PlyCYU endolysin targeting Streptococcus agalactiae exhibits a CHAP activity and a glucosaminidase domain mediating multimerization.

Bacteriophage endolysins are attractive alternatives to antibiotics owing to their rapid action, host specificity, and unlikeliness of resistance development. Here, bioinformatic analysis of Streptococcus suis prophage sequences identified an endolysin, named PlyCYU, containing two putative catalytic domains-an N-terminal amidase_5 and a C-terminal glucosaminidase (Lyz2) domain-with two CW_7 family cell wall binding motifs. PlyCYU exhibited bactericidal activity against Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis, with a minimum bactericidal concentration (MBC) range of 1.25 µM-40 µM, and retained bactericidal activity against S. agalactiae serotype II in ultra-high-temperature-processed milk (MBC 2.5 µM). Site-directed mutagenesis indicated that the amidase_5 domain was catalytically active and exhibited a cysteine-, histidine-dependent amidohydrolase/peptidase (CHAP) activity with the catalytic residues Cys34 and His99. Subdomain truncation analysis showed that PlyCYU214 and PlyCYU277, comprising the CHAP domain with one and two CW_7 motifs, respectively, conferred bactericidal activity, but lower than that of PlyCYU, while cyuLyz2 alone showed no activity. Notably, the bacteriolytic activity of PlyCYU277 was enhanced when cyuLyz2 was present. Agreeably, reducing sugars were detected in S. agalactiae lysis by PlyCYU and PlyCYU277 combined with cyuLyz2, but not by CHAP-inactive variants (PlyCYU-Cys34Ala/Ser and PlyCYU-His99Ala), PlyCYU277, and cyuLyz2 alone. This implied that cyuLyz2 action is CHAP dependent. Size exclusion chromatography (SEC) coupled with multi-angle light scattering and SEC-UV revealed PlyCYU and cyuLyz2 are homomultimers, whereas PlyCYU214 and PlyCYU277 are monomers. Therefore, the cyuLyz2 domain is important for the quaternary structure and the maximal activity of PlyCYU. Altogether, this study established PlyCYU endolysin as a potential antibiotic alternative against Streptococcus.IMPORTANCEStreptococcus agalactiae is a major pathogen responsible for severe neonatal infections, bovine mastitis, and streptococcosis in fish. The increasing prevalence of multidrug-resistant bacteria presses the urgent need to discover antibiotic alternatives. Bacteriophage-derived endolysins represent a promising solution due to their ability to specifically and rapidly kill target bacteria and be less likely to develop resistance. Here, we identified and characterized a novel endolysin, PlyCYU, with potent bactericidal activity against different Streptococcus species, including S. agalactiae, S. dysgalactiae, and S. uberis, isolated from bovine and fish sources. This study also demonstrated the relationships between the structure assembly and activity of PlyCYU. PlyCYU forms a multimer, facilitated by its glucosaminidase (cyuLyz2) domain, for maximal activity. Altogether, we revealed that PlyCYU is a promising candidate for development as an antibiotic alternative for Streptococcus infection treatment and food safety applications, as well as for advancing our understanding of endolysin.