Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications

Abstract

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

Fig 1

Morphology of Romulus and Remus. Shown are transmission electron microscopic images of myoviruses Romulus (A) and Remus (B) negatively stained with 2% (wt/vol) potassium phosphotungstate (pH 7.0). Scale bars represent 100 nm.

Fig 2

Phage-mediated biofilm degradation. S. aureus PS47 biofilms grown on pegs for 24 h were treated with 10⁹, 10⁸, or 10⁷ PFU of ISP, Romulus, or Remus for 24 h. Remaining biofilm mass was represented relative to the negative control, treatment with phage buffer. Equality of variances was analyzed using an F test; an asterisk marks significant values examined with a two-tailed Student t test (P < 0.05). Mean values and corresponding standard deviations were based on six independent experiments.

Fig 3

BLASTN genome comparison of the phages representing the three species identified within Twortlikevirus. The outer ring presents the ORFs of the circularized phage Remus genome (blue) and the numbering of every 10 ORFs. The two other rings display the BLASTN homology between Remus on one hand and K (red) and Twort (green) on the other hand. The inner ring shows the G+C content of the Remus genome (black). The three functional modules in the genome and some major predicted gene functions are indicated.

Fig 4

Split genes in Romulus and Remus. Shown is a graphical representation of the splicing events in the terminase large-subunit gene (A), the gene encoding the portal protein (B), the helicase gene (C), the ribonucleotide reductase large-subunit gene (D), the DNA polymerase gene (E), and the gene encoding a DNA repair protein (F) in phages Romulus and Remus compared to those in phages K, G1, ISP, and Twort or Polaribacter irgensii 23-P. The open reading frames are presented as boxes, and the size of the corresponding amino acid sequence is indicated within each box. Group I intron-encoded homing endonucleases are depicted in blue. The insertion sequence-encoded transposase is depicted in dark blue. P5 represents the predicted promoter preceding ORF14. In the cases of K, G1, and ISP, the ORF numbering corresponds to that of ISP. H, histidine; N, asparagine; T, threonine.

Fig 5

Coomassie G-250-stained SDS-PAGE gel of the structural proteome of bacteriophage Remus. A low-molecular-mass marker is indicated on the left. The numbers on the right correspond to the analyzed gel pieces of which the identified proteins are listed in Table 2. Prominent protein bands are indicated with capital letters.