The new SH3_T domain increases the structural and functional variability among SH3b-like CBDs from staphylococcal phage endolysins

Endolysins, proteins encoded by phages to lyse their hosts and release their progeny, have evolved to adapt to the structural features of each host. The endolysins from Staphylococcus-infecting phages typically feature complex architectures with two enzymatically active domains (EADs) and one cell wall-binding domain (CBD) belonging to the bacterial SH3 (SH3b) superfamily. This study focuses on three SH3b-like CBDs from exemplary staphylococcal phage endolysins (LysRODI, LysC1C, and LysIPLA5) that were structurally and functionally characterized. While RODI_CBD and C1C_CBD were assigned to the well-known SH3_5 family, a new family, SH3b_T, was identified using the CBD from LysIPLA5 as a model. GFP-fused CBDs were created to assess their differential binding to a collection of staphylococcal strains. IPLA5_CBD showed enhanced binding to Staphylococcus epidermidis, while RODI_CBD and C1C_CBD exhibited distinct binding profiles, with RODI_CBD targeting Staphylococcus aureus specifically and C1C_CBD displaying broad binding. Sequence comparisons suggested that a few differences in key amino acids could be responsible for the latter binding difference. The CBDs modulated the activity spectrum of synthetic EAD-CBD combinations in accordance with the previous binding profiles, but in a manner that was also dependent on the EAD present in the fusion protein. These results serve as a context for the diversity and versatility of SH3b domains in staphylococcal endolysins, providing insights on how (i) the CBDs from this superfamily have diverged to adapt to diverse bacterial ligands in spite of sharing a common fold; and (ii) the evolution of specificity relies on the EAD-CBD combination rather than solely the CBD. IMPORTANCEClinical management of bacterial infections is nowadays compromised by the rise in antimicrobial resistance. The development of new antimicrobial therapies with diverse modes of action is therefore of pivotal importance to complement the current standard of care. Phage endolysins are a new class of antibacterial agents based on rapid peptidoglycan degradation. The natural reservoir of phage endolysins offers a practically infinite diversity. This works reveals a broadly spread but still unknown phage endolysin domain targeting staphylococci while providing structural-functional insights that are paramount to understand the evolution of endolysins and how they can be applied as an antimicrobial.