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

Abstract

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.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

“…M. pneumoniae strain M129 (ATCC 29342) and Escherichia coli strains NovaBlue (Novagen, Darmstadt, Germany), BL21(DE3) (Novagen), and E. cloni (Lucigen, Middleton, WI), as well as A549 cells (human lung carcinoma cell line, ATCC CCL-185), were grown as described previously (25). The protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford, IL).…”

“…The animal experiments were approved by the ethical board of Landesdirektion Sachsen, Dresden, Germany (permit 24-9168.25-1/2011-1). Intranasal infection of guinea pigs with freshly harvested mycoplasmas, subcutaneous immunization with total proteins of M. pneumoniae and with recombinant proteins, booster immunization, and serum collection were performed as described previously (25).…”

“…Wells of 96-well plates were coated with the membrane and cytosolic protein fractions (15 g/ml) as described previously, incubated with the corresponding antisera against the glycolytic enzymes (1:250 each), and detected with peroxidase-conjugated secondary antibody (1:1,000). Sera to Eno of M. pneumoniae as cytosolic protein (25,26) and to C-terminal part (rP12) of the main P1 adhesin as membrane-associated protein (38) acted as controls. Additionally, guinea pig serum to total proteins of M. pneumoniae was included.…”

“…Furthermore, PdhB, one of the proteins that interact with various host factors (11), is also able to bind human fibronectin (27). Interestingly, PdhD and Eno of M. pneumoniae were also reported as binding partners of human Plg, but they do not occur on the surfaces of the bacteria (25,26). In addition to PdhA, PdhB, and GapA, other glycolytic enzymes such as lactate dehydrogenase (Ldh) are detected in the Triton X-100-insoluble fraction that represents the membrane-associated proteins of M. pneumoniae (28).…”

“…In M. pneumoniae, GapA was reported as a surface-associated protein that can bind human fibrinogen (24). Additionally, pyruvate dehydrogenase (Pdh) subunits A to C were found on the cell surface interacting with human Plg (25,26). In the case of PdhB, the protein complex activates Plg, resulting in the degradation of human fibrinogen (26).…”