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

## TL;DR

Researchers engineered a phage-derived endolysin to effectively kill Flavobacterium psychrophilum, a harmful bacteria in aquaculture, without relying on antibiotics.

## Contribution

The study introduces a modified endolysin that can bypass the outer membrane of Gram-negative bacteria using T9SS components.

## Key findings

- Ely174 endolysin reduced F. psychrophilum optical density from 0.8 to 0.2 in 6 minutes at 2.5 µg/mL.
- Fusion with the C-terminal domain of T9SS protein SprA allowed Ely174 to lyse untreated F. psychrophilum.
- Protein engineering improved Ely174's thermal stability and lytic activity threefold.

## 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.

Flavobacterium psychrophilum 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, F. psychrophilum 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.