Prokaryotic expression of Mycoplasma bovis LRR5 protein involved in the invasion of Mycoplasma bovis into host cells

Abstract:

Pathogenic mycoplasma has the ability to invade host cells, which is a key mechanism to their pathogenicity. Functional proteins that mediate the entry of mycoplasma may serve as potential drug or vaccine targets. [Objective] To clone and express the LRR5 protein encoded by MBOVPG45_0564 in Mycoplasma bovis and to explore its role in the invasion of host cells by M. bovis. [Methods] The NCBI database was used for the homology analysis of MBOVPG45_0564, and the structure of LRR5 protein was predicted by the Discovery Studio Client system. After prokaryotic expression of LRR5 protein, the mouse polyclonal antibody was prepared, and the subcellular localization of LRR5 protein was observed by immunoelectron microscopy. The invasion of M. bovis into embryonic bovine lung (EBL) cells after LRR5 antibody blocking was observed by plate counting and laser confocal microscopy. After conjugation of LRR5 protein to the surface of fluorescent microspheres, the entry of the microspheres into EBL cells was observed by laser confocal microscopy and a high-content live-cell imaging system. [Results] MBOVPG45_0564 was a conserved sequence in M. bovis and encoded LRR5, a membrane-associated protein with a typical crescent-like spatial conformation. Multiple repeating leucine motifs were assembled in a supercoiled manner to form a solenoid protein structural unit. After LRR5 antibody blocking, the invasion rate of M. bovis to EBL cells reduced (P<0.05), and the fluorescent microspheres conjugated with LRR5 protein could successfully enter EBL cells. [Conclusion] The LRR5 protein encoded by MBOVPG45_0564 is localized on the M. bovis membrane and plays a role in the invasion of M. bovis into host cells.