Tilapia hepcidin (TH)2-3 as a transgene in transgenic fish enhances resistance to Vibrio vulnificus infection and causes variations in immune-related genes after infection by different bacterial species

Hepcidin is an antimicrobial peptide (AMP) secreted by the liver during inflammation that plays a central role in mammalian iron homeostasis. But the function of hepcidin in fish is still not completely understood. We recently described three different hepcidins (named Wawa hepcidin (TH)1-5. TH2-2, and TH2-3) from tilapia Oreochromis mossambicus. the cDNA sequences were determined, the predicted peptides were synthesized, and the TH2-3 peptide showed antimicrobial activity against several bacteria We hypothesized that TH2-3 may have a biological function like an AMP in fishes and can be used as a transgene to boost resistance against bacterial infection. To examine the antimicrobial effects of TH2-3, we produced and engineered the overexpression of TH2-3 in zebrafish (Dam rerio) and the convict cichlid (Archocentrus nigrofasciatus) The microinjected plasmid also contained a green fluorescent protein (GFP) which was used as an indicator to trace germline transmission In vivo, transgenic TH2-3 fish (of the F3 generation) were challenged with Vibrio vulnificus (204) and Streptococcus agalactiae (SA) Results showed significant clearance of bacterial numbers of V vulnificus (204) but not of S agalactiae in transgenic TH2-3 fish. A gene expression study using a real-time RT-PCR revealed that transgenic TH2-3 zebrafish showed Increased endogenous expressions of Myd88, tumor necrosis factor-alpha, and TRAM1 in vivo After transgenic TH2-3 zebrafish were infected with V vulnificus (204), interleukin (IL)-10, IL-26, lysozyme, toll-like receptor (TLR)-4a, and Myd88 were upregulated, but IL-1 beta (at 12-24 h) and IL-15 (at 1-12 h) were downregulated post-infection. After transgenic TH2-3 zebrafish were Infected with S agalactiae, IL-1 beta (at 1-24 h), IL-15 (at 6 h), IL-22 (at 1-6 h), and TLR3 (at 1-24 h) were downregulated, but TLR4a (at 6-12 h) and c3b (at 12 h) were upregulated post-Infection. Our findings identify the TH2-3 transgene in transgenic fish as an active component of the host response to bacterial pathogens. These results suggest that using TH2-3 as a transgene in zebrafish can effectively inhibit bacterial growth. specifically the V vulnificus (204) strain for up to 24 h (C) 2010 Elsevier Ltd All rights reserved