Discovery of antimicrobial peptides clostrisin and cellulosin from Clostridium: insights into their structures, co-localized biosynthetic gene clusters, and antibiotic activity.

Antimicrobial resistance presents a substantial threat to global public health, demanding urgent attention and action. This study focuses on lanthipeptides, ribosomally encoded peptides that display significant structural diversity and hold promising potential as antibiotics. Genome mining was employed to locate biosynthetic gene clusters (BGCs) containing class II lanthipeptide synthetases encoded by lanM genes. A phylogenetic study analyzing homologous sequences of functional LanM sequences revealed a unique evolutionary clade of 17 LanM proteins associated with 12 Clostridium bacterial genomes. In silico exploration identified nine complete BGCs, including one super-cluster containing two co-localized operons from Clostridium cellulovorans 743B, that encode for two new peptides named clostrisin and cellulosin. Each operon was heterologously expressed in Escherichia coli. Molecular weights associated with the expected post-translational modifications of the purified lanthipeptide were confirmed by MS-MS/MS analysis for cellulosin, while clostrisin was not post-translationally modified. Both peptides demonstrated antimicrobial activity against multidrug-resistant bacteria, such as a clinical strain of Staphylococcus epidermidis MIQ43 and Pseudomonas aeruginosa PA14. This is the first report of lanthipeptides from the Clostridium genus produced with its native biosynthetic machinery, as well as chemically and biologically characterized. This study showcases the immense potential of genome mining in identifying new RiPP synthetases and associated bioactive peptides.

An artificial virus-like triblock protein shows low in vivo humoral immune response and high stability.

The use of viral vectors for in vivo gene therapy can be severely limited by their immunogenicity. Non-viral vectors may represent an alternative, however, reports analyzing their immunogenicity are still lacking. Here, we studied the humoral immune response in a murine model triggered by artificial virus-like particles (AVLPs) carrying plasmid or antisense DNA. The AVLPs were assembled using a family of modular proteins based on bioinspired collagen-like and silk-like sequences that produce virus-like particles. We compared our AVLPs against an Adeno Associated Virus 1 (AAV), a widely used viral vector for in vivo gene delivery that has been approved by the FDA and EMA for gene therapy. We found that a 1000-fold higher mass of AVLPs than AAV are necessary to obtain similar specific antibody titters. Furthermore, we studied the stability of AVLPs against relevant biological reagents such as heparin and fetal bovine serum to ensure nucleic acid protection in biological media. Our study demonstrates that the AVLPs are stable in physiological conditions and can overcome safety limitations such as immunogenicity. The scarce humoral immunogenicity and high stability found with AVLPs suggest that they have potential to be used as stealth non-viral gene delivery systems for in vivo studies or gene therapy.