Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits of Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.

Establishment of a low-tumorigenic MDCK cell line and study of differential molecular networks.

Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate.

Fructose-1,6-bisphosphate aldolase of Mycoplasma bovis is a plasminogen-binding adhesin.

Mycoplasma bovis is an extremely small cell wall-deficient pathogenic bacterium in the genus Mycoplasma that causes serious economic losses to the cattle industry worldwide. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in the glycolytic pathway, is a multifunctional protein in several pathogenic bacterial species, but its role in M. bovis remains unknown. Herein, the FBA gene of the M. bovis was amplified by PCR, and subcloned into the prokaryotic expression vector pET28a (+) to generate the pET28a-FBA plasmid for recombinant expression in Escherichia coli Transetta. Expression of the 34 kDa recombinant rMbFBA protein was confirmed by electrophoresis, and enzymatic activity assays based on conversion of NADH to NAD+ revealed Km and Vmax values of 48 µM and 43.8 µmoL/L/min, respectively. Rabbit anti-rMbFBA and anti-M. bovis serum were generated by inoculation with rMbFBA and M. bovis, and antigenicity and immunofluorescence assay demonstrated that FBA is an immunogenic protein expressed on the cell membrane in M. bovis cells. Enzyme-linked immunosorbent assays revealed equal distribution of FBA in the cell membrane and cytoplasm. Complement-dependent mycoplasmacidal assays showed that rabbit anti-rMbFBA serum killed 44.1% of M. bovis cells in the presence of complement. Binding and ELISA assays demonstrated that rMbFBA binds native bovine plasminogen and in a dose-dependent manner. Fluorescent microscopy revealed that pre-treatment with antibodies against rMbFBA decreased the adhesion of M. bovis to embryonic bovine lung (EBL) cells. Furthermore, adherence inhibition assays revealed 34.4% inhibition of M. bovis infection of EBL cells following treatment with rabbit anti-rMbFBA serum, suggesting rMbFBA participates in bacterial adhesion to EBL cells.

Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum.

The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD(+). Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein.

BACKGROUND: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated. RESULTS: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 µmol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum. CONCLUSION: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

Update on feline calicivirus: viral evolution, pathogenesis, epidemiology, prevention and control.

Feline calicivirus (FCV) is a prevalent and impactful viral pathogen affecting domestic cats. As an RNA virus, FCV exhibits high mutability and genetic plasticity, enabling its persistence within cat populations. Viral genetic diversity is associated with a broad spectrum of clinical manifestations, ranging from asymptomatic infections and mild oral and upper respiratory tract diseases to the potential development of virulent systemic, and even fatal conditions. This diversity poses distinctive challenges in diagnosis, treatment, and prevention of diseases caused by FCV. Over the past four decades, research has significantly deepened understanding of this pathogen, with an emphasis on molecular biology, evolutionary dynamics, vaccine development, and disease management strategies. This review discusses various facets of FCV, including its genomic structure, evolution, innate immunity, pathogenesis, epidemiology, and approaches to disease management. FCV remains a complex and evolving concern in feline health, requiring continuous research to enhance understanding of its genetic diversity, to improve vaccine efficacy, and to explore novel treatment options.

Blocking ACSL6 Compromises Autophagy via FLI1-Mediated Downregulation of COLs to Radiosensitize Lung Cancer.

Lung cancer (LC) is the leading cause of cancer-related mortality worldwide. Radiotherapy is the main component of LC treatment; however, its efficacy is often limited by radioresistance development, resulting in unsatisfactory clinical outcomes. Here, we found that LC radiosensitivity is up-regulated by decreased expression of long-chain acyl-CoA synthase 6 (ACSL6) after irradiation. Deletion of ACSL6 results in significant elevation of Friend leukemia integration 1 transcription factor (FLI1) and a marked decline of collagens (COLs). Blocking of ACSL6 impairs the tumor growth and upregulates FLI1, which reduces the levels of COLs and compromises irradiation-induced autophagy, leading to considerable therapeutic benefits during radiotherapy. Moreover, the direct interaction between ACSL6 and FLI1 and engagement between FLI1 and COLs indicates the involvement of the ACSL6-FLI1-COL axis. Finally, the potently adjusted autophagy flux reduces its otherwise contributive capability in surviving irradiation stress and leads to satisfactory radiosensitization for LC radiotherapy. These results demonstrate that enhanced ACSL6 expression promotes the aggressive performance of irradiated LC through increased FLI1-COL-mediated autophagy flux. Thus, the ACSL6-FLI1-Col-autophagy axis may be targeted to enhance the radiosensitivity of LC and improve the management of LC in radiotherapy.

High Concentration of FBS Can Save mTOR Down-Regulation Caused by Mycoplasmas bovis Infection.

Mycoplasmas bovis (M. bovis) is an important pathogen that causes a variety of diseases, such as bovine respiratory diseases and causes significant losses to the national cattle industry every year, seriously affecting the development of the cattle industry worldwide. The pathogenic mechanism of M. bovis infection is still unknown, which leads to the lack of timely diagnosis and treatment. In this study, embryonic bovine lung (EBL) cells, infected with M. bovis were collected for gene profiling and detection of marker genes in the mTOR signaling pathway. The result showed that M. bovis infection significantly inhibits EBL growth in a dose-dependent manner. The transcription profiling data uncovered that M. bovis infection repressed a series of gene expressions in EBL cells, which are mainly related to metabolic process and immune response. Notably, many marker genes in the PI3K-Akt-mTOR pathway showed down-regulation after M. bovis infection. Further evidence showed that M. bovis infection inhibits expression of mTOR signaling pathway marker genes in EBL cells, which are time dependent. To further understand the M. bovis-induced inhibitory effect of mTOR signaling pathway, this study employed FBS as a supplement for exogenous nutrients and found that addition of a high concentration of FBS can rescue M. bovis-induced cell damage. In addition, a high concentration of FBS can rescue down-regulated mTOR signaling, including increasing transcriptional expression and protein phosphorylation level of mTOR pathway marker genes. This study demonstrated that M. bovis infection leads to inhibition of the nutrient metabolic pathway mTOR in a time-dependent manner, which would be helpful to further understand M. bovis infection mechanism and develop a new efficient anti-mycoplasma strategy targeting mTOR signaling.

Mycoplasmas bovis P48 induces apoptosis in EBL cells via an endoplasmic reticulum stress-dependent signaling pathway.

Mycoplasma bovis (M. bovis) is a small bacterium that lacks a cell wall. M. bovis infection can result in chronic pneumonia and polyarthritis syndrome (CPPS), otitis media, conjunctivitis, and meningitis in feedlot cattle and mastitis in dairy cattle. To gain more understanding of the mechanism of M. bovis and host interaction, this study focused on P48, an important membrane protein involved in M. bovis adhesion, proliferation and virulence. In this study, exogenous P48 protein was introduced to explore its function in embryonic bovine lung (EBL) cells by recombinant vector and protein purification. We found that M. bovis infection inhibited EBL cells growth and enhanced apoptosis. Both intracellular and extracellular P48 protein treatment also induce apoptosis. Moreover, P48 activates endoplasmic reticulum (ER) stress response via increasing ER stress markers expression. To further explore the underlying mechanism, we performed inhibition experiments using ER stress inhibitor 4-PBA and specific siRNA interference against GRP78, and found that P48 protein modulated EBL cells apoptosis in an ER stress signaling-dependent manner. This study provided more data to further understand M. bovis infection mechanism and develop effective anti-mycoplasma strategy.

Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschussifanicus).

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD), and its infection results in mortality of 70-90% in farmed and wild rabbits. RHDV is thought to replicate strictly in rabbits. However, there are also reports showing that gene segments from the RHDV genome or antibodies against RHDV have been detected in other animals. Here, we report the detection and isolation of a RHDV from diseased Alpine musk deer (Moschussifanicus). The clinical manifestations in those deer were sudden death without clinical signs and hemorrhage in the internal organs. To identify the potential causative agents of the disease, we used sequence independent single primer amplification (SISPA) to detect gene segments from viruses in the tissue samples collected from the dead deer. From the obtained sequences, we identified some gene fragments showing very high nucleotide sequence similarity with RHDV genome. Furthermore, we identified caliciviral particles using an electron microscope in the samples. The new virus was designated as RHDV GS/YZ. We then designed primers based on the genome sequence of an RHDV strain CD/China to amplify and sequence the whole genome of the virus. The genome of the virus was determined to be 7437 nucleotides in length, sharing the highest genome sequence identity of 98.7% with a Chinese rabbit strain HB. The virus was assigned to the G2 genotype of RHDVs according to the phylogenetic analyses based on both the full-length genome and VP60 gene sequences. Animal experiments showed that GS/YZ infection in rabbits resulted in the macroscopic and microscopic lesions similar to that caused by the other RHDVs. This is the first report of RHDV isolated from Alpine musk deer, and our findings extended the epidemiology and host range of RHDV.

[Tandem expression of the major epitope domains of the Moschus chrysogaster hemorrhagic disease virus VP60 and its protective efficacy to rabbits].

Moschus chrysogaster (sifanicus) viral hemorrhagic disease (McVHD) is an acute and highly lethal infectious disease caused by Moschus chrysogaster hemorrhagic disease virus (McHDV) whose genome sequence is highly homologous with rabbit hemorrhagic disease virus. To screen the protective antigen of McHDV and set the basis for study of McVHD vaccine, the antigen epitope of major structural protein VP60 of McHDV was analyzed, and the specific primers were designed to obtain three amplified DNA sequences encoding the main antigen epitope of VP60 from McHDV by using RT-PCR. Then the three DNA fragments were sequenced and cloned to prokaryotic expression vector with pET-28a(+) by using overlap extension PCR, and finally the prokaryotic expression plasmid pET-truncated-VP60 was constructed. Subsequently, the pET-truncated-VP60 was transformed into Escherichia coli BL21(DE3), and the recombinant proteins were expressed by IPTG induction. Finally, the expressed protein was purified and applied to immunize that without immunizing with RHD vaccine, then the antiserum titers were evaluated by the hemagglutination inhibition test, and the immune-protective efficacy of the recombinant proteins was observed and analyzed through animal challenge test. The results showed that the multi-epitope DNA fragments of VP60 of McHDV was successfully expressed in the form of inclusion bodies in E. coli, and the relative molecular weight of recombinant proteins is about 45 kDa. After immunized with the recombinant proteins, 100% of New Zealand white rabbits were resistant to attack of McHDV, which indicates efficient immune-protective efficacy of chosen epitope recombinant protein. The study laid a foundation for the development of the new subunit vaccines of McVHD.

Whole-Genome Sequence of Corynebacterium xerosis Strain GS1, Isolated from Yak in Gansu Province, China.

We report here the isolation, sequencing of the complete closed genome, and annotation of Corynebacterium xerosis strain GS1. This strain was isolated from the liver lesion of a yak in Gansu Province, China. The genome consists of one chromosome with 2,738,835 bp and comprises 2,304 protein-coding genes.

Identification of a novel, small, conserved hypothetical protein involved in Brucella abortus virulence by modifying the expression of multiple genes.

Brucellosis is an important zoonotic disease worldwide, caused by Brucella spp., which are facultative intracellular bacteria with no classic virulence factors, as virulence is dependent on the ability to invade and replicate within host cells. In this study, we identified a novel gene bab_RS22045 that encodes a small highly conserved protein in Rhizobiales. To investigate the role of this gene, a deletion mutant and complement strain were constructed. Virulence testing showed that bab_RS22045 is necessary for Brucella virulence, and was designated as virulence-related hypothetical protein, VhpA. The results of a cell infection experiment showed that vhpA was not associated with Brucella adherence to and invasion of HeLa cells, or further intracellular survival within RAW264.7 cells. The results of sensitivity testing showed the vhpA mutant had similar sensitivity to hydrogen peroxide, polymyxin B, and sodium nitroprusside as the wild-type (WT) strain. Interestingly, RNA-seq analysis showed that deletion of the vhpA gene affected the expression patterns of multiple Brucella genes, and the main four up-regulated genes and five down-regulated genes were further confirmed using quantitative real-time PCR analysis. Subsequently, a series of over-expression strains were constructed, and virulence testing showed that over-expression of four up-regulated genes (bab_RS17930, bab_RS17925, bab_RS26460, and bab_RS30050) significantly reduced virulence of the WT strain, and over-expression of bab_RS18680 in the vhpA mutant partially restored virulence, suggesting that vhpA plays an important role in Brucella virulence by changing the expression patterns of multiple genes. Additionally, heterogeneous complementary analysis showed that the homologous vhpA genes of Sinorhizobium meliloti and Agrobacterium tumefaciens could not restore virulence of the vhpA mutant, although VhpA is a highly conserved protein in Rhizobiales. Overall, a novel, small, hypothetical gene was identified that is associated with B. abortus virulence, which highlights the roles of small encoding genes in Brucella virulence.

Development of a loop-mediated isothermal amplification targeting a gene within the pyruvate dehydrogenase complex, the pdhA gene, for rapid detection of Mycoplasma gallisepticum.

Mycoplasma gallisepticum infections impose a significant economic burden on the poultry industry. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized to detect M. gallisepticum based on a gene within the pyruvate dehydrogenase complex, the pdhA gene, which codes for the major subunit (E1α) in the complex. The reaction conditions were optimized, and the specificity was confirmed by successful amplification of several M. gallisepticum strains, while no amplification was detected with 20 other major bacterial and viral pathogens of poultry. Additionally, the LAMP assay achieved 10-fold higher sensitivity than an existing polymerase chain reaction (PCR) method. The LAMP assay was applied to swab samples collected from poultry farms and compared with PCR. The positive detection rate was 20.2% (37/183) by LAMP and 13.1% (24/183) by PCR. The LAMP assay could provide a cost-effective, quick, and sensitive method for the detection of M. gallisepticum.

Characterization of triosephosphate isomerase from Mycoplasma gallisepticum.

Triosephosphate isomerase (Tpi) is a glycolytic enzyme that is essential for efficient energy production in many pathogens. However, its function in Mycoplasma gallisepticum has not been fully elucidated. In this study, the mga0357 gene of M. gallisepticum, which encodes TpiA (MGTpiA), was amplified and expressed in Escherichia coli by IPTG induction. The purified recombinant MGTpiA protein exhibited catalytic activity that was similar to TPI from rabbit muscle, reducing NAD(+) to NADH. The MGTpiA was also found to be a surface-exposed protein by western blotting and immunofluorescence assays. In addition, cytadherence inhibition assays confirmed that the cytadherence of M. gallisepticum to the DF-1 cells was significantly inhibited by the anti-MGTpiA serum. The results of the study suggested that MGTpiA plays an important role in the metabolism and closely related to the M. gallisepticum pathogenicity.

[Cloning and immunological characteristics of canine IL-18 cDNA].

AIM: To clone the canine IL-18 cDNA and to explore the immunological effectiveness of canine IL-18 as an adjuvant of genetic vaccine. METHODS: Canine leukocytes were separated from peripheral blood stimulated with ConA for 5-12 h. The full length cDNA of canine IL-18 was amplified by RT-PCR using leukocyte mRNA as a template. IL-18 cDNA was cloned into pMD18-T. Sequencing result showed that the full length cDNA was 582 bp, encoding 193 amino acids, which was identical with that published. The eukaryotic expression vector pIIL18 was constructed. Dogs were inoculated in mixture form of pIGneo and pIIL18. RESULTS: Canine IL-18 cDNA was successfully cloned and eukaryotic expression vector pIIL18 was constructed. The immunological assay result showed that the anti-rabies virus-specific antibody level of the group immunized with mixture of pIIL18 and pIGneo was obviously lower than that of the group immunized with pIGneo alone, but the level of cellular immunity of the former was higher than the latter. CONCLUSION: Canine IL-18 can enhance cellular immunity but at the same time suppress humoral immunity, which lays the foundation for IL-18 as an adjuvant of genetic vaccine.