Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis.
Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host's homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn(2+), and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development.
Acosta
Daniel
D
Unidad de Biología Parasitaria, Instituto de Higiene, Facultad de Ciencias, Uruguay.
Cancela
Martín
M
Piacenza
Lucia
L
Roche
Leda
L
Carmona
Carlos
C
Tort
José F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology* / Q000517:prevention & control; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005210:Fasciola hepatica / Q000201:enzymology* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D005211:Fascioliasis / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D015870:Gene Expression; D007074:Immunoglobulin G / Q000097:blood; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000276:immunology*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011817:Rabbits; D012547:Schistosoma / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D000909:Antibodies, Helminth; D002413:Cations, Divalent; D003374:Coumarins; D018076:DNA, Complementary; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D008670:Metals; C068949:7-leucylamido-4-methylcoumarin; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex; D003545:Cysteine