A Novel Type I Crustin Isoform from Scylla olivacea and its Antibacterial Potential: Membrane Depolarization, Disruption, and Induction of Reactive Oxygen Species as Modes of Action.

Abstract

Availability of novel antimicrobial agents is an urgent necessity to combat the growing threat posed by multidrug-resistant bacteria, prompting exploration of marine antimicrobial peptides (AMPs) as potential solutions. Crustacean AMPs are equitably diverse in terms of structure and function, making them consistent templates for novel antimicrobials. From Scylla olivacea gill transcriptome cDNA, a putative Crustin AMP sequence of 333 nucleotides, encoding a 111 amino acid Crustin Type-I isoform was identified. The mature peptide encoding region was cloned and recombinantly expressed in E. coli using Luria Bertani (LB) broth, yielding approximately 0.9 mg/L peptide. This cationic (+6.25) peptide with amphipathic properties (34% hydrophobicity) exhibited antibacterial effects against Gram-positive and Gram-negative strains, with MIC 16 μM against Vibrio spp. The identified modes of actions included disruption of bacterial membranes, membrane potential dissipation, and induction of ROS. Scanning electron microscopy (SEM) analysis revealed bacterial lysis and structural damage. Being non-toxic to mammalian cells (CHO-K1) and non-haemolytic, So-Crustin qualifies to be safe for therapeutic applications. It was quite stable under different physio-chemical/biological conditions, including temperature, pH, NaCl concentrations and proteases like trypsin and proteinase K. This study emphasizes So-Crustin's potential as a safe and effective antibacterial agent.