Phage therapy has recently re-emerged as an alternative strategy for control of antimicrobial resistant (AMR) bacterial pathogens. Besides the whole phages, several of their encoded molecules also have antimicrobial properties. To develop a repository of therapeutic antimicrobials against AMR pathogens, identifying and characterizing novel phage endolysins is crucial. In the present study, an endolysin-encoding gene was identified in A. hydrophila phage CF7. The putative endolysin was named as CF7lysin. CF7lysin was 180 amino acids long, and its estimated molecular weight was 19.79 kDa. Hydrophobic amino acid residues were dominant at the N-terminal region of CF7lysin. Tertiary structure modelling revealed the globular structure of CF7lysin with one enzymatically active domain (EAD) and a major groove made up of highly conserved amino acid residues. After in silico characterization, in vivo expression of CF7lysin gene in A. hydrophila host was also confirmed with reverse-transcriptase PCR. Recombinant CF7lysin (rCF7lysin) completely lysed host cells when it was expressed in E. coli BL21 cells. Expression of rCF7lysin in E. coli was also confirmed by dot blot hybridization and bacterial growth curve assay.To confirm the role of N-terminal hydrophobic region in the holin-independent endolytic activity of CF7lysin, truncated CF7lysin gene (after removing the nucleotides encoding 24 N-terminal amino acid residues) was again cloned and expressed in E. coli. The truncated recombinant CF7lysin (trCF7lysin) did not cause host lysis during induced expression. The purified trCF7lysin protein showed lytic activity against Gram-positive bacteria during lawn assay. However, no lytic activity was observed against Gram-negative bacteria. The study provides important insights into the lytic activity of a novel phage endolysin. In the future, sequence manipulations in the endolysin gene sequence could be tried to enhance its lytic activity against Gram-positive and Gram-negative bacteria.
Keywords
Aquaculture; Bacterial pathogens; Antimicrobial resistance; Phage; Endolysin