α-Enolase, An Adhesion-related Factor Of Mycoplasma Bovis

Mycoplasmas, unlike bacteria, lack rigid cell walls and are bound to the host cell by the plasmamembrane. The adherence of mycoplasmas to the host cell initiates infection. Recently, there is a lot ofresearch on the mycoplasma surface adhesion-related fact, but how Mycoplasma adhere to the surfaceof host cell surface is not clear. So we do the research on the Mycoplasma bovis (M.bovis) surfaceadhesion-related fact.Recently, the α-enolase, has been found on many bacteria and it is closely related to bacterialadherence to the host cell. Therefore, we hypothesized that α-enolase exists in the surface of M.bovis,and M.bovis surface α-enolase is a plasminogen binding protein. Through analysising the whole genomesequence of M.bovis (Huibei), we found that α-enolase gene does exist in the genome of M.bovis, whichcontains two typical plasminogen-binding-site motifs of α-enolase. So we speculated that α-enolase ofM.bovis is a plasminogen-binding protein, which is related to adhersion of M.bovis to host cells. Thisspeculation should be further validated by experimentsTo further validate our speculation, the recombinant protein of M.bovis α-enolase (rMbEno), had amolecular weight of69kDa, was obtained by applying prokaryotic expression system. The purifiedrMbEno was used to generate rabbit polyclonal antibodies. This analysis, using anti-rMbEno antibodies,revealed a strong reactivity to a protein of approximately49kDa that is found in the whole, membraneand soluble cytosolic protein fractions, suggesting that MbEno is present in both membrane and solublecytosolic proteins of Mb cells. By the ligand blotting assays, we discovered that both themembrane-fraction proteins and the soluble cytosolic-fraction proteins of M. bovis, including α-enolase,interacted with Plg. Thus, more than two different M. bovis membrane-fraction proteins can bind Plg.Then, ELISA was performed to further confirm that enolase is involved in M. bovis binding to Plg.Adherence and inhibition of M. bovis was determined by a bacteriological assay. In this assay, M.bovis adherence to EBE cells was observed by Confocal laser scanning microscopy (CLSM), and theadherence and inhibition values was calculated by the presence of Plg and anti-rMbEno antibody. Tofurther validate the adherence and inhibition, the adherence and inhibition values were calculated. Theresult shows that after Plg pre-incubation, M. bovis adhered EBL cells rates was enhanced by11.9%.And the anti-enolase antibody that inhibited adherence was more effective than nonimmune rabbitantibody (P <0.05), when the EBL cells were treated with Plg.M. bovis α-enolase both the recombinant and the native proteins were subjected to aplasminogen-binding in vitro assay. And we demonstrate the ability of M. bovis α-enolase to bind Plgcould be of great importance for its establishment in the host, allowing the adhesion to the EBL. Wespeculate that α-enolase may be involved in various clinical and pathologic sequelae of M. bovis, suchas arthritis, tenosynovitis and meningitis.