Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss)

Authors: Shepherd, Brian; Ma, Hao; HAN, YUEH-CHIANG - Oak Ridge Institute For Science And Education (ORISE); Palti, Yniv; Gao, Guangtu; Liu, Sixin; Wiens, Gregory - Greg

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2020
Publication Date: 12/7/2020
Citation: Shepherd, B.S., Ma, H., Han, Y., Palti, Y., Gao, G., Liu, S., Wiens, G.D. 2020. Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology. 116 (103961). https://doi.org/10.1016/j.dci.2020.103961.
DOI: https://doi.org/10.1016/j.dci.2020.103961

Interpretive Summary: Antimicrobial peptides (AMPs) are short peptides/proteins that form key components as the first-line defense of the innate immune system in many species including mammals, lower vertebrates, fish, insects and plants. As such, AMPs exhibit abilities to defend their hosts against broad spectrum of infectious microbial pathogens (bacterial, viral, fungal and parasitic) and are thought to be promising alternatives to the use of antibiotics in human health and agriculture. Six new antimicrobial peptides (AMPs) with sequence homology to the Nk-lysin (Nkl) family of proteins have been characterized and their patterns of gene expression in rainbow trout (Oncorhynchus mykiss) described. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following challenge with aquaculture-relevant pathogens as well as by physiological stressors.

Technical Abstract: Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128-133 aa in length, and mature peptides of 109-111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a & b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2-3 fold) of five (nkl 2-4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (~57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.