Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium

Abstract

Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positive Enterococcus faecium is classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome of E. faecium allowed the identification of a putative PDE gene with predictive amidase activity (EfAmi1; EC 3.5.1.28) in a prophage-integrated sequence. EfAmi1 is composed by two domains: a N-terminal Zn2+-dependent N-acetylmuramoyl-l-alanine amidase-2 (NALAA-2) domain and a C-terminal domain with unknown structure and function. The full-length gene of EfAmi1 was cloned and expressed as a 6xHis-tagged protein in E. coli. EfAmi1 was produced as a soluble protein, purified, and its lytic and antimicrobial activities were investigated using turbidity reduction and Kirby–Bauer disk-diffusion assays against clinically isolated bacterial pathogens. The crystal structure of the N-terminal amidase-2 domain was determined using X-ray crystallography at 1.97 Å resolution. It adopts a globular fold with several α-helices surrounding a central five-stranded β-sheet. Sequence comparison revealed a cluster of conserved amino acids that defines a putative binding site for a buried zinc ion. The results of the present study suggest that EfAmi1 displays high lytic and antimicrobial activity and may represent a promising new antimicrobial in the post-antibiotic era.

Metagenomic analysis of hot spring soil for mining a novel thermostable enzybiotic

“…The acquired results suggest an efficient bactericidal activity and allowed the determination of the minimum bactericidal concentration (MBC) and the half-maximal lethal concentration (LC 50 ) of Ami1 against S. aureus , S. epidermidis , and E. coli DH5a, after 1.30 h, 3 h, and 6 h of incubation (Table 1 ). The bactericidal activity of Ami1 falls within the range expected for highly active endolysins that have been recently reported (Guo et al 2017 ; Wu et al 2018 ; Plotka et al 2019 ; Varotsou et al 2023 ; Premetis et al 2023b ).

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“…Genome search on the available Thermotalea metallivorans genomic dataset (BioProject accession: PRJNA294866, ID 294866) revealed an open reading frame (ORF) (Protein ID: WP_068556374.1), encoding an endolysin that belongs to the cell wall amidase type 2 (NALAA-2) (Vermassen et al 2019 ). Endolysins, which belong to the NALAA-2 family, have attracted significant attention because of their enhanced antimicrobial activity (Broendum et al 2018 ; Vermassen et al 2019 ; Premetis et al 2023b ). We selected to analyze endolysins since they have received much interest from the scientific community as potential powerful antimicrobials in the prophylaxis and treatment of bacterial infections due to their specificity, mode of action, potential for engineering, and lack of resistance mechanisms.…”

“…Prophages represent a valuable source of new genes, which are often associated with unique catalytic and functional features. Therefore, from a biotechnology point of view, prophages have attracted significant attention as they can expand the repertoire of available resources for the identification and discovery of novel endolysin sequences (Premetis et al 2023b ). Analysis of the primary sequence of Ami1 uncovered interesting features.…”

“…Endolysins or PG hydrolases constitute a specific group of enzybiotics encoded by bacteriophages and/or prophages in host cells at the end of the bacteriophage lytic cycle (Murray et al 2021 ; Love et al 2020 ). Previous studies have proven that endolysins exhibit lytic activity against the majority of resistant bacterial cells (Loeffler et al 2003 ; Harhala et al 2018 ; Abdelkader et al 2019 ; Dams and Briers 2019 ; Premetis et al 2023a , b , c ). Moreover, evaluation tests of endolysins have confirmed their non-toxic behavior after their administration in animal models (Rodríguez-Rubio et al 2013 ; Hermoso et al 2003 ; Kashani et al 2018 ; Guo et al 2017 ).…”