Antimicrobial peptides are a fundamental component of mollusks’ defense systems, though they remain a thinly investigated subject. Here, infection by Vibrio parahemolyticus triggered a significant increase in antimicrobial activity in oyster plasma. By using PBS-challenged oysters as a control, plasma peptides from immunologically challenged oysters were subjected to peptidomic profiling and in silico data mining to identify bioactive peptides. Thirty-five identified plasma peptides were up-regulated post infection, among which, six up-regulated peptides (URPs) showed a relatively high positive charge. URP20 was validated with significant antibacterial activity. Virtually, URP20 triggered aggregation of bacterial cells, accompanied by their membrane permeabilization. Interestingly, URP20 was found to be active against Gram-positive and Gram-negative foodborne pathogens as well as Candida albicans, with no cytotoxicity to mammalian cells and mice. Our study provides the first large-scale plasma peptidomic dataset that identifies novel bioactive peptides in marine mollusks. Further exploration of peptide diversity in marine invertebrates should prove a fruitful pursuit for designing novel AMPs with broad applications.

Marine antimicrobial peptides (AMPs) represent a promising source for combating infections, especially against antibiotic-resistant pathogens and traditionally challenging infections. However, traditional drug discovery methods face challenges such as time-consuming processes and high costs. Therefore, leveraging machine learning techniques to expedite the discovery of marine AMPs holds significant promise. Our study applies machine learning to develop marine AMPs, focusing on Crassostrea gigas mucus rich in antimicrobial components. We conducted proteome sequencing of C. gigas mucous proteins, used the iAMPCN model for peptide activity prediction, and evaluated the antimicrobial, hemolytic, and cytotoxic capabilities of six peptides. Proteomic analysis identified 4490 proteins, yielding about 43,000 peptides (8–50 amino acids). Peptide ranking based on length, hydrophobicity, and charge assessed antimicrobial potential, predicting 23 biological activities. Six peptides, distinguished by their high relative scores and promising biological activities, were chosen for bactericidal assay. Peptides P1 to P4 showed antimicrobial activity against E. coli, with P2 and P4 being particularly effective. All peptides inhibited S. aureus growth. P2 and P4 also exhibited significant anti-V. parahaemolyticus effects, while P1 and P3 were non-cytotoxic to HEK293T cells at detectable concentrations. Minimal hemolytic activity was observed for all peptides even at high concentrations. This study highlights the potent antimicrobial properties of naturally occurring oyster mucus peptides, emphasizing their low cytotoxicity and lack of hemolytic effects. Machine learning accurately predicted biological activity, showcasing its potential in peptide drug discovery.

Unlike other animals, marine invertebrates lack an adaptive immune system and instead rely on innate immunity as their first line of defense. A key component of this innate response is the production of biologically active molecules, particularly antimicrobial peptides (AMPs), which offer promising solutions to the escalating global crisis of antimicrobial resistance (AMR). This review comprehensively examines the sources, structural diversity, mechanisms of action, biological functions, and therapeutic potential of AMPs derived from a wide range of marine invertebrate phyla. These evolutionarily conserved peptides exhibit broad-spectrum antibacterial, antifungal, antiviral, antiparasitic, and even anticancer activities. The review also summarizes strategies for AMP isolation and production, ranging from natural extraction to recombinant expression and chemical synthesis, and outlines their potential biotechnological applications. Furthermore, we highlight the transformative role of artificial intelligence (AI) in accelerating AMP discovery, design, and production, including predictive modeling, de novo peptide generation, and optimization workflows. Despite significant progress, challenges remain in large-scale production, pharmacokinetic characterization, and functional validation. Addressing these gaps through integrative omics, structural biology, and AI-driven innovation will be crucial for unlocking the full therapeutic potential of marine invertebrate AMPs in combating infectious diseases and antimicrobial resistance.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 μM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

As an important mollusk in reef ecosystems, Tridacna squamosa forms pro-survival symbiotic relationships that hinge on an exquisite redox equilibrium between the host and the photosynthetic symbiont, zooxanthellae. The exact regulatory mechanisms thereof remain poorly understood. In this study, a novel Nfe2-like transcription factor in T. squamosa was identified and characterized with respect to its antioxidant and cytoprotective roles. Gene structure and phylogenetic analysis reveal that T. squamosa possesses a single transcription factor TsNfe2l in contrast to mammalian Nfe2l1 (Nrf1) and Nfe2l2 (Nrf2), belonging to protein members of the bZIP-NFE2 subfamily and functionally resembling the mammalian Nfe2l1. A conserved bZIP domain permits its binding to the antioxidant response element (ARE) in vitro and in HEK293T cells. Further analyses such as promoter prediction suggest that TsNfe2l target genes engage mainly in the regulation of multiple enzymes involved in antioxidation and allied pathways. Notably, TsNfe2l transcriptionally upregulates carnosine synthase (TsATPGD), which subsequently produces L-carnosine abundantly to shield cells from oxidative damage. Moreover, the blockage of TsNfe2l nucleic acid binding reduced the expression of TsATPGD and L-carnosine content in the gill, resulting in elevated lipid peroxidation. Collectively, our findings establish novel molecular insight into TsNfe2l as a critical regulator of redox homeostasis in T. squamosa through carnosine synthesis.