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.

Nine potential (fatty) alcohol dehydrogenase genes and one alcohol oxidase gene were identified in Yarrowia lipolytica by comparative sequence analysis. All relevant genes were deleted in Y. lipolytica H222ΔP which is lacking β-oxidation. Resulting transformants were tested for their ability to accumulate ω-hydroxy fatty acids and dicarboxylic acids in the culture medium. The deletion of eight alcohol dehydrogenase genes (FADH, ADH1-7), which may be involved in ω-oxidation, led only to a slightly increased accumulation of ω-hydroxy fatty acids, whereas the deletion of the fatty alcohol oxidase gene (FAO1), which has not been described yet in Y. lipolytica, exhibited a considerably higher effect. The combined deletion of the eight (fatty) alcohol dehydrogenase genes and the alcohol oxidase gene further reduced the formation of dicarboxylic acids. These results indicate that both (fatty) alcohol dehydrogenases and an alcohol oxidase are involved in ω-oxidation of long-chain fatty acids whereby latter plays the major role. This insight marks the first step toward the biotechnological production of long-chain ω-hydroxy fatty acids with the help of the nonconventional yeast Y. lipolytica. The overexpression of FAO1 can be further used to improve existing strains for the production of dicarboxylic acids.

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate D-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. Members of the class Mollicutes are known to be the smallest and simplest self-replicating prokaryotes. Among them, Mycoplasma pneumoniae is a common pathogen in humans. The cell wall-less bacterium causes infections of the respiratory tract, including severe interstitial pneumonia (1), and usually accounts for 5 to 10% of all cases of community-acquired pneumonia among pediatric (2) and adult (3) patients. Because of the timedependent incidence of infections, Ͼ25% of pneumonia cases in epidemic periods are attributed to this agent (4). In addition, a number of extrapulmonary manifestations and complications are described, affecting mainly the skin and the central nervous system (5).Due to the greatly reduced genome of M. pneumoniae (816 kbp, 689 open reading frames [ORFs]), the metabolic pathways and the repertoire of virulence factors are limited (6). However, M. pneumoniae has developed effective strategies to infect humans and allow long-term survival in the respiratory mucosa. These include the targeted adherence of the bacteria to epithelial cells via a specialized complex of adhesins and adhesion-related proteins (7) as the first step in colonization,...