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

Abstract

Flavobacterium psychrophilum is the pathogen of bacterial cold-water disease, causing enormous economic losses in aquaculture. Antibiotic therapy to control F. psychrophilum 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 F. psychrophilum bacteriophage was heterologously expressed. The optical density of Triton-pretreated F. psychrophilum 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 F. psychrophilum 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 Bacteroidetes. The fusion of endolysin Ely174 with the C-terminal domain of the T9SS component protein SprA enabled Ely174-CTDSprA to lyse untreated F. psychrophilum. 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.