Specificity of the SH3 domain in Staphylococcal endolysin Endo88 through in vitro and in silico mutagenesis

Bacteriophages produce endolysins at the end of their lytic cycle to degrade bacteria host cell walls and release progeny virions. Endolysins are being extensively studied as antimicrobial alternative due to rising antimicrobial resistance and as biosensors for their high specificity. Endolysins typically consist of enzymatically active domains (EADs) at the N-terminus, which degrade the bacterial peptidoglycan layer, and a C- terminal cell wall binding domain (CBD), which binds to specific peptidoglycan components and influences endolysin lytic host range. This study investigates the role of the CBD in lytic host range by analyzing the impact of amino acid modifications introduced through site-directed mutagenesis in the CBD of Endo88, an endolysin derived from Staphylococcus aureus phage 88. The mutations were designed based on two strategies; (i) comparison of primary structure of the SH3 domains with different host range, and (ii) comparison of the tertiary structure of predicted binding site of endolysins with different host range. Five mutant endolysins were generated, cloned, and expressed in Escherichia coli, and their lytic and binding activities against an array of bacteria were compared to those of the wild-type Endo88. Lytic activity was evaluated using turbidity reduction assay which was optimized by Biophysical characterization of Endo88 including different buffers, temperature and pH. It was ascertain that Endo88 had optimum lytic activity in Tris buffer without NaCl, was active at pH of 6.0-11.0 and possessed lytic activity up to 37°C but quickly loses activity at 45°C. The binding activity of endolysins to various bacteria was assessed via a GFP-bound CBD flow cytometry fluorescence assay. The results revealed that while the mutations affected lytic and binding activity significantly, it did not alter the host range of Endo88. This suggests that CBD modifications may not necessarily affect host specificity but do influence binding activity and even lytic efficiency, despite the CBD having no lytic activity and the Endo88 being a modular protein. Further, flow cytometry analysis indicated no direct correlation between binding and lytic activity. This aligns with findings from previous studies, which have shown that some endolysins can retain lytic activity even in the absence of a CBD. Although the CBD is generally considered essential for maximal lysis, the data from this study suggest that binding may not always be critical for determining the host range. Nevertheless, the reduction in lytic activity caused by CBD mutations points to the CBD's auxiliary role in enhancing overall lysis in endolysin. Molecular docking of two mutants and Endo88 provided insights into binding site interactions with various peptidoglycan structures, which could aid in predicting host range alterations through endolysin engineering. This study highlights the intricate relationship between the CBD and EAD in modulating endolysins efficiency. Understanding this interplay could be contributory in developing more effective endolysins to combat antimicrobial resistance, especially in developing targeted phage endolysin therapies and diagnostics via biosensor development. Finally, this study contributes to the limited knowledge on endolysin host range since most studies focusses on the lytic efficiency rather than the host-range.

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