A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein.

BACKGROUND: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species. RESULTS: We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model. CONCLUSIONS: We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.

Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection.

BACKGROUND: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. RESULTS: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. CONCLUSIONS: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.