Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae.

The obligate pathogenic mycoplasma species Mycoplasma pneumoniae uses a limited but effective repertoire of virulence factors to infect and colonize the human respiratory tract. Besides the development of a unique adhesion complex and the expression of tissue-damaging factors, surface-located glycolytic enzymes and their capacity to bind to components of the human extracellular matrix (ECM) support pathogen-host interactions. Here, we demonstrated that the glycolytic enzymes enolase (Mpn606) and pyruvate dehydrogenase subunit B (Mpn392; PDHB) of M. pneumoniae show concentration-dependent binding to human plasminogen. Monospecific polyclonal antisera against both recombinant proteins reduced the binding to plasminogen significantly. The surface location of PDHB but not of enolase was demonstrated using Triton X fractionation of M. pneumoniae total protein content, membrane fractionation, colony blotting, mild proteolysis of mycoplasma cells, and immunofluorescence tests. To characterize the binding site of plasminogen in surface-displaced PDHB, the mycoplasmal protein was separated into four recombinant proteins followed by investigation of the binding behaviour of peptides that overlap the protein part interacting with plasminogen. Spot analysis resulted in a novel region of 12 amino acids (FPAMFQIFTHAA, position 91 to 102 of PDHB), which is responsible exclusively for binding of human plasminogen and also interacts in a dose-dependent manner with this host protein. The data indicate that the plasminogen-binding enzymes enolase and especially the surface-associated PDHB may contribute to the pathogenesis of M. pneumoniae infections.