Identification and functional characterization of Ism1 as a new member of antimicrobial protein

Previous studies have demonstrated that Isthmin1 (Ism1) performs multiple biological functions and plays a role in immune responses. Nevertheless, the direct role of Ism1 in the context of bacterial infection remains poorly characterized. Here we characterized the anti-bacterial activity of Ism1 in zebrafish Danio rerio. The expression of zebrafish ism1 was found to be up-regulated upon the challenge with bacteria, as well as with lipoteichoic acid (LTA) and lipopolysaccharides (LPS), suggesting its involvement in the innate immune response. The recombinant Ism1 (rIsm1) could bind to both Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Aeromonas hydrophila), as well as the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces, including LTA, LPS and peptidoglycan (PGN), indicating that rIsm1 recognizes pathogens through these PAMPs. Furthermore, rIsm1 exhibited potent bactericidal activities against all tested bacteria. MIC/MBC assays showed that rIsm1 exhibited bactericidal activity against all four tested strains, with MIC values of 64 - 128 μg/ml and MBC/MIC ratios ≤4. Mechanistically, rIsm1 treatment was associated with disruption of cell membrane integrity, membrane depolarization, and increased intracellular reactive oxygen species (ROS) production. Moreover, in vivo bacterial clearance assays demonstrated that rIsm1 accelerated bacterial clearance in zebrafish embryos/larvae. Additionally, we observed extracellular localization of Ism1 in shrimp, zebrafish, and mice, suggesting a critical role in systemic immunity across different animal species. Collectively, our study demonstrates that zebrafish Ism1 serves as a key immunocompetent factor, defines its specific function and mode of action during immune responses, and provides new insights into the biological roles of Ism1 as well as the innate immunity in vertebrates.