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

ABSTRACT: Big defensins, ancestors of ?-defensins, are composed of a ?-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward ?-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the ?-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate ?-defensins.IMPORTANCE ?-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human ?-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the ?-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 ?-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 ?-defensin derivatives active at physiological and high salt concentrations.

SUBMITTER: Loth K

PROVIDER: S-EPMC6805989 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Loth Karine K Vergnes Agnès A Barreto Cairé C Voisin Sébastien N SN Meudal Hervé H Da Silva Jennifer J Bressan Albert A Belmadi Nawal N Bachère Evelyne E Aucagne Vincent V Cazevielle Chantal C Marchandin Hélène H Rosa Rafael Diego RD Bulet Philippe P Touqui Lhousseine L Delmas Agnès F AF Destoumieux-Garzón Delphine D

mBio 20191022 5

Big defensins, ancestors of β-defensins, are composed of a β-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward ...[more]

PMID: 31641083

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Project description:Big defensins are antimicrobial polypeptides believed to be the ancestors of ?-defensins, the most evolutionary conserved family of host defense peptides (HDPs) in vertebrates. Nevertheless, big defensins underwent several independent gene loss events during animal evolution, being only retained in a limited number of phylogenetically distant invertebrates. Here, we explore the evolutionary history of this fascinating HDP family and investigate its patchy distribution in extant metazoans. We highlight the presence of big defensins in various classes of lophotrochozoans, as well as in a few arthropods and basal chordates (amphioxus), mostly adapted to life in marine environments. Bivalve mollusks often display an expanded repertoire of big defensin sequences, which appear to be the product of independent lineage-specific gene tandem duplications, followed by a rapid molecular diversification of newly acquired gene copies. This ongoing evolutionary process could underpin the simultaneous presence of canonical big defensins and non-canonical (?-defensin-like) sequences in some species. The big defensin genes of mussels and oysters, two species target of in-depth studies, are subjected to gene presence/absence variation (PAV), i.e., they can be present or absent in the genomes of different individuals. Moreover, big defensins follow different patterns of gene expression within a given species and respond differently to microbial challenges, suggesting functional divergence. Consistently, current structural data show that big defensin sequence diversity affects the 3D structure and biophysical properties of these polypeptides. We discuss here the role of the N-terminal hydrophobic domain, lost during evolution toward ?-defensins, in the big defensin stability to high salt concentrations and its mechanism of action. Finally, we discuss the potential of big defensins as markers for animal health and for the nature-based design of novel therapeutics active at high salt concentrations.

Project description:BACKGROUND: Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. FINDINGS: Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate ?-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CS??-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. CONCLUSIONS: We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression.

Project description:Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic beta-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.

Project description:β-Defensins protect the respiratory tract against the myriad of microbial pathogens entering the airways with each breath. However, this potentially hostile environment is known to serve as a portal of entry for herpesviruses. The lack of suitable respiratory model systems has precluded understanding of how herpesvirus virions overcome the abundant mucosal β-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits β-defensins to invade its host and initiate viral spread. The equine β-defensins (eBDs) eBD1, -2, and -3 were produced and secreted along the upper respiratory tract. Despite the marked antimicrobial action of eBD2 and -3 against many bacterial and viral pathogens, EHV1 virions were resistant to eBDs through the action of the viral glycoprotein M envelope protein. Pretreatment of EHV1 virions with eBD2 and -3 increased the subsequent infection of rabbit kidney (RK13) cells, which was dependent on viral N-linked glycans. eBD2 and -3 also caused the aggregation of EHV1 virions on the cell surface of RK13 cells. Pretreatment of primary equine respiratory epithelial cells (EREC) with eBD1, -2, and -3 resulted in increased EHV1 virion binding to and infection of these cells. EHV1-infected EREC, in turn, showed an increased production of eBD2 and -3 compared to that seen in mock- and influenza virus-infected EREC. In addition, these eBDs attracted leukocytes, which are essential for EHV1 dissemination and which serve as latent infection reservoirs. These novel mechanisms provide new insights into herpesvirus respiratory tract infection and pathogenesis.IMPORTANCE How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of host-specific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine β-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution.

Project description:BACKGROUND:X-tox proteins are a family of immune-related proteins only found in Lepidoptera and characterized by imperfectly conserved tandem repeats of several defensin-like motifs. Previous phylogenetic analysis of X-tox genes supported the hypothesis that X-tox have evolved from defensins in a lineage-specific gene evolution restricted to Lepidoptera. In this paper, we performed a protein study in which we asked whether X-tox proteins have conserved the antimicrobial functions of their ancestral defensins and have evolved as defensin reservoirs. METHODOLOGY/PRINCIPAL FINDINGS:We followed the outcome of Spod-11-tox, an X-tox protein characterized in Spodoptera frugiperda, in bacteria-challenged larvae using both immunochemistry and antimicrobial assays. Three hours post infection, the Spod-11-tox protein was expressed in 80% of the two main classes of circulating hemocytes (granulocytes and plasmatocytes). Located in secretory granules of hemocytes, Spod-11-tox was never observed in contact with microorganisms entrapped within phagolyzosomes showing that Spod-11-tox is not involved in intracellular pathogen killing. In fact, the Spod-11-tox protein was found to be secreted into the hemolymph of experimentally challenged larvae. In order to determine antimicrobial properties of the Spod-11-tox protein, it was consequently fractionated according to a protocol frequently used for antimicrobial peptide purification. Over the course of purification, the anti-Spod-11-tox immunoreactivity was found to be dissociated from the antimicrobial activity. This indicates that Spod-11-tox is not processed into bioactive defensins in response to a microbial challenge. CONCLUSIONS/SIGNIFICANCE:Altogether, our results show that X-tox proteins have not evolved as defensin reservoirs and have lost the antimicrobial properties of the ancestral insect defensins. The lepidopteran X-tox protein family will provide a valuable and tractable model to improve our knowledge on the molecular evolution of defensins, a class of innate immune effectors largely distributed over the three eukaryotic kingdoms.

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

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

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