Diversity of the Antimicrobial Peptide Genes in Collembola

Abstract

Multidrug-resistant bacteria are a current health crisis threatening the world's population, and scientists are looking for new drugs to combat them. Antimicrobial peptides (AMPs), which are part of the organism's innate immune system, are a promising new drug class as they can disrupt bacterial cell membranes. This study explored antimicrobial peptide genes in collembola, a non-insect hexapod lineage that has survived in microbe-rich habitats for millions of years, and their antimicrobial peptides have not been thoroughly investigated. We used in silico analysis (homology-based gene identification, physicochemical and antimicrobial property prediction) to identify AMP genes from the genomes and transcriptomes of five collembola representing three main suborders: Entomobryomorpha (Orchesella cincta, Sinella curviseta), Poduromorpha (Holacanthella duospinosa, Anurida maritima), and Symphypleona (Sminthurus viridis). We identified 45 genes belonging to five AMP families, including (a) cysteine-rich peptides: diapausin, defensin, and Alo; (b) linear α-helical peptide without cysteine: cecropin; (c) glycine-rich peptide: diptericin. Frequent gene gains and losses were observed in their evolution. Based on the functions of their orthologs in insects, these AMPs potentially have broad activity against bacteria, fungi, and viruses. This study provides candidate collembolan AMPs for further functional analysis that could lead to medicinal use.

Keywords: AMP evolution; AMP gene identification; antimicrobial peptide; collembola immunity; drug discovery.

Figure 1

Overview of the AMP gene identification pipeline used in this study. Key steps include transcriptome assembly, AMP genes identification from transcriptomes and genomes, prediction of physicochemical properties, AMP activity, 3D structure, and phylogenetic analysis.

Figure 2

Collembola AMP gene families and the number of genes. Five collembola AMP gene families (diapausin, Alo, diptericin, cecropin, and defensin) are reported in this study. Phylogenetic relationships of five collembola species and the divergent time are inferred from TimeTree.org (Date access: 15 July 2022). Species with genome data are indicated with an asterisk.

Figure 3

Sequence alignment and phylogenetic relationships of collembola diapausin: (a) protein alignment showing disulfide bridges between six conserved cysteine residues; (b) Phylogenetic tree of arthropod diapausins. Branch supports (aLRT) higher than 0.9 were indicated with black dots on tree nodes (Abbreviation: Fcan = F. candida, Hduo = H. duospinosa, Amar = A. maritima, Ocin = O. cincta, Scur = Si. curviseta, Svir = Sm. viridis, Slit = Spodoptera littoralis, Evar = Eumeta variegata, Mart = Machimus arthriticus, Gatr = Gastrophysa atrocyanea).

Figure 4

Sequence alignment and phylogenetic relationships of collembola Alo peptides: (a) protein alignment showing disulfide bridges between six conserved cysteine residues; (b) Phylogenetic tree of arthropod Alo peptides. Branch supports (aLRT) higher than 0.9 were indicated with black dots on tree nodes (Abbreviation: Hduo = H. duospinosa, Amar = A. maritima, Ocin = O. cincta, Scur = Si. curviseta, Apla = Agrilus planipennis, Alon = Acrocinus longimanus, Cmac = Callosobruchus maculatus, Prha = Platymeris rhadamanthus, Agif = Aphidius gifuensis, Msca = Megaselia scalaris, Aori = Agelena orientalis, Aape = Agelenopsis aperta, Hcur = Hololena curta, Dtin = Dinothrombium tinctorium).

Figure 5

Sequence alignment and phylogenetic relationships of collembola Diptericin: (a) protein alignment; (b) Phylogenetic tree of arthropod diptericins. Branch supports (aLRT) higher than 0.9 were indicated with black dots on tree nodes (Abbreviation: Ocin = O. cincta, Scur = Si. curviseta, Svir = Sm. viridis Dmoj = Drosophila mojavensis, Dnav = D. navojoa, Dvir = D. virilis, Dgri = D. grimshawi, Dalb = D. albomicans, Dleb = D. lebanonensis, Bdor = Bactrocera dorsalis, Ccap = Ceratitis capitata, Dpse = D. pseudoobscura, Dper = D. persimilis, Dgua = D. guanche, Dwil = D. willistoni, Dana = D. ananassae, Drho = D. rhopaloa, Dkik = D. kikkawai, Dmel = D. melanogaster, Dsim = D. simulans, Dsec = D. sechellia, Pter = Protophormia terraenovae, Anas = Armadillidium nasatum, Avul = A. vulgare, Aven = Araneus ventricosus).

Figure 6

Sequence alignment and phylogenetic relationships of collembola cecropins: (a) protein alignment; (b) Phylogenetic tree of arthropod cecropins. Branch supports (aLRT) higher than 0.9 were indicated with black dots on tree nodes (Abbreviation: Scur = Si. curviseta, Hcun = Hyphantria cunea, Sexi = Spodoptera exigua, Slit = Spodoptera litura, Bman = Bombyx mandarina, Bmor = B. mori, Hcec = Hyalophora cecropia, Obru = Operophtera brumata, Dple = Danaus plexippus, Gmel = Galleria mellonella, Evar = Eumeta variegata, Rfer = Rhynchophorus ferrugineus, Agam = Anopheles gambiae, Adar = A. darlingi, Aalb = Aedes albopictus, Aaeg = A. aegypti).

Figure 7

Sequence alignment and phylogenetic relationships of collembola defensin: (a) protein alignment showing disulfide bridges between six conserved cysteine residues; (b) Phylogenetic tree of arthropod defensins. Branch supports (aLRT) higher than 0.9 were indicated with black dots on tree nodes (Abbreviation: Svir = Sm. viridis, Mmar = Mesobuthus martensii, Osav = Ornithodoros savignyi, Smim = Stegodyphus mimosarum, Tmer = Tropilaelaps mercedesae, Lheb = Leiurus hebraeus, Aaus = Androctonus australis, Dvar = Dermacentor variabilis, Rmic = Rhipicephalus microplus, Isca = Ixodes scapularis, Ocor = Ornithodoros coriaceus, Tdis = Tityus discrepans, Sper = Sarcophaga peregrina, Cvic = Calliphora vicina, Pter = Protophormia terraenovae, Tcur = Temnothorax curvispinosus, Dleb = Drosophila lebanonensis, Dmoj = D. mojavensis, Dvir = D. virilis, Hill = Hermetia illucens, Scal = Stomoxys calcitrans, Rfer = Rhynchophorus ferrugineus, Hsal = Harpegnathos saltator, Sory = Sitophilus oryzae, Tcas = Tribolium castaneum, Bdor = Bactrocera dorsalis, Aver = Asbolus verrucosus, Tmol = Tenebrio molitor, Zatr = Zophobas atratus, Apla = Agrilus planipennis).

Figure 8

Prediction of antimicrobial activity: (a) heat map for the antimicrobial prediction of collembolan AMPs based on three programs (ClassAMP, iAmpPred, and Campr3). The probability value ranges from 0 to 1 and is indicated by the degree of color (SVM = Support Vector Machine, RF = Random Forest, ANN = Artificial Neural Network, DA = Discriminant Analysis, AB = antibacterial property, AV = antiviral property, AF = antifungal property); (b) hemolytic activity and strain-specific antimicrobial prediction using DBAASP, where Gram-positive bacteria include E. coli, P. aeruginosa, and K. pneumoniae; Gram-negative bacteria include S. aureus and B. subtilis; fungi include C. albicans and S. cerevisiae.