title: The Mycoplasma gallisepticum α-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen.
abstract: The α-enolase protein is reported to be an adhesin in several pathogenic bacterial species, but its role in Mycoplasma gallisepticum is unknown. In this study, the M. gallisepticum α-enolase gene was adapted to heterologous expression in Escherichia coli by performing overlapping polymerase chain reaction with site-directed mutagenesis to introduce A960G and A1158G mutations in the nucleotide sequence. The full-length mutated gene was cloned into a pGEM-T Easy vector and subcloned into the expression vector pET32a(+) to construct the pET-rMGEno plasmid. The expression of rMGEno in E. coli strain DE3 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGEno exhibited α-enolase catalytic activity that it could reflect the conversion of NADH to NAD(+). Mouse antiserum to α-enolase was generated by immunization with rMGEno. Immunoblotting and immunofluorescence assay with the antiserum identified α-enolase on the surface of M. gallisepticum cells. Enzyme-linked immunosorbent assay characterized rMGEno as a chicken plasminogen binding protein. An adherence inhibition assay on immortalized chicken fibroblasts (DF-1) demonstrated more than 77% inhibition of adhesion in the presence of mouse antiserum, suggesting that α-enolase of M. gallisepticum participates in bacterial adhesion to DF-1 cells.
authors.affiliation: Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Chen
authors.forename: Shengqing
authors.initials: S
authors.lastname: Yu
authors.forename: Xinyue
authors.initials: X
authors.lastname: Shen
authors.forename: Danqing
authors.initials: D
authors.lastname: Chen
authors.forename: Xvsheng
authors.initials: X
authors.lastname: Qiu
authors.forename: Cuiping
authors.initials: C
authors.lastname: Song
authors.forename: Chan
authors.initials: C
authors.lastname: Ding
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000907:Antibodies, Bacterial; D001422:Bacterial Adhesion; D002478:Cells, Cultured; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000382:microbiology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D045686:Mycoplasma gallisepticum / Q000201:enzymology* / Q000472:pathogenicity*; D009243:NAD / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D008565:Membrane Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D009243:NAD; D010958:Plasminogen; D010751:Phosphopyruvate Hydratase