title: Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.
abstract: This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca(2+)-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca(2+) at 22-65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκβα and Bcl-2. In contrast, rMbovNase(Δ181-342) without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. dkyzhanghui@163.com.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. dkyzhanghui@163.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. zhaog0524@163.com.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. zhaog0524@163.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. guoyusi0626@163.com.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. guoyusi0626@163.com.
authors.forename: Yusi
authors.initials: Y
authors.lastname: Guo
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. drharishsanja@hotmail.com.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. drharishsanja@hotmail.com.
authors.forename: Harish
authors.initials: H
authors.lastname: Menghwar
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. chenyingyu@mail.hzau.edu.cn.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. chenyingyu@mail.hzau.edu.cn.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Wuhan 430070, China. chenyingyu@mail.hzau.edu.cn.|Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology, Huazhong Agricultural University, Wuhan 430070, China. chenyingyu@mail.hzau.edu.cn.
authors.forename: Yingyu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. chenhch@mail.hzau.edu.cn.|College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. chenhch@mail.hzau.edu.cn.|Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Wuhan 430070, China. chenhch@mail.hzau.edu.cn.|Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology, Huazhong Agricultural University, Wuhan 430070, China. chenhch@mail.hzau.edu.cn.
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural 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, 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: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D065206:Extracellular Traps / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D045682:Mycoplasma bovis / Q000201:enzymology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D013211:Staphylococcus aureus / Q000201:enzymology; D051996:Transcription Factor RelA / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D051996:Transcription Factor RelA; D004247:DNA; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D003851:Deoxyribonucleases; D008836:Micrococcal Nuclease
keywords: MBOV_RS02825; Mycoplasma bovis; TNASE_3 domain; apoptosis; binding and internalization; neutrophil extracellular traps (NETs); nuclease; secretory protein