Abstract
Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.
Highlights
In the exploration of antimicrobials to combat resistant bacterial infections, endolysins have gained attention as alternatives to conventional small molecular antibiotics (Schuch et al, 2014; Czaplewski et al, 2016; Jun et al, 2017; Totté et al, 2017)
We report the results from endolysin assembly of the enzymatically active domains (EAD) cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK)
Due to the RNAse activity of wild type barnase, which has been described to be toxic to bacterial cells used for production, a previously described enzymatically inactive variant (E73W) of barnase was used as fusion partner for the construction of a CHAP-BarnaseE73W fusion protein (Hartley, 1993; Ramachandran and Udgaonkar, 1996; Figures 1A–C)
Summary
In the exploration of antimicrobials to combat resistant bacterial infections, endolysins have gained attention as alternatives to conventional small molecular antibiotics (Schuch et al, 2014; Czaplewski et al, 2016; Jun et al, 2017; Totté et al, 2017). Endolysins are functionally and structurally similar to both bacterial exolysins and autolysins. The exolysins are produced by certain bacteria with the function of causing lysis of alien bacterial species (Schindler and Schuhardt, 1964), and autolysins regulate cell wall metabolism and population size (Rogers et al, 1980). Development of resistance to endolysins is considered unlikely since these enzymes cleave evolutionary conserved and essential structures in the cell wall with high specificity for particular bacteria (Pastagia et al, 2011; Rodríguez-Rubio et al, 2013). There are four endolysin-based products in ongoing clinical trials against staphylococcal infections (Totté et al, 2017), and one endolysin-based formulation has reached market approval as a medical device for topical application in treatment of less severe skin conditions such as rosacea, acne, and eczema (Herpers et al, 2014)
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