Adhesion Ability Study Of The Surface-localized Protein VpmaX In Mycoplasma Bovis Hubei-1

As a member of Moliicutes, Mycoplama bovis (M.bovis) can cause persistent pneumonia, arthritis, andmastitis in young calves. M. bovis infection is increasingly pervasive in China, and the strain M. bovisHubei-1was first isolated in the Hubei province of China, making it became the most importantpathogen that threatens the cattle industry.Because adhesion to the host cell is a prerequisite for the colonization and infection of the host, theidentification of adhesion proteins in pathogens is important for understanding the mechanisms of itspathogenesis. So far some surface proteins and lipoproteins in mycoplasmas have been identified andimplicated to play roles in cell adherence in Mycoplasma pneumonia and Mycoplasma agalactiae. Wescreened the entire genome of M.bovis Hubei-1and found a gene vpmaX, whose encoded protein mayhave the ability to adhere host cells.The690bp M.bovis Hubei-1vpmaX encodes a25kDa protein vpmaX, who wato GeneBank, but it is absolutely differently from the M.bovis PG45VspA protein. Sequence analysis revealed that vpmaX contains several repetitive units and a typicalbacterial lipoprotein signal sequence. The vpmaX gene was cloned and expressed in E. coli to obtainrecombinant vpmaX (rvpmaX). Western blot analysis using a rabbit antibody against rvpmaXdemonstrated that vpmaX is a membrane protein. Immunostaining visualized via confocal laserscanning microscopy showed that rvpmaX was able to adhere to fixed embryonic bovine lung cells(EBL), and this was also confirmed by a sandwich ELISA, devised to investigate interaction betweenrvpmaX and EBL membrane and cytoplasmic protein fractions.Antigenic variation occurred among Vsps of the M.bovis PG45. We found M. bovis grown in brothcontaining anti-rvpmaX serum for five passage expressed a new protein with a molecular mass of55kDa. Meanwhile, the vpmaX and the new observed55kDa protein still expressed even the organism wascultured under immune pressure for ten passages.The Vsps gene cluster exists in M.bovis PG45and HB0801, but it was completely deleted in M.bovisHubei-1. Our study found the vpmaX in Hubei-1shared some traits of Vsp family of PG45. Moreover,the adhesion assay verify that the vpmaX is an adhesion protein in M.bovis Hubei-1and it alsopossesses some ability of antigenic variation.