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

Abstract

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,...

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