Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage

Abstract

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional Cterminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Structural and Functional Insights Into Skl and Pal Endolysins, Two Cysteine-Amidases With Anti-pneumococcal Activity. Dithiothreitol (DTT) Effect on Lytic Activity

“…The search of structural homologues of N-Skl identified the CHAP domain of the Enterococcus faecalis phage IME-EF1 endolysin (CHAP IME−EF1 ; PDB entry 6IST) (Zhou et al, 2020) as the best possible template (full sequence coverage; 31% sequence identity and 62% similarity). The Ramachandran plot of the final model−obtained with the alignment of Figure 4A−showed that all residues were in allowed regions (88.4% in most favored regions) and according to VERIFY 3D 87% residues had a 3D-1D average score ≥ 0.2 (88.8% in 6IST template).…”

“…In addition, little is known about Pal and Skl three-dimensional (3D) structure, substrate recognition, and catalysis. Although the atomic structure of several CHAP domains has been solved (McGowan et al, 2012;Gu et al, 2014;Sanz-Gaitero et al, 2014;Zhou et al, 2020), the 3D structure of an Amidase_5 domain is yet unknown, though a distant relationship with the NlpC/P60 family (Pfam entry PF00877) has been proposed (Anantharaman and Aravind, 2003). CHAP and NlpC/P60 domains belong to the Peptidase_CA clan (CL0125), whose members are evolutionarily related to the papain-like cysteineprotease family (Anantharaman and Aravind, 2003;Bateman and Rawlings, 2003;Ridgen et al, 2003).…”

Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis

“…Generally, reports from recent years indicated a high biodiversity of bacteriophages specific for E. faecalis strains. Such reports can be exemplified by articles describing isolation and characterization of previously unknown phages of different properties [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ], genetic modification of known phages and their use in experimental phage therapy (including effects on biofilms) [ 46 , 47 ], assessment of phages in therapy using animal models [ 48 , 49 , 50 , 51 , 52 , 53 ], and cloning of phage genes coding for specific lysins and characterization of the gene products in the light of killing E. faecalis cells [ 54 , 55 , 56 , 57 , 58 , 59 , 60 ].…”

The Age of Phage: Friend or Foe in the New Dawn of Therapeutic and Biocontrol Applications?

“…Phage endolysins have been one of the most examined phage-encoded enzymes, owing to their essential role in mediating the release of phage progeny during the final stages of the phage life cycle, which in turn may be exploited to aid in the lysis and death of selected bacteria [319,320]. For example, endolysins such as LysIME-EF1 have exhibited efficient antibacterial activity against multiple strains of Enterococcus faecalis [321], while the endolysin Ts2631 of phage vB_Tsc2631 has been proposed as a model for future antimicrobial agents due to its intrinsic bactericidal activity and uncommon thermal stability [322]. Additionally, endolysin LysMK34 of phage PMK34 has exhibited intrinsic antibacterial activity up to 2.4 and 4.8 log reductions in Pseudomonas aeruginosa and Acinetobacter baumannii, as well as aiding to resensitize colistin-resistant bacterial strains [323].…”