title: Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2reducing enzyme and an adhesin.
abstract: Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+and reduce O2to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovisNOX-expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovisNOX-for cell adhesion and H2O2production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Xifang
authors.initials: X
authors.lastname: Zhu
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Yusi
authors.initials: Y
authors.lastname: Guo
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Chenfei
authors.initials: C
authors.lastname: He
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Farhan
authors.initials: F
authors.lastname: Anwar Khan
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Yingyu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.
authors.forename: Changmin
authors.initials: C
authors.lastname: Hu
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, China.|Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan, 430070, China. aizhen@mail.hzau.edu.cn.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China. aizhen@mail.hzau.edu.cn.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, China. aizhen@mail.hzau.edu.cn.|Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, 430070, China. aizhen@mail.hzau.edu.cn.
authors.forename: Aizhen
authors.initials: A
authors.lastname: Guo
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D002417:Cattle; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D045682:Mycoplasma bovis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*
chemical_list: D018829:Adhesins, Bacterial; D005353:Fibronectins; D009097:Multienzyme Complexes; D009243:NAD; D006861:Hydrogen Peroxide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D010100:Oxygen