Study on molecular mechanism of short-form progranulin PGRN1 gene from Mozambique tilapia in defense against Vibrio species

• Main Authors: Wu, Sheng-Han, 吳聖韓
• Other Authors: Gong, Hong-Yi
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/fg9zd2

博士 === 國立臺灣海洋大學 === 水產養殖學系 === 107 === Progranulin (PGRN) is a multi-functional growth factor that mediates cell proliferation, survival, migration, tumorigenesis, wound healing, development, and anti-inflammation activity. A short-form PGRN gene PGRN1 of Mozambique tilapia (Oreochromis mossambicus) expresses two transcripts, OmPGRN1 encoding a 206 a.a. PGRN with two granulin (GRN) units named OmGRN-A and OmGRN-B. And a novel alternatively spliced transcript encoding a novel, secreted GRN peptide composed of 20-a.a. ER signal peptide and 41-a.a. GRN unit named OmGRN-41. OmGRN-41 and OmPGRN1 were not only abundantly expressed in immune-related organs including spleen, head kidney, and intestine of Mozambique tilapia, but also were further induced in the spleen of Mozambique tilapia challenged with Vibrio vulnificus at 1 h post infection (hpi) and 6 hpi, respectively. In this study, we established three transgenic zebrafish lines expressing the secreted OmGRN-41, OmGRN-A and OmPGRN1 specifically in muscle. The relative percent of survival (RPS) was enhanced in adult transgenic zebrafish expressing OmGRN-41 (68%), OmGRN-A (32%) and OmPGRN1 (36%) compared with control transgenic zebrafish expressing AcGFP after challenge with V. vulnificus. And the secreted OmGRN-41 can induce the expression of innate immune response-related genes, such as TNFa, TNFb, IL-8, IL-1β, IL-6, IL-26, IL-21, IL-10, complement C3, lysozyme (Lyz) and the hepatic antimicrobial peptide hepcidin (HAMP), in adult transgenic zebrafish without V. vulnificus infection. In addition, the OmGRN-41 can enhance the innate immune response by further elevating TNFb, IL-1β, IL-8, IL-6, and HAMP expression in early responsive time to the V. vulnificus challenge in transgenic zebrafish. Furthermore, synthetic OmGRN-41 peptide was used to investigate its anti-bacterial activities against various bacterial pathogens. The results showed that synthetic OmGRN-41 had bactericidal activity against gram-negative Vibrio species including V. vulnificus (64 μM), V. alginolyticus (128 μM), V. harveyi (128 μM), V. parahaemolyticus (256 μΜ); and exhibited bacteriostatic activities to V. alginolyticus (64 μM in 8 hours), and V. harveyi (64 μM in 12 hours), and V. parahaemolyticus (64 μM in 8 hours, 128 μM in 12 hours). However, no anti-bacterial activities of synthetic OmGRN-41 was observed for gram-negative Aeromonas hydrophila, Edwardsiella tarda, and gram-positive Streptococcus agalactiae, and Streptococcus iniae. Time-kill curves showed that the Vibrio species was eradicated over 99% of bacteria by synthetic OmGRN-41 treatment for 2 hours, including V. vulnificus (64 μM in 1 hour), V. alginolyticus (128 μM in 1 hour), V. harveyi (128 μM in 2 hours) and V. parahaemolyticus (256 μM in 2 hours). Synthetic OmGRN-41 exerted the antimicrobial activity to V. vulnificus with MIC and MBC at 64 μM after treated in pH 2 to pH 10 or after heated for 1 hour at high temperature from 40℃ to 100℃. The TEM observed that the outer membrane of V. vulnificus was disrupted by synthetic OmGRN-41 leading to morphology rupture and loss of cytoplasmic contents. Additionally, little hemolytic activity (<3%) for the tilapia and sheep erythrocytes were detected after treated with 2 to 128 μM synthetic OmGRN-41. The in vivo experiments also confirmed that synthetic OmGRN-41 can effectively enhance the survival of Nile tilapia (Oreochromis niloticus) infected by V. vulnificus, and decreased the number of V. vulnificus in the liver of infected Nile tilapia. Finally, some candidate molecules interacting with OmGRN-41 were identified by the yeast two-hybrid system, such as vitellogenin, calmodulin, mitochondrial pyruvate carrier, hemopexin and so on. Our results suggest that the OmGRN-41 peptide is a novel bactericidal agent, especially for Vibrio species. OmGRN-41 as a new host defense peptide is not only an immune modulator but also an antimicrobial peptide. It indicates that OmGRN-41 can be applied to therapy for vibriosis in human or aquaculture animals.