Immunogenic and plasminogen-binding surface-associated alpha-enolase of Trichomonas vaginalis.
Trichomonas vaginalis is a protist that causes the most common human sexually transmitted infection. A T. vaginalis cDNA expression library was screened with pooled sera from patients with trichomoniasis. A highly reactive cDNA clone of 1,428 bp encoded a trichomonad protein of 472 amino acids with sequence identity to alpha-enolase (tv-eno1). The sequence alignment confirmed the highly conserved nature of the enzyme with 65% to 84% identity among organisms. The expression of tv-eno1 was up-regulated by contact of parasites with vaginal epithelial cells, and this is the first report demonstrating up-regulation by cytoadherence of a plasminogen-binding alpha-enolase in T. vaginalis. Immunofluorescence with monoclonal antibody of nonpermeabilized trichomonads showed tv-ENO1 on the surface. The recombinant tv-ENO1 was expressed in Escherichia coli as a glutathione S-transferase (GST)::tv-ENO1 fusion protein, which was cleaved using thrombin to obtain affinity-purified recombinant tv-ENO1 protein (tv-rENO1) detectable in immunoblots by sera of patients. Immobilized tv-rENO1 bound human plasminogen in a dose-dependent manner, and plasminogen binding by tv-rENO1 was confirmed in a ligand blot assay. The plasminogen-specific inhibitor epsilon-aminocaproic acid blocked the tv-rENO1-plasminogen association, indicating that lysines play a role in binding to tv-rENO1. Further, both parasites and tv-rENO1 activate plasminogen to plasmin that is mediated by tissue plasminogen activator. These data indicate that as with other bacterial pathogens, tv-ENO1 is an anchorless, surface-associated glycolytic enzyme of T. vaginalis.
Mundodi
V
V
Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Kucknoor
A S
AS
Alderete
J F
JF
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004847:Epithelial Cells / Q000469:parasitology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014246:Trichomonas vaginalis / Q000737:chemistry / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010958:Plasminogen; D010751:Phosphopyruvate Hydratase