Screening and functional validation of host interacting proteins for the key invasion protein LRR5 of Mycoplasma bovis.

Mycoplasma bovis induces a range of inflammatory conditions in cattle, causing substantial economic losses to the global cattle industry. The pathogen employs adhesion and invasion mechanisms to evade host immune responses, enabling long-term infection. Therefore, identifying invasion-related virulence factors and their potential host receptors is crucial for elucidating the pathogenic mechanisms of M. bovis and developing targeted control strategies. However, the virulence factors mediating M. bovis invasion, their corresponding host receptors, and the regulatory mechanisms of their interaction during invasion remain unclear. Given this knowledge gap, we hypothesize that M. bovis utilizes the virulence factor LRR5 to bind candidate host receptors, and that this interaction is a key regulatory factor for M. bovis invasion into host cells. Inhibition of host receptor expression leads to partial inhibition of M. bovis invasion. In this study, we used immunoprecipitation-mass spectrometry (IP-MS), we have identified vimentin (VIM) as a candidate host protein. Molecular docking via Discovery Studio Client predicted interaction sites between LRR5 and VIM, and LigPlus software visualized key binding residues. There might be 5 pairs of interacting amino acid combinations between the LRR5 and VIM proteins. Among them, LRR5 has 4 key amino acids, and VIM has 3 key amino acids. The pull-down assay conducted in vitro and the confocal microscopy experiment performed within EBL cells further confirmed the specific interaction between LRR5 and VIM. Functional validation through siRNA-mediated knockdown of VIM, combined with plate counting assays, demonstrated that reduced VIM expression inhibited the partial ability of PG45 to invade EBL cells. (P < 0.01). These results suggest that VIM could be a potential host receptor mediating M. bovis invasion, invasion providing a scientific basis for developing novel strategies, such as vaccines, to control M. bovis infections.