The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets

Abstract

β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus. Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations.

Functional Insights From the Evolutionary Diversification of Big Defensins

“…Basic or dibasic sites (Arg-Arg or Lys-Arg) are found between the two structural domains of big defensins. The proteolytic cleavage of the native Tt-BigDef at this dibasic site, experimentally achieved (12), generated two fragments with distinct antimicrobial activities, as also observed for the two synthetic domains of Cg-BigDef1 (21). The covalent association of Cg-BigDef1 domains is synergistic and essential for salt-stable antimicrobial activity (21).…”

“…Recently, in the scallop A. purpuratus, Ap-BigDef1 was localized not only inside hemocytes but also in the digestive gland, mantle and gill tissues from challenged scallops (30). With the recent developments to produce big defensins in large amounts (21), new perspectives are now open for understanding the biology of these HDPs, from tissue distribution to function across multiple species.…”

“…In species harboring the canonical big defensin structure (horseshoe crab, amphioxus, scallop and oyster), big defensins play immune functions: actually, they have antimicrobial activities at the physiological salt concentrations of their marine host, indicating they likely contribute to the defense against infections (12,14,17,21). The oyster Cg-BigDef1 remains the only big defensin produced in sufficient amounts to establish a large activity spectrum (21). It shows a broad range of bactericidal activities against reference, environmental, and clinical strains, including strains multiresistant to antibiotics.…”

Big DefensinApBD1 from the scallopArgopecten purpuratusis an antimicrobial peptide which entraps bacteria through nanonets formation

“…As the β-defensin like C-terminal domain was not properly folded in rApBD1, we did not perform the standard antimicrobial assays used to determine the minimal inhibitory concentrations of cysteine-stabilized AMPs (Yang et al 2000;Seo et al 2005;Gueguen et al 2009). However, rApBD1 possesses an integral N-terminal domain enabling to study the potential aggregation properties of this peptide and compare them to the bacterially-triggered formation of nanonets that was described for Cg-BigDef1 (Loth et al 2019). We therefore assessed the potential formation of rApBD1 aggregates upon contact with bacteria.…”

“…Studies related to the antimicrobial activities of big defensins have included assays testing the native polypeptide isolated from the horseshoe crab hemocytes (Saito et al 1995), recombinant polypeptide produced in bacteria (Teng, Gao and Zhang 2012) and yeast (Zhao et al 2007), and synthetic peptides obtained by chemical native ligation (Loth et al 2019). In those studies, all big defensins showed antibacterial activity against Gram-positive, Gram-negative bacteria and fungi.…”