The C-terminal domain of T9SS component protein SprA assists <i>Flavobacterium psychrophilum</i> bacteriophage endolysin Ely174 to lyse Gram-negative bacteria

<i>Flavobacterium psychrophilum</i> is the pathogen of bacterial cold-water disease, causing enormous economic losses in aquaculture. Antibiotic therapy to control <i>F. psychrophilum</i> risks the development of drug-resistant strains. The use of phage-derived endolysins as effective substitutes for antibiotics is a current research hotspot. In this study, the endolysin Ely174 of <i>F. psychrophilum</i> bacteriophage was heterologously expressed. The optical density of Triton-pretreated <i>F. psychrophilum</i> at 600 nm was decreased from 0.8 to 0.2 in approximately 6 min using 2.5 µg/mL endolysin Ely174. In addition to its wide pH tolerance range, Ely174 also displayed a broad host spectrum. The presence of Mg²⁺, Ca²⁺ and Na⁺ in the reaction system enhanced its bactericidal activity. The lytic activity of Ely174 was increased threefold by random mutagenesis. The thermal stability of cold-adapted Ely174 has been improved by rational design. The final superimposing mutation resulted in variant A39H/P48I/E144A, which lysed pretreated <i>F. psychrophilum</i> rapidly even after thermal treatment at 50°C for 2 h. The type IX secretion system (T9SS) is involved in the pathogenic mechanisms of bacteria in the phylum <i>Bacteroidetes</i>. The fusion of endolysin Ely174 with the C-terminal domain of the T9SS component protein SprA enabled Ely174-CTD_SprA to lyse untreated <i>F. psychrophilum</i>. Recombination with ABC transport permease could help endolysin Ely174 overcome the outer membrane barrier of Gram-negative bacteria. These results indicate the potential of endolysin Ely174 as an antibacterial agent in aquaculture and the food industry. Protein engineering of the endolysin Ely174 also provides a new perspective for the control of T9SS-containing bacteria.IMPORTANCE<i>Flavobacterium psychrophilum</i> is a major pathogenic bacterium of salmonids. The antibiotic treatments used to control bacterial diseases in aquaculture are associated with food safety and environmental pollution issues. The application of phage-derived endolysins is a promising approach to replace antibiotics and prevent the emergence of drug-resistant bacteria. In this study, <i>F. psychrophilum</i> bacteriophage endolysin Ely174 exhibited bactericidal activity in different environments, including extreme pH, bovine serum, and cations. The lytic activity and thermal stability of endolysin Ely174 were improved by a combination of random mutagenesis and rational design. The protein engineering of endolysin Ely174 with a type IX secretion system element or functional protein enabled Ely174 to enter the peptidoglycan layer of Gram-negative bacteria and exert its function in the absence of outer membrane permeabilizers. The study findings expand our understanding of the control of T9SS-containing pathogens and provide more possibilities for further engineering modifications of endolysins.