The SH3b cell wall–binding domain of SpM23_B peptidoglycan hydrolase has antimicrobial activity

Bacterial peptidoglycan hydrolases (PGHs) are enzymes that degrade the peptidoglycan layer of bacterial cell walls and are essential for cell wall remodeling during growth, division, and cell wall turnover. PGHs commonly comprise an enzymatically active domain (EAD) and a cell wall–binding domain (CBD). Src Homology 3b (SH3b) domains are widely recognized as bacterial surface-targeting modules that enhance the activity and specificity of PGHs. However, their potential antimicrobial functions independent of the catalytic domain remain largely unexplored. Here, we investigated the antimicrobial properties of CBD_Sp_B, an SH3b domain derived from the SpM23_B PGH of Staphylococcus pettenkoferi . CBD_Sp_B exhibited potent bactericidal activity against all tested bacterial strains and, unexpectedly, also showed antifungal activity against multiple Candida species, including the WHO critical-priority pathogen Candida auris . Electron microscopy revealed pronounced ultrastructural alterations in both bacterial and fungal cells following treatment. SYTOX Green assays showed no significant membrane permeabilization, whereas DiSC 3 (5) measurements demonstrated a loss of bacterial membrane potential, indicating membrane depolarization as a component of its antimicrobial activity. Further analysis identified the C-terminal region of CBD_Sp_B as a key determinant of its antimicrobial function. However, its activity was strongly dependent on ionic strength and was abolished at NaCl concentrations ≥ 25 mM, limiting applications under physiological conditions. Our findings reveal a previously unrecognized antimicrobial function of an SH3b domain, extending its biological role beyond bacterial cell-wall binding. The broad-spectrum bactericidal and antifungal activity of CBD_Sp_B highlights its potential as a novel antimicrobial scaffold, particularly for topical and non-clinical applications under low-ionic-strength conditions, while also providing a basis for engineering salt-tolerant derivatives.