatically increased the M. bovis P1 and P150 intracellular survival, particularly for P150. These data suggest that P150 infection upregulates CHOP expression, which can increase apoptosis and mediate a crosstalk between ER stress and apoptosis during infection, and hence, contribute to high cytotoxicity and low intracellular survival.

Secreted MbovP0145 Promotes IL-8 Expression through Its Interactive ß-Actin and MAPK Activation and Contributes to Neutrophil Migration.

Mycoplasma bovis (M. bovis) is an important pathogen of cattle responsible for huge economic losses in the dairy and beef industries worldwide. The proteins secreted by M. bovis are mainly related to its adhesion, invasion, virulence, and intracellular survival and play a role in mycoplasma-host interactions. In our previous study, we found MbovP0145, a secreted protein present in the M. bovis secretome, but little is known about its function. In this study, we assessed the inflammatory characteristics and underlined mechanism of this inflammation of recombinant MbovP0145 (rMbovP0145). For this, bovine lung epithelial cells (EBL) were stimulated by rMbovP0145 to see the IL-8 production in a time- and dose-dependent manner. We observed that rMbovP0145 increased the production of IL-8 via ERK1/2 and P38 pathway activation. Further, the effect of the M. bovis ΔMbov_0145 mutant and its complementary strain on IL-8 mRNA expression was also confirmed. A pulldown assay of the GST-tagged MbovP0145 protein with mass spectrometry demonstrated that ß-actin could specifically interact with rMbovP0145 to mediate the IL-8 signaling. As knockdown of ß-actin expression with RNA interference in EBL cells decreased the mRNA expression of IL-8 and the phosphorylated ERK1/2 and P38 proteins, whereas disrupted actin polymerization by cytochalasin D led to a significantly higher IL-8 expression and MAPK phosphorylation in rMbovP0145-stimulated cells. Compared to M. bovis HB0801 and its complementary strain, the culture supernatant of EBL cells infected with the M. bovis ΔMbov_0145 mutant induced less neutrophil migration to the lower chamber in a transwell system. In conclusion, MbovP0145 promoted IL-8 expression by interacting with ß-actin through activation of the MAPK pathway, thus contributing to neutrophil migration.

Comparative Secretome Analyses of Mycoplasma bovis Virulent and Attenuated Strains Revealed MbovP0145 as a Promising Diagnostic Biomarker.

Mycoplasmas are successful pathogens both in humans as well as in animals. In cattle, Mycoplasma bovis (M. bovis) is known to be responsible for serious health complications, including pneumonia, mastitis, and arthritis. However, M. bovis pathogenesis remains unclear. Secreted proteins of M. bovis could influence infection and modify host defense signaling pathways after they enter their extracellular space in the host micro-environment. Therefore, this study was aimed to compare the secretomes of M. bovis HB0801 virulent (P1) and attenuated (P150) strains and identify potential pathogenesis-related secreted proteins and biomarkers. The cells of P1 and P150 strains were grown in pleuropneumonia-like organism medium to log phase and then transferred to phosphate-buffered saline for 2 h. Then, the supernatant was analyzed by using label-free quantitative proteomics, and 477 potential secreted proteins were identified. Combined with the bioinformatics prediction, we found that 178 proteins were commonly secreted by the P1 and P150 strains, and 49 of them were encoded by mycoplasmal core genes. Additionally, 79 proteins were found to have a different abundance between the P1 and P150 strains. Among these proteins, 34 were more abundant and uniquely expressed in P1, indicating a possible association with the virulence of M. bovis. Three differentially secreted proteins, MbovP0145, MbovP0725, and MbovP0174, as well as one equally secreted protein, MbovP0481, as positive control and one protein of inner membrane, MbovP0310, as negative control were, respectively, cloned, expressed, and evaluated for antigenicity, subcellular location, and the secretion nature with their mouse antisera by western blotting and colony immunoblotting assay. Among them, MbovP0145 was confirmed to be more secreted by P1 than P150 strain, highly reactive with the antisera from naturally infected and P1 experimentally infected cattle but not with the P150 vaccinated calves, indicating its potential as a diagnostic antigen. In conclusion, these findings may represent the most extensive compilation of potentially secreted proteins in mycoplasma species and the largest number of differentially secreted proteins between the virulent and attenuated M. bovis strains to date and provide new insights into M. bovis pathogenesis and diagnosis.

An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis: Securing survival in cell culture.

Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogen Mycoplasma bovis as a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reduced M. bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursors de novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival of M. bovis by securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.