Abstract

BackgroundStaphylococcus 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.ResultsWe 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.ConclusionsWe 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.

Highlights

  • Staphylococcus aureus is a major cause of nosocomial and community-acquired infections

  • The methicillin-resistant S. aureus (MRSA) strain B911 was used for bactericidal activity assays, and RN4220 was used for zymograms.Plasmid pET21a (Novagen, USA) was used for cloning and the constructs were expressed under the control of a T7 promoter

  • In addition to the endolysin gene in the lysis cassette encoded by open reading frames 30/32 [26], we found another gene encoding a muralytic enzyme near the genes encoding structural tail proteins (Figure 1)

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Summary

Introduction

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Peptidoglycan-degrading enzymes or murein hydrolases have the ability to digest bacterial cell walls Such enzymes from bacteriophages represent a unique class of antibacterial agents because of their ability to cleave bacterial peptidoglycan in a species-specific or genusspecific manner. Holins are transmembrane proteins that create lesions in the cytoplasmic membrane through which peptidoglycan-degrading enzymes (endolysins) gain access to the peptidoglycan layer [4,5] Bacteriophages encode another peptidoglycan-degrading enzyme involved in the initial stages of infection that facilitates phage DNA injection into the host cell. These proteins, which are distinct from endolysins, aid in the rapid lysis of host cells by a phenomenon referred to as “lysis from without” upon infection with high multiplicities of phage [6]. Enzymes involved in DNA injection are an integral component of the virion structure of many phages [7,8,9]

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