LytU is a bridge-link isopeptidase that contributes to vancomycin resistance in Staphylococcus aureus

Emergence of antimicrobial resistance is a pressing global issue. Although myriads of mechanisms for survival against antimicrobial pressure have surfaced, one key strategy is the structural modification of peptidoglycan, the main defense line in Gram-positive bacteria e.g., notorious pathogen Staphylococcus aureus . Here we show that the recently identified peptidoglycan hydrolase (PGH) LytU is an unusual catalyst in the lysostaphin (M23B) family of zinc-endopeptidases that hydrolyze the glycyl-glycine peptide bond in S. aureus . Unlike any other PGH recognized thus far, LytU hydrolyzes the isopeptide bond between the lysine ε amino group and pentaglycine, thus functioning as a bridge-link hydrolase. We discovered that LytM and LytU are functionally non-redundant, and they contribute to the cell wall remodeling at different stages of PG maturation. Additionally, we demonstrate that LytU particularly targets the PG precursor, pGly-Lipid II, and its overexpression leads to growth inhibition and a less densely cross-linked cell wall, which increases susceptibility to lysostaphin. Interestingly, LytU expression enhances resistance to the glycopeptide vancomycin, the drug of last resort against S. aureus .