Isolation and Characterization of a Novel Virulent Phage ASG01 of Aeromonas salmonicida and Its Cell Wall Hydrolase Activity

Abstract

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic.

Keywords: antibacterial agent; aquaculture; endolysin; heterologous expression; site-directed mutation.

Figure 1

Isolation, morphology and biological properties of ASG01. (A) Plaques formed on Aeromonas salmonicida DBFF01 agar plate. (B) Transmission electron micrographs reveal the morphology of phages. Scale bar = 100 nm. (C) Determination of optimal multiplicity of infection (MOI) of phage of ASG01. (D) Stability analysis of ASG01 temperature (30–80 °C). (E) Stability analysis of ASG01 pH (1–14).

Figure 2

Genome sequencing analysis of the phage ASG01. (A) Graphical representation of the phage ASG01. Circles show (from the outside to the inside) (1) the coding seqs counter-clockwise; (2) the coding seqs clockwise; (3) GC skew. Values greater than zero are in green, while those lower than zero are in purple; (4) G + C% content; (5) physical map scaled in kb. (B) Neighbor-joining phylogenetic tree based on the complete genome sequence of phage ASG01. (C) The genome annotation of ASG01.

Figure 3

Bioinformatic analysis of Cwh protein. (A) Signal peptide prediction. (B) Hydrophili-hydrophobic analysis. (C) Secondary structure prediction. (D) Tertiary structure prediction.

Figure 4

Analysis of antimicrobial activity and key active sites of Cwh. (A) Antimicrobial activity of Cwh by OD₆₀₀, (B) analysis of antimicrobial activity of Cwh by means of cell counting in Rosetta (pLys), (C) conserved amino acid residue analysis based on Uniprot data alignment, the arrows represent the eleven conserved amino acids in Cwh, and (D) lysis activity assay of the mutant protein in Cwh protein. Statistical analysis was performed with GraphPad (**, p < 0.01; ***, p <  0.001, ns, not significant).

Figure 5

Analysis of antimicrobial activity of Cwh in pathogen (As, Aeromonas salmonicida; Ah, Aeromonas hydrophila). (A) Construction of the engineered strain. (B) Scanning electron microscope observation of cells; un, untreated; in, Cwh expressed. (C) Antimicrobial activity of Cwh by means of cell counting in pathogens.