Abstract
Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting Klebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of Klebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP–linker–CBD–EAD) and the inverse configuration (CBD–EAD–linker–OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions.
Highlights
Lysins are bacteriophage-encoded enzymes that degrade bacterial peptidoglycan. They include virion-associated peptidoglycan hydrolases (VAPGHs) and endolysins, which are essential in the process of phage genome injection and phage progeny release, respectively [1]
(2020) screened a library of 174 and 322 variants of engineered lysins and other recombinant peptidoglycan hydrolases for activity against Staphylococcus aureus under raw milk and intracellular conditions, respectively [7,10]
VersaTile addresses the need of continuously evolving research questions and the investigation of new design rules for these antibacterial enzymes
Summary
Lysins are bacteriophage-encoded enzymes that degrade bacterial peptidoglycan. They include virion-associated peptidoglycan hydrolases (VAPGHs) and endolysins, which are essential in the process of phage genome injection and phage progeny release, respectively [1]. (2020) screened a library of 174 and 322 variants of engineered lysins and other recombinant peptidoglycan hydrolases for activity against Staphylococcus aureus under raw milk and intracellular conditions, respectively [7,10] These libraries of modular or truncated variants were all constructed with traditional restriction/ligation techniques, which are time-consuming and labor-intensive. To eliminate this technical hurdle, we have recently introduced a rapid DNA assembly method for an efficient combinatorial assembly of lysin domains, coined “the VersaTile technique”. K. pneumoniae strains producing extended-spectrum beta-lactamases (ESBLs) are increasingly prevalent or have evolved to carbapenem-resistant Enterobacteriaceae (CRE) [12]
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