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Identification and characterization of a novel lysin-like endopeptidase from the Vibrio cholerae predator phage ICP1
Laura Espinosa-Barrera1, Oscar Linares-Vergara2, Lina A Zermeño-Cervantes3
1Laboratorio de Biología Sintética, Estructural y Molecular, Facultad de Ingeniería Mecánica y Eléctrica, Universidad de Colima, Coquimatlán, Colima, 28400, Mexico; Unidad de Biología Integrativa, Centro de Investigación Científica de Yucatán, Merida, Yucatán, 97205, Mexico.
Abstract:
International Centre for Diarrheal Disease Research, Bangladesh cholera phage 1 (ICP1)-type vibriophages are specialized predators of the Gram-negative (G-) pathogen Vibrio cholerae. However, the muralytic lysins critical to the lytic mechanism of these vibriophages remain uncharacterized. In the present study, we identified a highly conserved peptidoglycan (PG) NlpC/P60 peptidase-encoding gene (osLys) through data mining of 85 ICP1 vibriophage genomes collected during seasonal cholera outbreaks. OsLys clustered within a monophyletic group of modular lysin-like endopeptidases from phages infecting Vibrio parahaemolyticus. Structural modeling revealed an N-terminal cell-wall-binding domain (CBD) accommodating two PG-binding motifs (Glu30-Thr37 and Asp54-Thr61), followed by a C-terminal catalytic cysteine-histidine-dependent amido-hydrolase/peptidase (CHAP) domain with the conserved catalytic triad Cys126/His176/Asn196. Functional analysis of OsLys revealed that it showed optimal PG hydrolytic activity (120 ± 0.3 U/μg) under mesophilic (25-40 °C) and neutral pH conditions, and its activity was enhanced by Ca2+ and Mg2+ ions and diminished by Zn2+ and Mn2+ ions. OsLys showed no PG hydrolytic activity in natural seawater or saline (NaCl >150 mM), supporting a primarily intracellular function. Nonetheless, OsLys displayed both muralytic activity against PG-exposing G-pathogens and bacteriostatic activity when applied exogenously. To our knowledge, OsLys is the first functionally characterized enzymatic lytic effector derived from a phage infecting V. cholerae.
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