Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) is an anchorless, multifunctional protein displayed on the surface of several fungi and Gram-positive pathogens, which contributes to their adhesion and virulence. To date a role for extracellular GAPDH in the pathogenesis of Gram-negative bacteria has not been described. The aim of this study was to analyze the extracellular localization of GAPDH in enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains and to examine its interaction with host components that could be related to the infection mechanism. Recombinant E. coli GAPDH was purified and polyclonal antibodies were obtained. Western blotting and immunoelectron microscopy showed that GAPDH is located on the bacterial surface and released to the culture medium of EHEC and EPEC strains. GAPDH export in these Gram-negative pathogens depends on the external medium, is not mediated by vesicles and leads to an extracellular active enzyme. Non-pathogenic E. coli strains do not secrete GAPDH. Two-dimensional electrophoresis analysis showed that in E. coli GAPDH is present at least in two major forms with different isoelectric points. Of these forms, the more basic is secreted. Purified GAPDH was found to bind human plasminogen and fibrinogen in Far-Western blot and ELISA-based assays. In addition, GAPDH remained associated with colonic Caco-2 epithelial cells after adhesion of EHEC or EPEC. These observations indicate that exported GAPDH may act as a virulence factor which could contribute to EHEC and EPEC pathogenesis. This is the first description of an extracellular localization for this enzyme, with a function other than its glycolytic role in Gram-negative pathogens.
Egea
L
L
Department of Biochemistry, School of Pharmacy, University of Barcelona, Av. Diagonal, 643. E-08028-Barcelona, Spain.
Aguilera
L
L
Giménez
R
R
Sorolla
M A
MA
Aguilar
J
J
Badía
J
J
Baldoma
L
L
D000595:Amino Acid Sequence; D015153:Blotting, Western; D018938:Caco-2 Cells; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005340:Fibrinogen / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014774:Virulence / Q000235:genetics
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005340:Fibrinogen; D010958:Plasminogen; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases