Characterization and evaluation of the anti-streptococcal activity of a novel zoocin A-like endopeptidase for potential veterinary applications

The increasing prevalence of antimicrobial resistance among streptococci necessitates the development of alternative antibacterial strategies against veterinary pathogens. Here, we report the first comprehensive characterization of pre-ZooA_agal, a zoocin A-like bacteriolytic enzyme derived from Streptococcus agalactiae strain QMA0326. Both the proenzyme containing signal peptide (SP) pre-ZooA_agal and its mature variant containing the catalytic and target recognition domains (ZooA_agal) were successfully expressed and purified following the optimization of expression conditions. The enzyme displayed high selectivity toward bacteria possessing the A11.5 peptidoglycan chemotype (A1α-L-Lys-L-Ala2), which is common in streptococcal species, such as S. dysgalactiae, S. agalactiae or S. uberis, exhibiting a minimum bactericidal concentration of 0.5 μM and rapid dose-dependent lysis. Pre-ZooA_agal showed strong bacteriolytic activity across a broad pH range (6.0-11.0) and remained active in NaCl concentrations up to 500 mM. Its enzymatic activity was sustained in serum and milk and enhanced in the presence of Ca2+ and Mg2+ ions. Electron microscopy observations confirmed extensive structural damage to susceptible bacterial cells following enzyme treatment. Importantly, no toxicity of both pre-ZooA_agal and ZooA_agal was observed in vertebrate models. These findings highlight pre-ZooA_agal and ZooA_agal as a stable and highly selective enzybiotics with potential for the targeted control of streptococcal infections relevant to veterinary medicine.