This study aimed to develop a genetically stable, high-brightness fluorescent reporter system for Mycoplasma pneumoniae (MP) and to demonstrate its utility in investigating host-pathogen interactions. The system is based on the mNeonGreen fluorescent protein. Its expression level was enhanced by utilizing the promoter of the MP tuf gene, while transposition efficiency was improved by codon optimization of the transposase gene. To facilitate the selection of recombinant mycoplasmas containing the transposon, strategies involving antibiotic resistance gene and promoter optimization were employed. Using random transposition via a miniTn4001 transposon vector, we achieved the efficient and stable integration of both mNeonGreen and an antibiotic resistance gene into the MP genome. Experimental results indicated that MP colonies electrotransformed with the chloramphenicol-resistant transposon exhibited normal morphology, whereas those with the tetracycline-resistant transposon showed abnormal morphology and growth defects. To evaluate the practicality of the resulting mNeonGreen-expressing recombinant MP strain (M129-mNeonGreen) for monitoring MP infection, particularly cellular adherence/invasion, we conducted infection assays using human bronchial epithelial cells (BEAS-2B) and human cervical adenocarcinoma cells (HeLa). Specific green fluorescence was observed by confocal microscopy when M129-mNeonGreen adhered to both BEAS-2B and HeLa cells, which was absent in cells infected with the wild-type M129 strain, confirming the utility of the recombinant strain for detecting MP infection. Furthermore, to validate the in vivo tracking capability of M129-mNeonGreen, BALB/c mice were infected with the recombinant strain. Frozen section analysis of lung tissues revealed specific green fluorescence signals three days post-infection, demonstrating the successful application of this reporter strain for in vivo tracing in a mouse model. In conclusion, this study successfully established a bright and genetically stable fluorescent reporter system for MP, providing a valuable tool for elucidating the in vivo and in vitro functionalities of MP.

Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Avibacterium paragallinarum (APG) are respiratory-borne bacterial pathogens that severely harm the poultry industry. The clinical symptoms caused by them share many similarities, such as respiratory disease, growth retardation, and decreased egg production. They are not suitable for rapid diagnosis through isolation and culture and often need to detect nucleic acids or antibodies for differential diagnosis. In this study, bioinformatics analyses were used, and six specific coding genes were identified as being shared among all the APG strains that were absent in other species with published genome sequences. Combined with MG- and MS-specific genes identified in previous studies, we established a set of single-plex and multiplex qPCR assays for the rapid differential diagnosis of these three pathogens. The results indicated that the correlation coefficients (R2) of the standard curve established in these methods were not less than 0.999, and the amplification efficiencies (E) were between 90 % and 110 %. In terms of specificity, with the exception of the amplification curve and CT value generated in the positive control, other related pathogens, chicken cells, and empty plasmid did not amplify. In terms of sensitivity, the 100 % detection sensitivity of MG single-plex qPCR, MS single-plex qPCR, APG single-plex qPCR, and MG-MS duplex qPCR established in this study was 5 copies/reaction. The 100 % detection sensitivity of MG-MS-APG triplex qPCR was 5 copies/reaction in both the MG and MS detection channels and 10 copies/reaction in the APG detection channel. The detection rate of triplex qPCR in the APG detection channel at 5 copies/reaction was 80 %. The intra-group and inter-group variation coefficients of the qPCR methods established in this study were all within 2 % in the repeatability evaluation. In terms of the coincidence rate of clinical sample testing, the qPCR methods showed 100 % detection consistency for the clinical samples tested. The established qPCR methods exhibited good specificity, sensitivity, and repeatability, which provide powerful technical support for the rapid and efficient differential diagnosis of MG, MS, and APG simultaneously.

LigiLactobacillus saerimneri (L. sae) has shown considerable promise as a probiotic in recent years, particularly in poultry production. Comprehensive evaluation of its genetic functions, safety profile, and immunogenicity is essential prior to practical application. Our previous study demonstrated that the chicken-derived strain L. sae M-11 colonizes effectively and exhibits a favorable safety profile at adequate dosages. In this study, we further evaluated the potential of L. sae M-11 by analyzing its genetic basis for intestinal adaptation, metabolic features, safety risks, and suitability as a delivery vector. Comparative genomic analysis revealed that L. sae has evolved distinctive genetic features and functional specialization that may facilitate host adaptation. Genomic stability assessments and virulence factor screening confirmed that L. sae M-11 poses no substantial health risks. Furthermore, based on transmembrane protein predictions, the LPQTGE-motif protein was identified as a cell wall anchor in genetically engineered L. sae M-11 using immunoelectron microscopy. Notably, this delivery system selectively activated peripheral blood monocyte-derived dendritic cells (PB-MoDCs) in vitro, as evidenced by the up-regulation of maturation markers (CD83, CD80), pro-inflammatory cytokines (IL-1β, IL-6), Th1-associated IL-12, and the chemokine CXCLi1. However, it exhibited a limited antigen presentation capacity, indicated by low expression levels of CD40, MHCII, DEC205, TNF-α, and IFN-γ. The prospects and challenges associated with the application of L. sae M-11 have been discussed. Overall, these findings support the potential development of L. sae M-11 as a microbial cell factory and mucosal delivery vector.

Mycoplasma synoviae infection has caused serious economic losses to the poultry industry worldwide. The molecular mechanism by which M. synoviae colonizes the synovium and induces synovitis is unclear. In this study, desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry analyses were used to screen M. synoviae membrane proteins that bind the membrane proteins of synovial sheath cells (SSCs). Among the 128 screened proteins, elongation factor G (EF-G) of M. synoviae was identified as a surface-located protein using colony blotting and dual fluorescence analyses. The immunogenicity of EF-G was confirmed by the preparation of a rabbit polyclonal antibody. EF-G was identified as a cytoadhesin that directly binds to SSCs using indirect immunofluorescence assay and ELISA plate binding assay. In addition, antibody adhesion inhibition and protein adhesion inhibition demonstrated that EF-G could significantly promote the adhesion of M. synoviae to SSCs. Co-IP, GST pull-down, bacterial two-hybridization, and ELISA plate binding assays were performed to demonstrate the binding of EF-G and vimentin in vivo and in vitro. Antibody adhesion inhibition, protein adhesion inhibition, and siRNA interference adhesion inhibition assays demonstrated that vimentin significantly affected M. synoviae adhesion to SSCs. These studies indicate that two interacting proteins, EF-G, a novel cytoadhesin, and vimentin, an important cell surface receptor, play important roles in the adhesion of M. synoviae to SSCs, laying a foundation for subsequent studies on the mechanism of M. synoviae-induced synovitis and providing meaningful targets for screening target drugs against M. synoviae infection.

利用CD-HIT-EST快速聚类分析和BLASTN同源性比对共鉴定出17个滑液支原体(Mycoplasma synoviae)特有基因,并从中选取保守基因VY93_RS02885作为分子诊断的靶标基因.根据该基因核苷酸序列设计了1对引物,建立了滑液支原体的SYBR green qPCR检测方法.结果表明,该方法对不同浓度的模板和对应的CT值具有较好的线性关系,R2值为0.9988;该方法的特异性较好,BLASTN比对,检测引物对仅能匹配滑液支原体的特有基因VY93_RS02885,结果显示,该方法能特异性地检测滑液支原体,与其他常见的引起禽关节炎的细菌/支原体性病原无交叉反应;该方法的敏感性较好,100％阳性检测的靶标基因最小拷贝数为10;该方法的重复性较好,对3个不同浓度的模板进行组间和组内重复检测,变异系数≤1.1727％.此外,该方法对临床样品的检出率与传统检测方法相符.因此,本研究建立的针对滑液支原体特有基因的SYBR green qPCR检测方法具有用时短、特异性高、准确性高、敏感性高、可重复性强的优点,可为滑液支原体的快速诊断和流行病学调查提供技术手段.

Mycoplasma synoviae is an important pathogen of poultry, causing significant economic losses in this industry. Analysis of the unique genes and shared genes among different M. synoviae strains and among related species is helpful for studying the molecular pathogenesis of M. synoviae and provides valuable molecular diagnostic targets to facilitate the identification of M. synoviae species. We selected a total of 46 strains, including six M. synoviae strains, from 25 major animal (including avian) Mycoplasma species/subspecies that had complete genome sequences and annotation information published in GenBank, and used them for comparative genomic analysis. After analysis, 16 common genes were found in the 46 strains. Thirteen single-copy core genes and the 16s rRNA genes were used for genetic evolutionary analysis. M. synoviae was found to have a distant evolutionary relationship not only with other arthritis-causing mycoplasmas, but also with another major avian pathogen, Mycoplasma gallisepticum, that shares the major virulence factor vlhA with M. synoviae. Subsequently, six unique coding genes were identified as shared among these M. synoviae strains that are absent in other species with published genome sequences. Two of the genes were found to be located in the genetically stable regions of the genomes of M. synoviae and were determined to be present in all M. synoviae isolated strains (n = 20) and M. synoviae-positive clinical samples (n = 48) preserved in our laboratory. These two genes were used as molecular diagnostic targets for which SYBR green quantitative PCR detection methods were designed. The two quantitative PCR methods exhibited good reproducibility and high specificity when tested on positive plasmid controls and genomic DNA extracted from different M. synoviae strains, other major avian pathogenic bacteria/mycoplasmas, and low pathogenic Mycoplasma species. The detection limit for the two genes was 10 copies or less per reaction. The clinical sensitivity and specificity of the quantitative PCR methods were both 100% based on testing chicken hock joint samples with positive or negative M. synoviae infection. This research provides a foundation for the study of species-specific differences and molecular diagnosis of M. synoviae.

Mycoplasma synoviae (MS) infection causes infectious synovitis and arthritis with hyperplasia of synovial cells in the chicken joint. However, its mechanism is unknown. We used primary chicken synovial fibroblast (CSF) as the research object to study the role of MS in the proliferation of MS-infected CSF and determine the mechanisms involved. Using integrated transcriptomic and proteomic analyses of the interaction between CSF and MS, we screened a proliferation-regulated factor, serum amyloid A (SAA), that may regulate proliferation of MS-infected CSF. SAA appears to be associated with MS-induced CSF proliferation. To study the role of SAA in MS-induced CSF proliferation, a eukaryotic expression vector overexpressing SAA and a small interfering RNA (siRNA) targeting Saa were constructed to manipulate the expression of SAA. Cell proliferation and apoptosis were detected via cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), or terminal deoxyribonucleotidyl transferase-mediated dUTP nick-dnd labeling (TUNEL) assays, respectively. Western blot analysis was used to examine the protein expression level of SAA, cyclin E1, and cyclin-dependent kinase 2 (CDK2). In vitro, MS significantly promoted the proliferation of CSF and increased the production of SAA. Overexpression of SAA accelerated the proliferative ability of CSF, whereas knockdown of SAA depressed the proliferative ability of CSF. A TUNEL assay indicated that MS did not induce apoptosis. Silencing of SAA suppressed the expression of cyclin E1 and CDK2. These results suggest that MS may upregulate the expression of SAA, accelerate the cell cycle, and promote proliferation of CSF.

Here, we report the complete genome sequence of Mycoplasma synoviae HN01, a virulent epidemic strain isolated from a sick chicken with synovitis in Henan Province, China. HN01 is the Asian source of an M. synoviae strain that is completely sequenced, genome annotated, and published with relevant data.

Mycoplasma synoviae (MS) is an important avian pathogen causing considerable economic hardship in the poultry industry. A major inflammation caused by MS is synovitis that occurs in the synovial tendon sheath and joint synovium. However, the overall appearance of pathological changes in the tendon sheath and surrounding tissues caused by MS infection at the level of pathological tissue sections was poor. Studies on the role of MS and synovial sheath cells (SSCs) interaction in the development of synovitis have not been carried out. Through histopathological observation, our study found that a major MS-induced pathological change of the tendon sheath synovium was extensive scattered and focal inflammatory cell infiltration of the tendon sheath synovial layer. In vitro research experiments revealed that the CFU numbers of MS adherent and invading SSC, the levels of expression of various pattern recognition receptors, inflammatory cytokines, and chemokines coding genes, such as IL-1β, IL-6, IL-8, CCL-20, RANTES, MIP-1β, TLR7, and TLR15 in SSCs, and chemotaxis of macrophages were significantly increased when the multiplicity of infection (MOI) of MS to SSC were increased tenfold. The expression level of IL-12p40 in SSC was significantly higher when the MOIs of MS to SSC were increased by a factor of 100. The interaction between MS and SSC can activate macrophages, which was manifested by a significant increase in the expression of IL-1β, IL-6, IL-8, CCL-20, RANTES, MIP-1β, and CXCL-13. This study systematically demonstrated that the interaction of MS with chicken SSC contributes to the inflammatory response caused by the robust expression of related cytokines and macrophage chemotaxis. These findings are helpful in elucidating the molecular mechanism of MS-induced synovitis in chickens.

Mycoplasma synoviae (MS), which causes respiratory disease, eggshell apex abnormalities, infectious synovitis, and arthritis in avian species, has become an economically detrimental poultry pathogen in recent years. In China, the disease is characterized by infectious synovitis and arthritis. However, the mechanism by which MS causes infectious synovitis and arthritis remains unknown. Increasing evidence suggests that synovial fibroblasts (SF) play a key role in the pathogenesis of arthritis. Here, both RNA sequencing and tandem mass tag analyses are utilized to compare the response of primary chicken SF (CSF) following infection with and without MS. The host response between non-infected and infected cells was remarkably different at both the mRNA and protein levels. In total, 2,347 differentially expressed genes (DEGs) (upregulated, n = 1,137; downregulated, n = 1,210) and 221 differentially expressed proteins (DEPs) (upregulated, n = 129; downregulated, n = 92) were detected in the infected group. A correlation analysis indicated a moderate positive correlation between the mRNA and protein level changes in MS-infected CSF. At both the transcriptomic and proteomic levels, 149 DEGs were identified; 88 genes were upregulated and 61 genes were downregulated in CSF. Additionally, part of these regulated genes and their protein products were grouped into seven categories: proliferation-related and apoptosis-related factors, inflammatory mediators, proangiogenic factors, antiangiogenic factors, matrix metalloproteinases, and other arthritis-related proteins. These proteins may be involved in the pathogenesis of MS-induced arthritis in chickens. To our knowledge, this is the first integrated analysis on the mechanism of CSF-MS interactions that combined transcriptomic and proteomic technologies. In this study, many key candidate genes and their protein products related to MS-induced infectious synovitis and arthritis were identified.

The anti-phagocytic abilities of bacteria often affect bacterial pathogenicity. Here, random mutant library of Streptococcus equi subsp. zooepidemicus (SEZ) was constructed using transposon mutagenesis. After careful screening, 30 transposon mutants with different transposon insertion sites were identified by conducting quantitative phagocytosis and insertion-site confirmation assays, whose anti-phagocytic abilities were significantly reduced relative to the wild-type strain. Insertion sites of 19 strains were monocistronic, including genes coding membrane proteins, transporters, and enzymes with unknown pathological function, such as sadM, adhP, purD, guaA, alpha-galactosidase coding gene, ABC transporter permease coding gene, metallo-beta-lactamase coding gene, and three secreted enzyme coding genes spuZ, slaB, and endoS, as well as known virulence factor coding genes, such as hasA and szM. The insertion sites of another 11 strains were polycistronic. We focused on four monocistronic-mutant strains: MhtpZ, MspuZ, MslaB, and MendoS. The anti-phagocytic abilities of not only the mutants that were precoincubated with the recombinant proteins, but also the complement strains were significantly more pronounced than those of all four corresponding mutants. The polyclonal antiserum against SlaB or EndoS also significantly decreased the anti-phagocytic capacity of wild-type SEZ. All four mutants exhibited significantly decreased viability in whole blood and reduced lethality in mice relative to the wild-type strain. Thus, we identified a variety of new anti-phagocytic factors, particularly multiple SEZ secreted enzymes. These factors are instrumental in the phagocytic resistance of SEZ in the absence of opsonin. Our results provide a framework for further studies of SEZ pathogenesis and relevant vaccine development for novel potential targets.

Streptococcus suis serotype 2 (SS2) is an important zoonotic agent in swine and humans. Anti-phagocytosis and survival in phagocytic cells and whole blood is essential for bacteria to be pathogenic. In this study, the host specificity determinant specificity subunit (coded by hsdS) of the Type I Restriction-Modification system and two peptidoglycan-binding proteins (coded by lysM and lysM', respectively), which were simultaneously found to be subjected to transcript-level influence by hsdS, were identified to facilitate the anti-phagocytosis of SS2 to a microglia cell line BV2. Furthermore, they significantly enhanced its survival in BV2, whole blood, and a peroxidation environment (H2O2) (p < 0.05), yet not in the acidic condition based on statistical analysis of the characteristic differences between gene mutants and wild-type SS2. In contrast, another specificity subunit, coded by hsdS', that belonged to the same Type I Restriction-Modification system, only significantly reduced the survival ability of SS2 in the acidic condition when in the form of a gene-deleted mutant (p < 0.05), but it did not significantly influence the survival ability in other conditions mentioned above or have enhanced anti-phagocytosis action when compared with wild-type SS2. In addition, the mutation of hsdS significantly enhanced the secretion of nitric oxide and TNF-α by BV2 with SS2 incubation (p < 0.05). The SS2 was tested, and it failed to stimulate BV2 to produce IFN-γ. These results demonstrated that hsdS contributed to bacterial anti-phagocytosis and survival in adverse host environments through positively impacting the transcription of two peptidoglycan-binding protein genes, enhancing resistance to reactive oxygen species, and reducing the secretion of TNF-α and nitric oxide by phagocytes. These findings revealed new mechanisms of SS2 pathogenesis.