title: Mycoplasma gallisepticum MGA_0676 is a membrane-associated cytotoxic nuclease with a staphylococcal nuclease region essential for nuclear translocation and apoptosis induction in chicken cells.
abstract: Mycoplasma gallisepticum can infect a wide variety of birds including the commercial poultry. M. gallisepticum MGA_0676 is a putative lipoprotein, which is similar to bacterial thermostable nucleases. But the possible pathogenic effect of M. gallisepticum MGA_0676 has not been investigated so far. In the present study, we cloned the MGA_0676 gene after deletion of the amino-terminal signal sequence and mutagenesis of the Mycoplasma TGA tryptophan codons to TGG and expressed recombinant MGA_0676 protein in Escherichia coli. We identified and characterized MGA_0676 as a Ca(2+)-dependent cytotoxic nuclease of M. gallisepticum with a staphylococcal nuclease (SNc) region that displays the hallmarks of nucleases. Membrane protein immunoblot analysis and immunogold electron microscopy revealed that MGA_0676 locates on the membrane surface of M. gallisepticum. Furthermore, apoptosis assay using annexin V-FITC and propidium iodide (annexin V/PI) indicated that MGA_0676 played significant roles in apoptosis induction and pathological damages in chicken cells. Moreover, confocal microscopy showed that MGA_0676 localizes in the nuclei of host cells. Besides, after the SNc region was deleted, MGA_0676 lost its ability of nuclear localization, nuclease activity, and cytotoxicity, which revealed that the SNc region is essential for nuclear translocation and induction of apoptosis in chicken cells. The above results suggest that MGA_0676 is an important virulence factor in cellular pathology and may play a unique role in the life cycle events of M. gallisepticum.
authors.affiliation: Laboratory of Rapid Diagnostic Technology for Animal Disease, College of Veterinary Medicine, China Agricultural University, 100193, Beijing, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.forename: Fei
authors.initials: F
authors.lastname: Jiang
authors.forename: Yuewei
authors.initials: Y
authors.lastname: Zhang
authors.forename: Saeed A
authors.initials: SA
authors.lastname: El-Ashram
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Sun
authors.forename: Jinyan
authors.initials: J
authors.lastname: He
authors.forename: Junjun
authors.initials: J
authors.lastname: Shen
authors.forename: Wenxue
authors.initials: W
authors.lastname: Wu
authors.forename: Jinxiang
authors.initials: J
authors.lastname: Li
mesh_terms: D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002645:Chickens; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D045686:Mycoplasma gallisepticum / Q000201:enzymology*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D037521:Virulence Factors; D003851:Deoxyribonucleases