title: A recombinant enolase from Anisakis simplex is differentially recognized in natural human and mouse experimental infections.
abstract: A 1,963-bp cDNA was isolated from an Anisakis simplex cDNA library by immunoscreening with a hyperimmune rabbit serum raised against a crude extract of A. simplex L3 larvae. The open reading frame encodes a putative protein of 436 amino acid residues, which exhibits high similarity (70-80%) to enolase molecules from various other organisms, including helminth parasites. After subcloning and expression of the A. simplex cDNA in PGEX-4T-3, the resulting glutathione S-transferase fusion protein, purified by glutathione-Sepharose-4B chromatography, showed functional enolase activity. The immunogenicity of the recombinant A. simplex enolase was analyzed by immunoblotting using sera obtained from (a) mice immunized with crude extracts (CE) of A. simplex, or other nematode species, (b) mice immunized with excretory-secretory (ES) antigens from A. simplex, or (c) mice infected with L3 larvae by the intraperitoneal route. In addition, we used ELISA, to investigate the presence of IgG1 and IgE antibodies against this molecule in sera from patients infected with A. simplex. Mouse sera obtained after infection with L3 or raised against CE antigens, but not sera raised against ES antigens, showed strong reactivity with the recombinant A. simplex enolase. We also obtained good reactivity in Western blotting with sera from mice immunized with CE antigens from Ascaris suum and Toxocara canis, but not with sera from mice immunized with CE antigens from Trichuris muris, Trichinella spiralis or Hysterothylacium aduncum. In contrast to the experimental infections/immunizations in mice, we were unable to detect anti-enolase IgE antibodies in sera from human patients infected with A.simplex (15 sera), and the levels of anti-enolase IgG1 antibodies in these sera were low and apparently nonspecific. These results seem to indicate that, during natural infection in humans, A. simplex larvae do not offer sufficient antigenic stimulus to induce anti-enolase antibodies.
authors.affiliation: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda Pozuelo Km 2,2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Esperanza
authors.initials: E
authors.lastname: Rodríguez
authors.forename: Fernanda
authors.initials: F
authors.lastname: Romarís
authors.forename: Sonia
authors.initials: S
authors.lastname: Lorenzo
authors.forename: Javier
authors.initials: J
authors.lastname: Moreno
authors.forename: Pedro
authors.initials: P
authors.lastname: Bonay
authors.forename: Florencio M
authors.initials: FM
authors.lastname: Ubeira
authors.forename: Teresa
authors.initials: T
authors.lastname: Gárate
mesh_terms: D000818:Animals; D017129:Anisakiasis / Q000276:immunology*; D017163:Anisakis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D000909:Antibodies, Helminth / Q000097:blood; D001200:Ascaris / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D007814:Larva / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D017166:Toxocara canis / Q000276:immunology; D017160:Trichinella spiralis / Q000276:immunology; D014258:Trichuris / Q000276:immunology
chemical_list: D000909:Antibodies, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E; D010751:Phosphopyruvate Hydratase