RESUMO
Growth Hormone-Releasing Peptide 6 (GHRP-6) (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2) is an agonist of the growth hormone secretagogue receptor. GHRP-6 mimics the effect of ghrelin. The present study focuses on the immunomodulatory effects of GHRP-6 in tilapia with and without the presence of Pseudomonas aeruginosa infection. GHRP-6 up-regulated the transcription levels of three piscidin-like antimicrobial peptides (Oreochromicins I, II, and III) and granzyme in a tissue-dependent manner. Antimicrobial activity stimulation in serum (lysozyme and anti-protease activity) was also confirmed. Besides, GHRP-6 enhanced the in vitro antimicrobial activity against P. aeruginosa in tilapia gills mucus and serum samples and decreased the bacterial load in vivo after infection with this Gram-negative bacterium. Our results evidenced, for the first time, a direct link between a growth hormone secretagogue ghrelin mimetic in fish and the enhancement of antimicrobial peptides transcription, which suggests that this secretagogue is capable to lead the activation of microbicidal activity in tilapia. Thus, these results open new possibilities for GHRP-6 application in aquaculture to stimulate the teleost immune system as an alternative treatment against opportunistic bacteria.
Assuntos
Anti-Infecciosos , Ciclídeos , Tilápia , Animais , Peptídeos Antimicrobianos , Grelina , Hormônio do Crescimento , SecretagogosRESUMO
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.
Assuntos
Antígenos/imunologia , Copépodes/imunologia , Doenças dos Peixes/prevenção & controle , Salmo salar/parasitologia , Vacinas , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Antígenos/química , Antígenos/genética , Aquicultura , Sequência de Bases , Chile , Clonagem Molecular , Copépodes/química , Copépodes/genética , DNA Complementar/química , Feminino , Doenças dos Peixes/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Noruega , Filogenia , Conformação Proteica , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , TilápiaRESUMO
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chile's salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccination-challenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.
Assuntos
Copépodes/genética , Copépodes/parasitologia , Ectoparasitoses/veterinária , Doenças dos Peixes/prevenção & controle , Doenças Parasitárias em Animais/prevenção & controle , Vacinação/métodos , Animais , Copépodes/imunologia , Ectoparasitoses/imunologia , Ectoparasitoses/prevenção & controle , Doenças dos Peixes/imunologia , Inativação Gênica , Dados de Sequência Molecular , Doenças Parasitárias em Animais/imunologia , Análise de Sequência de DNA , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
A DNA fragment, carrying the Candida utilis HIS3 gene, has been isolated from a genomic DNA library by complementation of the E. coli hisB mutant. Its nucleotide sequence was determined and it predicts a single open reading frame of 675 bp (224 aa). The deduced amino acid sequence is highly homologous to other yeast and fungi HIS3 genes.