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1.
Dev Comp Immunol ; 56: 13-24, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26615008

RESUMEN

Galectins, a family of ß-galactoside-binding lectins, are pattern recognition receptors that recognize pathogen-associated molecular patterns and are subsequently involved in the opsonization, phagocytosis, complement activation, and killing of microbes. Here, we report a novel galectin related protein (GRP) identified from rock bream (Oplegnathus fasciatus), designated OfGal like B. The cDNA of OfGal like B is 517 bp with an open reading frame (ORF) of 438 bp, encoding 145 amino acids, with a single carbohydrate recognition domain (CRD). However, only two of the seven critical residues responsible for carbohydrate recognition were identified in the CRD. There was no signal peptide identified in the OfGal like B protein. The genomic structure of OfGal like B, determined using a bacterial artificial chromosome (BAC) genomic library, consists of four exons and three introns. Homology assessment, multiple sequence alignment, and phylogenetic analysis indicated that OfGal like B is an evolutionarily conserved lectin that is closely related to the proto-type galectins. OfGal like B mRNA was constitutively expressed in a wide range of tissues in healthy rock breams. When challenged with bacterial or viral stimulants, OfGal like B was up-regulated in the gills and spleen of rock breams, indicating that it likely plays an important role during bacterial and viral infections. Furthermore, recombinant OfGal like B (rOfGal like B) lacked carbohydrate-binding activity but was able to recognize and agglutinate bacteria, including Streptococcus iniae, Listeria monocytogenes, Vibrio tapetis, Escherichia coli, and Edwardsiella tarda, and a ciliate parasite, Miamiensis avidus. These results collectively suggest that OfGal like B is involved in pathogen recognition and plays a significant role(s) in the innate defense mechanism of rock bream.


Asunto(s)
Proteínas de Peces/inmunología , Galectinas/inmunología , Pruebas de Aglutinación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/aislamiento & purificación , Peces/clasificación , Galectinas/química , Galectinas/genética , Galectinas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia
2.
Fish Shellfish Immunol ; 48: 154-68, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26549174

RESUMEN

The piscidin family comprises a group of antimicrobial peptides (AMPs) that are vital components of teleost innate immunity. Piscidins protect the host from pathogens, through multifaceted roles as immunomodulators and anti-infective peptides. The present study reports the identification, and characterization of a putative piscidin homolog, Of-Pis1, from rock bream (Oplegnathus fasciatus). A combined genomic and transcriptomic approach revealed that the Of-Pis1 gene comprises 1396 nucleotides (nt), four exons, and three introns. The cDNA with the 213 nt open reading frame encoded a 70-amino acid preprotein consisting of a signal peptide, a mature peptide, and a prodomain. Predicted mature Of-Pis1 was assumed to be a membrane-active AMP, based on the prediction of an amphipathic α-helical conformation with a net charge of +4. In addition, Of-Pis1 demonstrated significant similarities with other piscidin family members in terms of gene structure, sequence homology, and evolutionary relationship. Examination by quantitative real-time PCR (qPCR) of basal transcription of Of-Pis1 in the tissues of naïve rock bream, revealed predominant transcript levels in the gills, followed by the spleen, intestine, skin, and head kidney. In gill tissues, the temporally induced mRNA expression of Of-Pis1, upon in vivo injection trials with lipopolysaccharide (LPS); polyinosinic:polycytidylic acid (poly I:C); and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus (RBIV), was weak. In contrast, in vivo flagellin administration led to a robust upregulation of Of-Pis1 in different tissues. Antimicrobial potency was determined by employing recombinant (rOf-Pis1), and synthetic (pOf-Pis1) peptides, in in vitro assays. Recombinant overexpression inhibited the growth of bacteria expressing the rOf-Pis1 protein in a growth delay assay. The broad antimicrobial spectrum of pOf-Pis1 was evidenced by its potent activity against an array of microbes, including bacteria, fungi, and parasitic species. In addition, pOf-Pis1 showed no significant hemolytic toxicity against human erythrocytes. Collectively, the data presented in the current study improve our understanding of the piscidin AMP family, and the contribution of Of-Pis1 to the rock bream immunity.


Asunto(s)
Péptidos Catiónicos Antimicrobianos , Proteínas de Peces , Perciformes , Animales , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Células Cultivadas , Eritrocitos/efectos de los fármacos , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Hemólisis/efectos de los fármacos , Humanos , Oligohimenóforos/efectos de los fármacos , Oligohimenóforos/crecimiento & desarrollo , Perciformes/genética , Perciformes/inmunología , Perciformes/microbiología , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Secuencia de ADN
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