Interrogation of Autolysins as a Source of Novel Bacteriolytic Agents

Staphylococcus aureus is a formidable pathogen, and its high morbidity and mortality rates are compounded by its ability to quickly develop antibiotic resistance. The current antimicrobial pipeline is inadequate to curb this mounting public health threat, and a novel approach to treating Staphylococcal disease is necessary. Bacteriolytic enzymes, namely phage lysins, have provided a promising paradigm shift in the treatment of bacterial infections. These enzymes lead to cell lysis by degrading the peptidoglycan of bacterial cell walls, and have been shown to be efficacious both in vitro and in vivo with no resistance yet observed.;We posit that autolysins, bacterial enzymes important for processes such as cell division, may be a fruitful source of lysins in addition to phage. We first examined the lytic potential of S. aureus autolysin LytM, and found (in accordance with previous literature) its low activity to be unsuitable for therapeutic use. Many lytic enzymes are modular in nature and contain two domains: a cell wall binding domain (CWBD) that targets the peptidoglycan and a catalytic domain that hydrolyzes peptidoglycan bonds. We hypothesized that LytM's slow kinetics were due to the lack of a CWBD and corrected this deficiency by fusing the LytM catalytic domain to the CWBD of lysostaphin, a well-characterized staphylolytic enzyme. Chimerization of LytM increased its activity 540-fold, putting it on par with other bacteriolysins, and allowed it to effectively kill S. aureus biofilms. To more broadly test the viability of autolysins as lytic agents, five novel S. aureus autolysins were identified from its genome and expressed recombinantly. Similar lysostaphin-autolysin chimeras were also constructed to assess the effect of CWBD engineering on this class of enzymes. Four novel autolysins displayed lytic activity against S. aureus. The most active lysin exhibited a 160-fold increase in activity as a chimera and was able to lyse lysostaphin-resistant cells. This study provides a framework for the use of autolysins as enzybiotics, and acts as a proof-of-principal for the importance of CWBD engineering for improved lytic kinetics of these enzymes.