A multifunctional enolase mediates cytoadhesion and interaction with host plasminogen and fibronectin in Mycoplasma hyorhinis

In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread ofM. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. RecombinantM. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4  nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collec tively, these results imply that enolase moonlights as an important adhesin ofM. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.
Source: Veterinary Research - Category: Veterinary Research Source Type: research