Diversity, structure-function relationships and evolution of cell wall-binding domains of staphylococcal phage endolysins

In response to the antibiotic resistance crisis, enzyme-based antibiotics like bacteriophage endolysins offer a promising alternative. In their natural context, endolysins lyse bacterial hosts by degrading peptidoglycan at the end of the replication cycle. They have evolved complex modular architectures, particularly in Gram-positive bacteria, featuring variable enzymatically active domains (EADs) and cell wall-binding domains (CBDs). These domains can be combinatorially shuffled to enhance antibacterial properties. CBDs are commonly seen as an important driver for the specificity of wild-type and engineered endolysins, as seen in Listeria and pneumococcal endolysins. This study explores the structural diversity and functional behavior of CBDs in endolysins from staphylococcal phages. Analysis of 182 CBDs reveals greater diversity than expected, classified into three families within the SH3b fold: SH3b_P1 (including the well-known SH3_5 family), and the new SH3b_P2 and SH3b_T families. Experimental specificity profiles of 24 CBDs using eGFP-CBD fusions against various staphylococcal species and strains challenge the notion of high host specificity within the staphylococcal context. Instead, CBDs exhibit a broader and more variable specificity and co-evolve with their accompanying EADs for functional synergy. This work provides insights for rational endolysin engineering and highlights the importance of understanding structure-function relationships to enhance their therapeutic potential.