Project Number: 6028-31630-009-003-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2019
End Date: Jul 31, 2021
Objective 1: Produce catfish Interleukin-22 gene (cfIL-22) fusion protein variants using a plant-based expression platform and develop assays for establishing protein activity. Objective 2: To characterize the in vitro activity of cfIL-22 using catfish primary cells from the gill and skin. Objective 3: Evaluate the ability of the cfIL-22 as a therapeutant to protect catfish in two disease challenge models.
Approach for Objective 1: To establish the bioactivity of recombinant catfish IL-22 constructs. Previously developed constructs for cfIL-22 and cfIL-22:protein tag fusions will be used in the study. These IL-22 constructs will be expressed in a plant-based vector and tested for activity as it is not known a priori the impacts of fusion tags on protein expression or function. Bioassays will be performed in established fish cell line channel catfish ovary fibroblasts. Approach for Objective 2: To establish the function of cfIL-22 in gill and skin primary cells. In mammalian systems, IL-22 has been shown to have activity in expression and secretion of antimicrobial peptides (AMPs); expression and secretion of tissue repair proteins (TRPs); and activation of the Signal Transducer and Activator of Transcription 3 (STAT3) cell signaling pathway. Thus we will examine activity of cfIL-22 on AMPs, TRPS and STAT3 using fish gill and skin primary cells and methods generated in Objective 1. Approach for Objective 3: To examine cfIL-22 construct in a diseased animal model as potential therapeutant. An Ichthyophthirius multifiliis challenge will be performed to establish cfIL-22 activity in active wound healing. A second disease challenge will be performed using Flavobacterium columnare to test the antimicrobial activity of cfIL-22. The cfIL-22 construct will be administered at times throughout the challenge deemed most beneficial. In both challenges, mucosal tissues will be sampled over time-course of infection with and without cfIL-22 treatment and RNA sequencing performed to evaluate activation or suppression of gene networks and pathways important with cfIL-22 stimulation.