Biochimica et Biophysica Acta (BBA) - General Subjects
A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes
Graphical abstract
Introduction
Antimicrobial peptides (AMPs) have become recognized as important components of the innate immune system in a variety of organisms, ranging from plants to animals [1], [2]. In invertebrates, which lack the so-called acquired immunity, the role of AMPs is believed to be even stronger. Most eukaryotic antimicrobial peptides are positively charged and contain hydrophobic amino acids. Although highly diverse in their primary structures, AMPs share a common feature of amphipathicity. Most cationic AMPs are membrane-active: they insert into and destabilize microbial membranes by pore formation or detergent effect [3]. Alternatively, AMPs can translocate across membranes and/or inhibit fundamental metabolic functions of microorganisms [4]. AMPs have been tentatively classified into three main classes according to their amino acid compositions and three-dimensional structures: (i) linear peptides tending to adopt α-helical amphipathic structures, (ii) peptides stabilized by disulfide bonds, which form β-sheet or mixed α-helix/β-sheet structures, and (iii) peptides with an over-representation of particular amino acids such as Arg, Gly, Pro or Trp, which adopt extended structures [1].
Major progress has been made over the past decade in the antimicrobial response of economically relevant species of marine molluscs and crustaceans [5], [6]. In particular, due to their major production worldwide and to the huge economic losses caused by infectious diseases, penaeid shrimps have been the subject of a significant investigative effort. Viruses and to a lesser extent bacteria are the most serious shrimp pathogens [7], [8], fungal diseases (mainly due to Fusarium) being less frequent, but severe and deleterious [9]. Several families of gene-encoded peptides displaying antifungal activities have been reported in shrimp, which could contribute to their innate capacity to fight fungal infections. Penaeidins, which have been identified in penaeid shrimp only, are both active against bacteria and filamentous fungi [10], [11]. Their antifungal activity is believed to be mediated by their ability to bind chitin [12]. In addition to penaeidins, antilipopolysaccharide factors (ALFs), which form a functionally divergent family of AMPs from crustaceans [13], are essential in shrimp defense against fungal, bacterial and viral infections [14], [15]. The three-dimensional structures of both penaeidins and ALFs have been determined [16], [17]. Finally, stylicins, which form a family of LPS-binding AMPs isolated from penaeid shrimp, were shown to display potent activities against filamentous fungi [18].
Besides gene-encoded AMPs, a 23 amino acid-antifungal peptide termed PvHCt (Fig. 1) was shown to participate to penaeid shrimp antimicrobial defense. PvHCt results from the proteolytic cleavage of the C-terminal end of the Litopenaeus vannamei hemocyanin, the major protein of shrimp plasma, in response to a microbial challenge [19]. PvHCt displays a high content in histidine (5 out of 23 residues), and also contains 3 anionic residues. Although most of the known eukaryotic AMPs are gene-encoded cationic peptides, there are now a substantial number of anionic AMPs, which like PvHCt are encrypted within the primary sequence of precursor proteins and released by proteolysis in response to infection [20]. PvHCt corresponds to the most acidic C-terminal sequence of L. vannamei hemocyanins (gene accession number AHY8647) (Fig. 1). Generation of AMPs by cleavage of hemocyanin C-terminus was also described in the crayfish Pacifastacus leniusculus, with a 16 amino acid antibacterial peptide called astacidin-1 [21]. Contrary to astacidin-1, PvHCt from L. vannamei and the longer PsHCt-1 and -2 from Litopenaeus stylirostris are antifungal but not antibacterial [19]. To our knowledge, this family of antifungal peptides is the only one to be characterized in penaeid shrimp species. PvHCt is the smallest antimicrobial peptide isolated from L. vannamei plasma. It has a broad spectrum of antifungal activity with minimum inhibitory concentrations (MICs) in the range 3–50 μM. In particular, it inhibits the spore germination of the shrimp pathogen Fusarium oxysporum [19]. While the mechanisms underlying the antibacterial activity of AMPs have often been studied in details, much less attention has been paid to the mechanism of action of antifungal peptides from animal species. Most of the current knowledge comes from plant defensins, a family of cysteine-stabilized αβ defensins that display a broad diversity of antifungal mechanisms of action [22].
Here we elucidated the mechanism underlying the strictly antifungal activity exerted by the shrimp hemocyanin-derived peptide PvHCt. By a combination of circular dichroism (CD) and 1H NMR techniques, we showed that PvHCt is an amphipathic α-helical peptide that acquires its three-dimensional structure under membrane-mimicking conditions. We showed that PvHCt strongly binds to the surface of F. oxysporum hyphae and induces local membrane damages that result in cytoplasm gradual degeneration and fungal cell death.
Section snippets
Materials
Egg phosphatidylcholine (PC), ergosterol, cholesterol and melittin from venom of the honey bee Apis mellifera were from Sigma-Aldrich (France). Carboxyfluorescein (CF) was from Eastman Kodak (France). All reagents used were of analytical grade.
Synthetic peptides
Synthetic peptides (purity > 98%) either unprotected (PvHCt; MW = 2750 kDa) or acetylated at the N-terminus and amidated at the C-terminus (Ace-PvHCt-NH2; MW = 2791 kDa) were purchased from Genecust and Genepep, respectively. Peptide purity and mass identity
PvHCt adopts a helical conformation in membrane-mimicking media
The structure of PvHCt, the 23 residue C-terminal peptide resulting from cleavage of the shrimp L. vannamei hemocyanin, was studied using two synthetic peptides, PvHCt and Ace-PvHCt-NH2. PvHCt is a free N- and C-terminus peptide devoid of posttranslational modification, which is identical to the original natural peptide and was used previously for the initial study of its spectrum of activity [19]. Ace-PvHCt-NH2 is the N-acetylated and C-amidated analog of PvHCt, which was used here to examine
Discussion
We characterized here the three-dimensional structure and antifungal mechanism of action of PvHCt, a hemocyanin-derived antifungal peptide released in shrimp plasma in response to infection. Results from the present article showed that PvHCt adopts an α-helical structure in membrane-mimicking media and specifically inserts into fungal membranes thereby creating severe damages to fungal spores and hyphae leading to cytoplasm degeneration and ultimately cell death.
Conflict of interest
We declare no conflict of interest.
Author contributions
SR and DDG have contributed equally to this work. They designed and managed the overall project. EB and JP helped design the study. VWP and AB performed NMR measurements and molecular modeling. VWP performed CD experiments. JLR performed fluorescence microscopy experiments. CG purified the peptides and acquired the liposome permeabilization data. VWP, JD and CD acquired the electron microscopy data. SR and DDG wrote the manuscript with the help of the other authors.
Transparency document
Acknowledgments
Work carried out in Paris was supported by funding from the Muséum National d'Histoire Naturelle (MNHN) and the CNRS, and work based in Montpellier was funded by Ifremer and the CNRS. We are pleased to thank Dr. Guillaume Charrière and the Montpellier RIO Imaging platform for their precious help in fluorescence microscopy experiments. We are grateful to M. Vandervennet and G. Gastine for their technical assistance in microbiology. We also thank the Analytical Platform of the National Museum of
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Present address: Laboratoire de Recherche sur la Réparation et la Transcription dans les cellules Souches (LRTS), Institut de recherche en Radiobiologie Cellulaire et Moléculaire (IRCM), Direction des Sciences du Vivant, Commissariat à l'Energie Atomique (CEA), 92265 Fontenay-aux-Roses Cedex, France.
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Both authors have equally contributed to the present article.