Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent

Abstract

Despite intense efforts by the medical and pharmaceutical communities, Staphylococcus aureus continues to be a pervasive pathogen that causes a myriad of diseases and a high level of morbidity and mortality among infected patients. Thus, discovering or designing novel therapeutics able to kill both drug-resistant and drug-sensitive S. aureus remains a top priority. Bacteriolytic enzymes, mostly from phage, have shown great promise in preclinical studies, but little consideration has been given to cis-acting autolytic enzymes derived from the pathogen itself. Here, we use the S. aureus autolysin LytM as a proof-of-principal to demonstrate the antibacterial potential of endogenous peptidoglycan degrading enzymes. While native LytM is only marginally bactericidal, fusion of LytM to the lysostaphin cell wall binding domain enhances its anti-staphylococcal activity approximately 540-fold, placing it on par with many phage lysins currently in preclinical development. The potential to therapeutically co-opt a pathogen’s endogenous peptidoglycan recycling machinery opens the door to a previously untapped reservoir of antibacterial drug candidates.

Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity

“…The 5′ ends of the genes were appended with an NcoI restriction site and the 3′ ends with a Bam HI restriction site. These restriction sites were then used to clone into a modified pET26b vector (EMD Millipore, Darmstadt, Germany), which coded for a 5′-hexahistidine tag followed by a serine-methionine-alanine linker (Osipovitch and Griswold 2014). …”

“…The 5′ primer appended a 5′ Nco I site with an internal Bam HI site, and the 3′ primer appended a Hind III site. Nco I and Hind III were used to clone the LST cell wall binding domain (LBD) into the modified PET26b vector described above (Osipovitch and Griswold 2014). This vector was then used to clone all fusions using Nco I and Bam HI, retaining the 5′ hexahistidine and serine-methionine-alanine linker and including a glycine-serine linker between the autolysin catalytic domain and the LBD.…”

“…Proteins were expressed and purified in the manner previously described (Osipovitch and Griswold 2014), with minor changes as noted in Table S2. Protein purity was assessed by mixing samples with 4× Laemmli Sample Buffer (Bio-Rad, Hercules, CA) and running them on a 12% SDS-PAGE gel (Bio-Rad).…”

“…Thus, the LST CWBD presents one potential solution to the issue of suboptimal autolysin targeting. Indeed, in a prior pilot study, the lytic activity of LytM, which again lacks a CWBD, was enhanced more than 500-fold upon fusion to the LST CWBD (Osipovitch and Griswold 2014). That early success prompted us to more broadly survey the S. aureus proteome for autolysins that might be leveraged as antibacterial agents.…”

“…In contrast, the SH3 CWBD of LST binds specifically to the pentaglycine crosslinks in S. aureus peptidoglycan, and as a result it has been shown to bind ubiquitously throughout the bacterial cell wall (Grundling and Schneewind 2006). Notably, truncation of the LST's CWBD results in a substantial reduction in lytic activity (Gargis et al 2010; Osipovitch and Griswold 2014), a fact that underscores the critical nature of lysin targeting to the bacterial cell surface.…”