# Identification and analysis of the crucial holin domain and sites and the bactericidal activity of a holin–endolysin lysis cassette from phage PZL-Ah152 against Aeromonas hydrophila

## TL;DR

This study identifies key parts of a phage protein that can kill antibiotic-resistant bacteria and shows it works in fish.

## Contribution

The study identifies critical residues in a holin protein and demonstrates the broad-spectrum antibacterial activity of a holin–endolysin fusion protein.

## Key findings

- The C-terminal domain and specific amino acid residues in Hol 46 are crucial for cell-penetrating activity.
- The Hol 46_Lys 17 fusion protein shows broad antibacterial effects against multiple bacterial species.
- Transcriptomic and animal studies confirm the efficacy and safety of the fusion protein in treating infections.

## Abstract

With the increasing prevalence of antibiotic-resistant bacteria, phage therapy has garnered significant attention. Holin and lysin play essential roles in the phage-induced lysis of bacteria. This study investigated the functions of the holin protein Hol 46 and the lysozyme protein Lys 17 from the phage PZL-Ah152 and the mechanisms underlying the action of the fusion protein Hol 46_Lys 17. Assays, including membrane protein extraction tests and fluorescence microscopy, verified that the Hol 46 protein localized to the cell membrane and significantly inhibited the growth of Aeromonas hydrophila Ah152. We determined that the Hol 46 C-terminal domain and glutamic acid residue at position 66, along with the lysine residues at positions 63 and 64, were critical for its cell-penetrating activity. Furthermore, the Hol 46_Lys 17 fusion protein was developed to combine the membrane-disrupting capacity of Hol 46 with the lytic action of Lys 17. Hol 46_Lys 17 exhibited not only broader antibacterial effects against Aeromonas (24/38) but also against Escherichia coli (3/12) and Salmonella (5/29). Transcriptomic studies revealed that treatment with Hol 46_Lys 17 led to significant changes in the expression of genes related to flagellar synthesis, bacterial chemotaxis, and TCSs. Finally, animal studies were performed to confirm the safety and effectiveness of Hol 46_Lys 17 in treating intestinal infections caused by A. hydrophila in crucian carp. Our data showed that phage lytic system-related proteins hold great potential for the treatment of infections caused by antibiotic-resistant bacteria.

As a zoonotic and fish-pathogenic bacterium, Aeromonas hydrophila causes significant harm worldwide. Owing to the emergence of A. hydrophila strains with multidrug resistance, phage therapy has garnered extensive attention. Holin and lysin, phage-derived antibacterial proteins, play crucial roles in antimicrobial activity. The glutamic acid at position 66 and lysine residues at positions 63 and 64 in the C-terminal domain of the Hol 46 protein from phage PZL-Ah152 were essential for its A. hydrophila cell-penetrating activity. The Hol 46_Lys 17 fusion protein exhibited broad-spectrum antibacterial activity, including effects against Salmonella and Escherichia coli. Transcriptomic assays further revealed the effects of Hol 46_Lys 17 on A. hydrophila Ah152 at the molecular level. In vivo studies confirmed its efficacy and safety for the treatment of intestinal infections in crucian carp.