First experimentally characterised Proteus phage endolysins have distinct metal cofactor dependencies.

Proteus mirabilis is a Gram-negative uropathogenic bacterial species responsible for many catheter-associated urinary tract infections (CAUTIs). Due to the growing rates of antimicrobial resistance among CAUTI pathogens, novel antimicrobial solutions are urgently needed. Bacteriophage endolysins are an emerging class of antimicrobial agents, but no endolysins from P. mirabilis phages have been reported to date. Here, we describe two new Proteus phage endolysins, LysPM1 and LysPM2, containing Peptidase_M15_3 and CHAP domains, respectively. We experimentally confirmed their antibacterial activity against several Proteus spp. when used in combination with outer membrane permeabilizers, including EDTA, citric acid, and chloroform, as well as against frozen bacteria. Both enzymes have optimum activity at pH 7-8 and retain activity at temperatures up to 60 °C. Both endolysins completely lost their lytic activity upon treatment with EDTA, suggesting that divalent cations participate in the catalytic mechanism; the addition of Ca2⁺ and Zn2⁺ to LysPM1, and Ca2⁺ and Mn2⁺ to LysPM2 partially restored the activity. Our study confirms LysPM1 and LysPM2 as the first experimentally characterized Proteus phage endolysins, with LysPM2 also being the first experimentally characterized Gram-negative endolysin with a single CHAP domain. These findings could guide further development of phage-derived lytic enzymes for treatment of Proteus infections. KEY POINTS: • LysPM1 & LysPM2 are the first experimentally characterized Proteus phage endolysins. • Both are single-domain endolysins: Peptidase_M15_3 in LysPM1 and CHAP in LysPM2. • Metal cofactors are required for their activity: Zn2⁺ for LysPM1, Ca2⁺ for LysPM2.