ENO1 from Mycoplasma bovis Disrupts Host Glycolysis and Inflammation by Binding ACTB
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells and Strains
2.2. Construction of Plasmids and Interfering Fragments
2.3. Transfection of Plasmids and Infection with M. bovis
2.4. Detection of Glucose, Lactic Acid, ATP, and ROS Levels
2.5. Western Blot
2.6. Immunofluorescence Experiment
2.7. GST Pull-Down
2.8. Co-Immunoprecipitation (Co-IP)
2.9. Key Interaction Amino Acid Prediction
2.10. RNA Extraction and RT-qPCR
2.11. Counting of Intracellular M. bovis
2.12. Quantitative and Statistical Analysis
3. Result
3.1. ENO1 and M. bovis Infection Induce Glycolysis in Host Cells
3.2. Interaction of ENO1 with Host Cell ACTB
3.3. ENO1 Regulates Glycolytic Reprogramming by Modulating ACTB
3.4. ENO1 Promotes the Inflammatory Response of Host Cells by Regulating ACTB
3.5. The 117Glu and 372Arg Residues of ACTB Are Critical Interaction Amino Acid Sites
3.6. M. bovis Infection Regulates Host Cell Glycolysis Through ACTB
3.7. ACTB-Mediated Regulation of Inflammatory Response and Intracellular Survival of M. bovis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Nicholas, R.A.J.; Ayling, R.D. Mycoplasma bovis: Disease, diagnosis, and control. Res. Vet. Sci. 2003, 74, 105–112. [Google Scholar] [CrossRef] [PubMed]
- Zhenhong, S.; Ping, F.; Kai, W.; Haiyan, Z.; Yuewei, Z.; Jian, X.; Fei, J.; Xu, L.; Wei, X.; Wenxue, W. Identification of novel immunogenic proteins from Mycoplasma bovis and establishment of an indirect ELISA based on recombinant E1 beta subunit of the pyruvate dehydrogenase complex. PLoS ONE 2014, 9, e88328. [Google Scholar] [CrossRef]
- Mustafa, R.; Qi, J.; Ba, X.; Chen, Y.; Hu, C. In vitro quinolones susceptibility analysis of chinese Mycoplasma bovis isolates and their phylogenetic scenarios based upon QRDRs of DNA topoisomerases revealing a unique transition in ParC. Pak. Vet. J. 2013, 33, 364–369. [Google Scholar]
- Pholpunthin, P.; Fukuyo, Y.; Matsuoka, K.; Nimura, Y. Immunomodulatory effect of Mycoplasma bovis in experimentally infected calves. Bull. Vet. Inst. Pulawy 2013, 57, 499–506. [Google Scholar]
- Dudek, K.; Nicholas, R.J.; Szacawa, E.; Bednarek, D. Mycoplasma bovis infections—occurrence, diagnosis and control. Pathogens 2020, 9, 640. [Google Scholar] [CrossRef]
- Shahriar, F.M.; Clark, E.G.; Janzen, E.; West, K.; Wobeser, G. Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia. Can. Vet. J. 2002, 43, 863. [Google Scholar] [PubMed]
- Mehinagic, K.; Pilo, P.; Vidondo, B.; Stokar-Regenscheit, N. Coinfection of swiss cattle with bovine parainfluenza virus 3 and Mycoplasma bovisat acute and chronic stages of bovine respiratory disease complex. J. Vet. Diagn. Investig. 2019, 31, 674–680. [Google Scholar] [CrossRef]
- Prysliak, T.; Merwe, J.V.D.; Lawman, Z.; Wilson, D.; Townsend, H.; PerezCasal, J. Respiratory disease caused by Mycoplasma bovis is enhanced by exposure to bovine herpes virus 1 (BHV-1) but not to bovine viral diarrhea virus (BVDV) type 2. Can. Vet. J. 2011, 52, 1195. [Google Scholar]
- Booker, C.W.; Abutarbush, S.M.; Morley, P.S.; Jim, G.K.; Pittman, T.J.; Schunicht, O.C.; Perrett, T.; Wildman, B.K.; Fenton, R.K.; Guichon, P.T. Microbiological and histopathological findings in cases of fatal bovine respiratory disease of feedlot cattle in Western Canada. Can. Vet. J. La Rev. Vet. Canadienne. 2008, 49, 473–481. [Google Scholar]
- Oliveira, T.E.S.; Pelaquim, I.F.; Flores, E.F.; Massi, R.P.; Headley, S.A. Mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows. Transbound. Emerg. Dis. 2019, 86, 82–93. [Google Scholar] [CrossRef]
- Maunsell, F.P.; Woolums, A.R.; Francoz, D.; Rosenbusch, R.F.; Step, D.L.; Wilson, D.J.; Janzen, E.D. Mycoplasma bovis infections in cattle. J. Vet. Intern. Med. 2011, 25, 772–783. [Google Scholar] [CrossRef]
- Song, Z.; Li, Y.; Liu, Y.; Xin, J.; Sun, W. α-Enolase, an adhesion-related factor of Mycoplasma bovis. PLoS ONE 2012, 7, e38836. [Google Scholar] [CrossRef]
- Li, L.; Lu, H.; Zhang, X.; Whiteway, M.; Wu, H.; Tan, S.; Zang, J.; Tian, S.; Zhen, C.; Meng, X.; et al. Baicalein acts against candida albicans by targeting Eno1 and inhibiting glycolysis. Microbiol. Spectr. 2022, 10, e0208522. [Google Scholar] [CrossRef]
- Zhu, X.; Baranowski, E.; Hao, Z.; Li, X.; Zhao, G.; Dong, Y.; Chen, Y.; Hu, C.; Chen, H.; Citti, C. An atypical GdpP enzyme linking cyclic nucleotide metabolism to osmotic tolerance and gene regulation in Mycoplasma bovis. Front. Microbiol. 2023, 14, 12. [Google Scholar] [CrossRef]
- Shi, Y.; Liu, J.; Zhang, R.; Zhang, M.; Cui, H.; Wang, L.; Cui, Y.; Wang, W.; Sun, Y.; Wang, C. Targeting endothelial ENO1 (Alpha-Enolase)-PI3K-Akt-mTOR axis alleviates hypoxic pulmonary hypertension. Hypertension 2023, 80, 1035–1047. [Google Scholar] [CrossRef]
- Zhang, Y.; Chang, L.; Xin, X.; Qiao, Y.; Qiao, W.; Ping, J.; Xia, J.; Su, J. Influenza a virus-induced glycolysis facilitates virus replication by activating ROS/HIF-1α pathway. Free Radic. Biol. Med. 2024, 225, 910–924. [Google Scholar] [CrossRef] [PubMed]
- Erlich, J.R.; To, E.E.; Liong, S.; Brooks, R.; Vlahos, R.; O’Leary, J.J.; Brooks, D.A.; Selemidis, S. Targeting evolutionary conserved oxidative stress and immunometabolic pathways for the treatment of respiratory infectious diseases. Antioxid. Redox Signal. 2020, 32, 993–1013. [Google Scholar] [CrossRef]
- Nicholas, S.A.; Coughlan, K.; Yasinska, I.; Lall, G.S.; Gibbs, B.F.; Calzolai, L.; Sumbayev, V.V. Dysfunctional mitochondria contain endogenous high-affinity human Toll-like receptor 4 (TLR4) ligands and induce TLR4-mediated inflammatory reactions. Int. J. Biochem. Cell Biol. 2011, 43, 674–681. [Google Scholar] [CrossRef] [PubMed]
- Ogryzko, N.V.; Lewis, A.; Wilson, H.; Meijer, A.; Renshaw, S.; Elks, P. Hif-1α–induced expression of Il-1β protects against Mycobacterial infection in zebrafish. J. Immunol. Author Choice 2018, 202, 494–502. [Google Scholar] [CrossRef]
- Finucane, O.; Sugrue, J.; Rubio, A.; Lynch, M. The NLRP3 inflammasome modulates glycolysis by increasing PFKFB3 in an IL-1β-dependent manner in macrophages. Sci. Rep. 2019, 9, 4034. [Google Scholar] [CrossRef] [PubMed]
- Duan, Q.; Dong, X.; Ma, Y.; Liu, C.; Zhang, M.; Ma, Y. WIN55212-2 inhibits glycolysis and alleviates acute lung injury in septic mice by regulating the mTOR/HIF-1α/PFKFB3 signaling pathway. Chin. J. Pathophysiol. 2024, 40, 521–526. [Google Scholar]
- Ihara, F.; Nishikawa, Y. Toxoplasma gondii manipulates host cell signaling pathways via its secreted effector molecules. Parasitol. Int. 2021, 83, 102368. [Google Scholar] [CrossRef] [PubMed]
- Moreira, D.; Rodrigues, V.; Abengozar, M.; Rivas, L.; Silvestre, R. Leishmania infantum Modulates Host Macrophage Mitochondrial Metabolism by Hijacking the SIRT1-AMPK Axis. PLoS Pathog. 2015, 11, e1004684. [Google Scholar] [CrossRef] [PubMed]
- Zhao, G.; Zhang, H.; Chen, X.; Zhu, X.; Guo, Y.; He, C.; Anwar Khan, F.; Chen, Y.; Hu, C.; Chen, H.; et al. Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin. Sci. Rep. 2017, 7, 44. [Google Scholar] [CrossRef] [PubMed]
- Adamu, J.Y.; Mitiku, F.; Hartley, C.A.; Sansom, F.M.; Tivendale, K.A. Mycoplasma bovis mbfN Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components. J. Bacteriol. 2020, 203, e00154-20. [Google Scholar] [CrossRef] [PubMed]
- Huang, J.; Zhu, H.; Wang, J.; Guo, Y.; Zhi, Y.; Wei, H.; Li, H.; Guo, A.; Liu, D.; Chen, X. Fructose-1,6-bisphosphate aldolase is involved in Mycoplasma bovis colonization as a fibronectin-binding adhesin. Res. Vet. Sci. 2019, 124, 70–78. [Google Scholar] [CrossRef]
- Gao, X.; Bao, S.; Xing, X.; Fu, X.; Zhang, Y.; Xue, H.; Wen, F.; Wei, Y. Fructose-1,6-bisphosphate aldolase of Mycoplasma bovis is a plasminogen-binding adhesin. Microb. Pathog. 2018, 124, 230–237. [Google Scholar] [CrossRef]
- Guo, Y.; Zhu, H.; Wang, J.; Huang, J.; Khan, F.A.; Zhang, J.; Guo, A.; Chen, X. TrmFO, a fibronectin-binding adhesin of Mycoplasma bovis. Int. J. Mol. Sci. 2017, 18, 1732. [Google Scholar] [CrossRef]
- Adamu, J.Y.; Wawegama, N.K.; Condello, A.K.; Marenda, M.S.; Markham, P.F.; Browning, G.F.; Tivendale, K.A. Mycoplasma bovis membrane protein MilA is a multifunctional lipase with novel lipid and glycosaminoglycan binding activity. Infect. Immun. 2020, 88, e00945-19. [Google Scholar] [CrossRef]
- Chen, X.; Huang, J.; Zhu, H.; Guo, Y.; Khan, F.A.; Menghwar, H.; Zhao, G.; Guo, A. P27 (MBOV_RS03440) is a novel fibronectin binding adhesin of Mycoplasma bovis. Int. J. Med. Microbiol. 2018, 308, 848–857. [Google Scholar] [CrossRef]
- Liu, S.; Li, Z.; Lan, S.; Hao, H.; Jin, X.; Liang, J.; Baz, A.A.; Yan, X.; Gao, P.; Chen, S.; et al. LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2. Vet. Res. 2023, 54, 107. [Google Scholar] [CrossRef]
- Lan, S.; Li, Z.; Hao, H.; Liu, S.; Huang, Z.; Bai, Y.; Li, Y.; Yan, X.; Gao, P.; Chen, S. A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells. FASEB J. 2023, 37, 19. [Google Scholar] [CrossRef]
- Zou, X.; Li, Y.; Wang, Y.; Zhou, Y.; Xin, J. Molecular Cloning and Characterization of a Surface-Localized Adhesion Protein in Mycoplasma bovis Hubei-1 Strain. PLoS ONE 2013, 8, e69644. [Google Scholar] [CrossRef] [PubMed]
- Sachse, K.; Grajetzki, C.; Rosengarten, R.; Hänel, I.; Pfützner, H. Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells. Zentralblatt Bakteriol. Int. J. Med. Microbiol. 1996, 284, 80–92. [Google Scholar] [CrossRef] [PubMed]
- Thomas, A.; Sachse, K.; Farnir, F.; Dizier, I.; Mainil, J.; Linden, A. Adherence of Mycoplasma bovis to bovine bronchial epithelial cells. Microb. Pathog. 2003, 34, 141–148. [Google Scholar] [CrossRef]
- Sachse, K.; Helbig, J.H.; Lysnyansky, I.; Grajetzki, C.; Müller, W.; Jacobs, E.; Yogev, D. Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins. Infect. Immun. 2000, 68, 680–687. [Google Scholar] [CrossRef]
- Zhu, X.; Dong, Y.; Baranowski, E.; Li, X.; Zhao, G.; Hao, Z.; Zhang, H.; Chen, Y.; Hu, C.; Chen, H.; et al. Mbov_0503 encodes a novel cytoadhesin that facilitates Mycoplasma bovis interaction with tight junctions. Microorganisms 2020, 8, 164. [Google Scholar] [CrossRef]
- Thomas, A.; Leprince, P.; Dizier, I.; Ball, H.; Gevaert, K.; Damme, J.V.; Mainil, J.; Linden, A. Identification by two-dimensional electrophoresis of a new adhesin expressed by a low-passaged strain of Mycoplasma bovis. Res. Microbiol. 2005, 156, 713–718. [Google Scholar] [CrossRef] [PubMed]
- Zhao, P.; He, Y.; Zhang, X.; Zhang, N.Z.; Zhang, K.S. Identification of novel immunogenic proteins in Mycoplasma capricolum subsp. capripneumoniae strain M1601. J. Vet. Med. Sci. 2012, 74, 1109–1115. [Google Scholar] [CrossRef]
- Poukkula, M.; Kremneva, E.; Serlachius, M.; Lappalainen, P. Actin-depolymerizing factor homology domain: A conserved fold performing diverse roles in cytoskeletal dynamics. Cytoskeleton 2011, 68, 471–490. [Google Scholar] [CrossRef]
- Snyder, B.N.; Cho, Y.J.; Qian, Y.; Coad, J.E.; Flynn, D.C.; Cunnick, J.M. AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes. Eur. J. Cell Biol. 2011, 90, 376–389. [Google Scholar] [CrossRef] [PubMed]
- Li, Y.; Ma, H.; Shi, C.; Feng, F.; Yang, L. Mutant ACTB mRNA 3′-UTR promotes hepatocellular carcinoma development by regulating miR-1 and miR-29a. Cell. Signal. 2019, 67, 109479. [Google Scholar] [CrossRef]
- Hackett, E.E.; Charles-Messance, H.; Leary, S.M.; Gleeson, L.E.; Sheedy, F.J. Mycobacterium tuberculosis limits host glycolysis and IL-1β by restriction of PFK-M via mricroRNA-21. Cell Rep. 2020, 30, 124–136.e124. [Google Scholar] [CrossRef] [PubMed]
- Cao, S.; Han, X.; Deng, X.; Guo, J.; Liu, L.; Zhang, Y.; Suleimenov, M.; Zhao, T.; Li, W.; Ding, J.; et al. Brucella secretory protein VceA promotes FOXO1 entry into the nucleus to shift host cell metabolism toward glycolysis. Acta Biochim. Biophys. Sin. 2024, 57, 805. [Google Scholar]
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Li, R.-R.; Yu, X.-J.; Liang, J.-Y.; Sheng, J.-L.; Zhang, H.; Chen, C.-F.; Ma, Z.-C.; Wang, Y. ENO1 from Mycoplasma bovis Disrupts Host Glycolysis and Inflammation by Binding ACTB. Biomolecules 2025, 15, 1107. https://doi.org/10.3390/biom15081107
Li R-R, Yu X-J, Liang J-Y, Sheng J-L, Zhang H, Chen C-F, Ma Z-C, Wang Y. ENO1 from Mycoplasma bovis Disrupts Host Glycolysis and Inflammation by Binding ACTB. Biomolecules. 2025; 15(8):1107. https://doi.org/10.3390/biom15081107
Chicago/Turabian StyleLi, Rui-Rui, Xiao-Jiao Yu, Jia-Yin Liang, Jin-Liang Sheng, Hui Zhang, Chuang-Fu Chen, Zhong-Chen Ma, and Yong Wang. 2025. "ENO1 from Mycoplasma bovis Disrupts Host Glycolysis and Inflammation by Binding ACTB" Biomolecules 15, no. 8: 1107. https://doi.org/10.3390/biom15081107
APA StyleLi, R.-R., Yu, X.-J., Liang, J.-Y., Sheng, J.-L., Zhang, H., Chen, C.-F., Ma, Z.-C., & Wang, Y. (2025). ENO1 from Mycoplasma bovis Disrupts Host Glycolysis and Inflammation by Binding ACTB. Biomolecules, 15(8), 1107. https://doi.org/10.3390/biom15081107