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Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

ABSTRACT: The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen.

SUBMITTER: Grundel A

PROVIDER: S-EPMC4433191 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

Gründel Anne A Friedrich Kathleen K Pfeiffer Melanie M Jacobs Enno E Dumke Roger R

PloS one 20150515 5

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhes ...[more]

PMID: 25978044

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Project description:Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. Cytoadherence is the critical stage for mycoplasma infection, and the associated proteins are important for mycoplasma pathogenesis. Many glycolytic enzymes are localized on the cell surface and can bind the extracellular matrix of host cells. In this study, the M. gallisepticum pyruvate dehydrogenase E1 alpha subunit (PDHA) and beta subunit (PDHB) were expressed in Escherichia coli, and their enzymatic activities were identified based on 2,6-dichlorophenol indophenol reduction. When recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were mixed at a 1:1 molar ratio, they exhibited strong enzymatic activity. Alone, rPDHA and rPDHB exhibited no or weak enzymatic activity. Further experiments indicated that both PDHA and PDHB were surface-exposed immunogenic proteins of M. gallisepticum. Bactericidal assays showed that the mouse anti-rPDHA and anti-rPDHB sera killed 48.0% and 75.1% of mycoplasmas respectively. A combination of rPDHA and rPDHB antisera had a mean bactericidal rate of 65.2%, indicating that rPDHA and rPDHB were protective antigens, and combining the two sera did not interfere with bactericidal activity. Indirect immunofluorescence and surface display assays showed that both PDHA and PDHB adhered to DF-1 chicken embryo fibroblast cells and adherence was significantly inhibited by antisera against PDHA and PDHB. Adherence inhibition of M. gallisepticum to DF-1 chicken embryo fibroblast cells was 30.2% for mouse anti-rPDHA serum, 45.1% for mouse anti-rPDHB serum and 72.5% for a combination of rPDHA and rPDHB antisera, suggesting that rPDHA and rPDHB antisera may have synergistically interfered with M. gallisepticum cytoadherence. Plasminogen (Plg)-binding assays further demonstrated that both PDHA and PDHB were Plg-binding proteins, which may have contributed to bacterial colonization. Our results clarified the enzymatic activity of M. gallisepticum PDHA and PDHB and demonstrated these compounds as Plg-binding proteins involved in cytoadherence.

Dataset Information

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

Publications

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

Similar Datasets

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