RESUMEN
Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.
Asunto(s)
Bacteriófagos/química , Endopeptidasas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Peptidoglicano/metabolismo , Vibrio parahaemolyticus/virología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Organismos Acuáticos , Bacteriófagos/clasificación , Bacteriófagos/genética , Bacteriófagos/metabolismo , Sitios de Unión , Calcio/química , Calcio/farmacología , Cationes Bivalentes , Endopeptidasas/química , Endopeptidasas/genética , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Filogenia , Unión Proteica/efectos de los fármacos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Cloruro de Sodio/química , Cloruro de Sodio/farmacología , Especificidad por Sustrato , Proteínas Virales/química , Proteínas Virales/genética , Zinc/química , Zinc/farmacologíaRESUMEN
Vibrio parahaemolyticus is the causative bacterium of acute hepatopancreatic necrosis disease (AHPND) in white shrimp Litopenaeus vannamei. This bacterium secretes protein toxins whose genes are encoded in an auto-transmissible plasmid called pVA1. The presence of this plasmid in V. parahaemolyticus is determinant for disease development. Its propagation is not only linked to bacterial colonisation capacity but also to horizontal gene transfer mechanisms. Nevertheless, the active uptake of plasmid, which is known as natural genetic transformation (NGT), has not yet been proposed as a possible acquisition mechanism of the pVA1 plasmid among Vibrio species. Previous studies suggest that some Vibrio species have the ability to undergo NGT in the presence of chitin. Therefore, the objective of this study was to evaluate the induction of NGT mediated by chitin in V. parahaemolyticus (ATCC-17802) through its ability to incorporate and express the pVA1 plasmid. The results showed that a reference strain that does not initially contain the plasmid can incorporate the plasmid under the appropriate transformation conditions, and cause mortality in white shrimp similar to that observed for pathogenic strains isolated from infectious outbreaks. Given the management and conditions of a shrimp farm with large amounts of chitinous exoskeletons, it is feasible that NGT could be a possible acquisition mechanism of plasmid pVA1 among Vibrio species, turning a non-causative strain of V. parahaemolyticus into a causative strain. With this study, we have expanded the knowledge of the pathogenesis process mediated by NGT and the understanding of the possible propagation mechanisms of emerging diseases in the aquaculture sector.