RESUMO
Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture teleosts and cause high lethality and economic loss, especially Larimichthys crocea. Current methods of controlling or preventing this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. The antiparasitic activity of some antimicrobial peptides (AMPs) attracted extensive attention of scholars. In the study, a novel piscidin 5-like type 4 (termed Lc-P5L4) excavated from comparative transcriptome of C. irritans - immuned L. crocea was identified and characterized. Sequence analysis shows the full-length cDNA of Lc-P5L4 is 539 bp containing an open reading frame (ORF) of 198 bp which encodes a peptide of 65 amino acid residues. The genome consists of three exons and two introns which exist in its ORF, and all the exon-intron boundaries are in accordance with classical GT-AG rule (GT/intron/AG). Multiple alignments indicate the signal peptides share highly conserved identity, while mature peptides are more diverse. Phylogenetic analysis displays Lc-P5L4 clusters together with other members of piscidin 5-like family. Next, quantitative Real-time PCR (qRT-PCR) detection found C. irritans infection could upregulate Lc-P5L4 expression level in all tested tissues significantly, it appeared earliest upregulation in the theronts infection stage in the head kidney; the expression contents reached to maximum level in the intestine, gill and muscle during trophonts falling off stage; while it was just upregulated during secondary bacterial infection stage in the liver and spleen. The data showed Lc-P5L4 upregulation time points were in accordance with different infection stages. With recombinant Lc-P5L4 (rLc-P5L4) obtained through Escherichia coli system, in vitro assay showed rLc-P5L4 could cause cilia deactivation, cell bodiesclumping and sticking to each other, then cell membrane rupture and contents leakage. The data illustrated Lc-P5L4 played critical roles in the immune defense against C. irritans infection, and provided another proof that piscidins exhibit multiple anti- C. irritans features.
Assuntos
Antiparasitários/metabolismo , Cilióforos/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perciformes/genética , Perciformes/metabolismo , Aminoácidos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/parasitologia , Infecções por Cilióforos/genética , Infecções por Cilióforos/metabolismo , Infecções por Cilióforos/parasitologia , DNA Complementar/genética , Éxons/genética , Doenças dos Peixes/genética , Doenças dos Peixes/metabolismo , Doenças dos Peixes/parasitologia , Genoma/genética , Íntrons/genética , Fígado/metabolismo , Fígado/parasitologia , Fases de Leitura Aberta/genética , Perciformes/parasitologia , Filogenia , Baço/metabolismo , Baço/parasitologia , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
Live-attenuated parasite vaccines are being explored as potential vaccine candidates since other approaches of vaccination have not produced an effective vaccine so far. In order for live-attenuated parasite vaccines to be tested in preclinical studies and possibly in clinical studies, the safety and immunogenicity of these organisms must be rigorously evaluated. Here we describe methods to test persistence in the immunized host and immunogenicity, and to identify biomarkers of vaccine safety and efficacy with particular reference to genetically attenuated Leishmania parasites.