Novel MRSA-targeting phage MetB16: Genomic features, structural insights, and therapeutic applications

Abstract

Background/aim: Recent reports have indicated that multidrug-resistant strains of S. aureus, including methicillin-resistant strains, may pose a significant threat to public health and global economic stability.

Materials and methods: In this study, we present the isolation and comprehensive characterization of a novel phage, derived from clinically isolated MRSA strains.

Results: MetB16 exhibited an incubation period of approximately 20 min, a lysis period of around 45 min, and a burst size of 127 Plaque Forming Units (PFU)/cell. The phage demonstrated remarkable biological stability across a pH spectrum of 4.0-9.0 and maintained integrity within a temperature range of 37 and -80 °C. Scanning transmission electron microscopy and phylogenetic analyzes classified MetB16 as belonging to the Triavirus genus, representing a novel species within the Triaviruses. Whole-genome sequencing revealed a 45,295 bp-long genome size with a G + C content of 33.34%. Notably, bioinformatic analyses identified random integration sites within the MRSA genome. Functional annotation of the genome uncovered 72 open reading frames (ORFs), of which 34 encoded hypothetical proteins of unknown function, and these ORFs were associated with phage structure, packaging, host lysis, DNA metabolism, and additional functions. To elucidate the therapeutic potential of temperate phages, detailed structural analyses were conducted on key proteins, including holin, endolysin, and minor tail proteins of MetB16.

Conclusion: This study provides for the first time, the preliminary studies on the biological properties of MetB16 and comprehensive data facilitating an in-depth analysis of the mechanism underlying phage-host interactions, serving as a valuable reference for the evaluation of temperate phages in phage therapy.

Keywords: Methicillin-resistant Staphylococcus aureus (MRSA); modeling phage protein; phage bioinformatics; prophage induction.

Figure 1

The spot test results (A) of phage filtrates obtained after specified durations of UV exposure of MRSA cultures, the plaque morphology of the selected phage filtrate (B), and STEM analysis (C) are presented.

Figure 2

The results of the host range (A), optimal MOI (B), adsorption curve (C),one-step growth graph (D), stability under certain temperature (E) and pH values (F) are shown. Statistically significant differences determined by the Friedman test were marked with an asterisk (*): p < 0.05.

Figure 3

Genomic circular map of phage MetB16. Various colors are used to represent the coding sequences (CDS) based on their predicted functions: phage proteins (grey), terminase (salmon pink), capsid proteins (green), lytic enzymes (light green), tail proteins (blue), DNA metabolism, replication, repair, and binding-related proteins (light purple), hypothetical proteins (pink) and Integrase gene (orange). The GC content skew is depicted in the middle circle with a light blue color, while the inner circle illustrates the GC skew using lilac and dark purple colors for above and below averages, respectively.

Figure 4

Circular comparative map of protein-coding genes between phage MetB16 and closely related phages (Staphylococcus phage phiSa2wa_st1, Staphylococcus phage vB_SauS_320, and Staphylococcus phage vB_SauS_690).

Figure 5

Proteomic tree of phage MetB16 (A) Circular proteomic tree displaying genome-wide similarities among phage MetB16 (highlighted with a red star), top BLASTn matches, and closely related reference phage genomes. (B) Rectangular proteomic tree illustrating phage MetB16 and the top 100 phages with the highest ViPTree SG scores.

Figure 6

Phylogeny of phage MetB16. (A) The VICTOR genome-based phylogenetic tree was constructed by comparing phage MetB16, other Caudoviricetes phages, and closely related phages using Viptree. The phages were organized into 2 families and 7 genera, further grouped into 95 species. The GC content of the phage genomes is depicted in varying shades of purple, while the genome size is represented by horizontal black lines on the right side. (B) The VIRIDIC heatmap illustrates the intergenomic similarity between phage MetB16 and closely related phages, as classified by VICTOR. Three alignment capacity factors were considered to calculate the relatedness between the phages: intergenomic similarities (shown in hues of purple to lilac), the percentage of aligned genome sequence in a pair (indicated by shades of pink), and their length ratio (displayed in shades of black).

Figure 7

Modeling of MetB16 Holin, Lysin and Minor Tail Protein. (A) Predicted IDDT per position. Residues scoring ≥90 on pIDDT indicate an exceptionally high confidence level, while those scoring between 90 and 70 are deemed to have high confidence. Residues scoring between 70 and 50 on the pLDDT metric exhibit low confidence, and those below 50 indicate extremely low confidence. (B) The Prediction Aligned Error (PAE) score for the top-ranked model illustrates the calculated error in predicted distances between pairs of residues. The positional indices of individual amino acids are represented on both axes. The uncertainty in the predicted distance between any two amino acids is color-coded from blue (0 Å) to red (30 Å), as depicted in the accompanying color bar. (C) New cartoon representation model illustrating the structure of proteins incorporates the pLDDT scores per position. These scores are integrated as the b-factor in the PDB file provided by AF2, represented in rainbow colors ranging from red (indicating high confidence) to blue (suggesting low confidence). Based on Interpro domain predictions, the pertinent residue is highlighted in black, while the domains are marked in red.

Figure 8

Structure Assessment of MetB16 Holin, Lysin and Minor Tail Protein. (A) The Ramachandran plot of the predicted structure from top ranked models illustrates the distribution of phi (φ) and psi (ψ) dihedral angles for each residue in the protein. This plot provides insights into the conformational quality and stereochemical accuracy of the protein models. Light green indicates that 99.7% of the data falls within the first contour line, medium green represents 95.0% within the second contour line, and dark green signifies 80.0% within the third contour line. (B) Each residue in the model is assigned a local quality score (x-axis), indicating its expected similarity to the native structure (y-axis). Residues with scores below 0.6 are typically indicative of low quality. (C) Plot is represented by the normalized QMEAN score (y-axis), contrasted with scores attained by high-resolution crystal structures. Elevated values suggest that the model shares a comparable quality to experimental structures of similar dimensions.