A Family of CSαβ Defensins and Defensin-Like Peptides from the Migratory Locust, <i>Locusta migratoria</i>, and Their Expression Dynamics during Mycosis and Nosemosis

<div><p>Insect defensins are effector components of the innate defense system. During infection, these peptides may play a role in the control of pathogens by providing protective antimicrobial barriers between epithelial cells and the hemocoel. The cDNAs encoding four defensins of the migratory locust, <i>Locusta migratoria</i>, designated LmDEF 1, 3–5, were identified for the first time by transcriptome-targeted analysis. Three of the members of this CSαβ defensin family, LmDEF 1, 3, and 5, were detected in locust tissues. The pro regions of their sequences have little-shared identities with other insect defensins, though the predicted mature peptides align well with other insect defensins. Phylogenetic analysis indicates a completely novel position of both LmDEF 1 and 3, compared to defensins from hymenopterans. The expression patterns of the genes encoding LmDEFs in the fat body and salivary glands were studied in response to immune-challenge by the microsporidian pathogen <i>Nosema locustae</i> and the fungus <i>Metarhizium anisopliae</i> after feeding or topical application, respectively. Focusing on <i>Nosema</i>-induced immunity, qRT-PCR was employed to quantify the transcript levels of <i>LmDEFs</i>. A higher transcript abundance of <i>LmDEF5</i> was distributed more or less uniformly throughout the fat body along time. A very low baseline transcription of both <i>LmDEFs</i> 1 and 3 in naïve insects was indicated, and that transcription increases with time or is latent in the fat body or salivary glands of infected nymphs. In the salivary glands, expression of <i>LmDEF3</i> was 20-40-times higher than in the fat body post-microbial infection. A very low expression of <i>LmDEF3</i> could be detected in the fat body, but eventually increased with time up to a maximum at day 15. Delayed induction of transcription of these peptides in the fat body and salivary glands 5–15 days post-activation and the differential expression patterns suggest that the fat body/salivary glands of this species are active in the immune response against pathogens. The ability of <i>N</i>. <i>locustae</i> to induce salivary glands as well as fat body expression of defensins raises the possibility that these AMPs might play a key role in the development and/or tolerance of parasitic infections.</p></div

Structural homology of LmDEFs to known antiprotozoal defensin.

<p>a, Alignment of LmDEF1, -3, and -5 with the antiprotozoal <i>Phlebotomus duboscqi</i> defensin (PhdDEF: Boulanger <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161585#pone.0161585.ref011" target="_blank">11</a>]). Percentage identity of LmDEFs to PhdDEF are shown at the end of each individual sequence; ^a denote the % identity with PhdDEF full peptide excluding the signal peptide, while ^b is denoting that % with the PhDEF mature active peptide. The amino acid residues are colored according to their physicochemical properties (red: small+ hydrophobic [incl. aromatic -Y]; blue: acidic; magenta: basic–H; green: hydroxyl + sulfhydryl + amine + G). The symbols under the alignment indicate: (*) identical sites; (:) conserved sites; (.) less conserved sites. The boxes indicate the six conserved cysteines; the conserved disulfide bridges are shown by # above these boxes (1–1; 2–2; 3–3). The active peptide cleavage site in PhdDEF is marked with a triangle; while the prodefensin is marked by a line. b, Three-dimensional <i>in silico</i> structure of “active” PhdDEF based on PDB entry 1icaA (defensin A of <i>Protophormia terraenovae</i>) as a template. The homology modeling was carried out with RaptorX; 40(100%) residues were modeled (<i>p</i>-value 9.45e-05). c, The secondary structure elements of PhdDEF predicted by ProFunc server for the purpose of comparison between it and LmDEFs (reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161585#pone.0161585.g002" target="_blank">Fig 2</a>). It comprises 1 sheet, 1 beta hairpin, 1 beta bulge, 2 strands, 1 helix, 4 beta turns, and 3 disulfides.</p

Multiple alignments of newly identified LmDEFs with other insect CSαβ defensins.

<p>Residues conserved in >50% of proteins are shaded. The numbers to the right refer to the position of the last residue of each line. Signal peptides are underlined. Bars indicate gaps to optimize the alignments. The six conserved cysteine residues involved in disulfide bridges are gray shaded. Possible activation peptide cleavage sites are marked with a triangle; enzymatic processing sites (e.g. -KR↓) to release the mature peptides are double underlined. The latter was predicted and/or determined based on various bioinformatical tools (PeptideCutter prediction, the cleaver package, and others) and similarity searches with mature insect defensins from GenBank and reported in the literature. The secondary elements (loop, α-helix, and β-sheet) of insect defensins are indicated as follow: β1, β2, β3, and β4 are regions for potential β turns; A1 and A2 are regions for potential β strands; H is a region for potential α-helix; C1, C2, and C3 are the potential disulfide linkages; S are the regions for bends; T are regions for hydrogen-bonded turns of CSαβ defensins (Following Dassanayake <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161585#pone.0161585.ref010" target="_blank">10</a>]). Accession numbers for the selected insect defensins: AmelDEF, <i>Apis mellifera</i>: [GeneBank: NP_001011616.2]; NvitDEF, <i>Nasonia vitripennis</i>: [GeneBank: NP_001159944.1]; PhcapDEF, <i>Pediculus humanus corporis</i>: [GeneBank: XP_002432619.1]; PaptDEF, <i>Pyrrhocoris apterus</i>: [GeneBank: AGI17576.1]; RproDEF, <i>Rhodnius prolixus</i> [GeneBank: AAO74626.1]; DmelDEF, <i>Drosophila melanogaster</i> [GeneBank: AAO72500.1]; AgamDEF, <i>Anopheles gambiae</i> [GeneBank: ABB00983.1]; AcupDEF, <i>Anomala cuprea</i> [GeneBank: BAD77967.1]; OrhiDEF, <i>Oryctes rhinoceros</i> [GeneBank: BAA36401.1]; BmorDEF, <i>Bombyx mori</i> [GeneBank: BAG48202.1]; SexiDEF, <i>Spodoptera exigua</i> [GeneBank: AEW24427.1].</p

Tissue specificity and developmental expression patterns of <i>LmDEFs</i>.

<p>a, spatiotemporal expression of <i>LmDEFs</i> in <i>N</i>. <i>locustae</i> infected fat body and salivary gland cells. b, same, but with <i>M</i>. <i>anisopliae</i>. M, DNA ladder; h, healthy insect; 1i, 3i, 5i,7i, 10i, and 15i the RNA of tissues collected on the 1^st, 3^rd, 5^th, 7^th, 10^th, and 15^th days after inoculation with pathogens; <i>L</i>.<i>m</i>. actin, locust actin gene. <i>N</i>.<i>l</i>., <i>Nosema</i> spores and <i>M</i>.<i>a</i>, <i>Metarhizium</i> hyphae as positive controls with RT-PCR. The locust actin gene was used as a control for the integrity of the cDNA templates. Amplification products were analyzed on agarose gels and visualized by UV illumination after ethidium bromide staining. All tissues were dissected from gregarious locusts.</p

Real-time quantitative PCR profile of <i>LmDEF</i> transcripts following <i>N</i>. <i>locustae</i> infection in the fat body (FB) and salivary glands (SG) in relation to time.

<p>Transcript accumulation values were normalized to the constitutively expressed β-actin gene and expressed as a function of the reference condition according to the 2^−ΔΔC_T method. The bars indicate the relative changes in RNA levels compared with the average expression of each gene under non-infected control conditions. The error bars indicate the standard errors of the means. The results are the means of at least three independent experiments. The statistical annotations, including the letters indicating significance, among treatments are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161585#pone.0161585.s004" target="_blank">S4 Fig</a>.</p

Identity and similarity values among the four putative locust defensins.

<p>Identity and similarity values among the four putative locust defensins.</p

Phylogenetic analysis.

<p>The relationship between LmDEFs with other insect defensin was inferred using the Neighbor-Joining method. <i>Ixodes ricinus</i> defensin 2 (tick) was used as the out-group. LmDEFs are marked with solid squares. Accession numbers are written next to insect species. Only representatives for defensin-2 were used. More closely related insect defensins within this group were removed to facilitate phylogenetic analysis and representation; therefore, only different lineages of the defensin2 family were shown. Bootstrap (1000 replicates) values are indicated for each root. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 26 amino acid sequences that were aligned by Kalign. All ambiguous positions were removed for each sequence pair. The evolutionary analysis was conducted with MEGA6.</p

The expression pattern of <i>LmDEFs</i> in the fat body and salivary glands in locusts infected with <i>Nosema</i> on the 1^st, 3^rd, 10^th, and 15^th days post-infection in comparison to control at each time.

<p>PCR products, using qRT-PCR primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161585#pone.0161585.s006" target="_blank">S2 Table</a>), were ran on 1.2% agarose gels and visualized by ethidium bromide staining. M, DNA-ladder; numbers preceding characters are gene numbers in healthy locust (h), or infected locust (i); A-actin gene.</p

Homology modeling of LmDEFs.

<p>a, Representation of the homology-derived solution structure of LmDEFs (a1-a4). α-helix (red), strand or β-sheet (blue), and coil (gray); the images were optimized with the Protein Picture Generator v1.21. Secondary structure elements were predicted with ProFunc server at EMBL-EBI; key: Helix-Strand: purple; helices labelled H1, H2,…and strands by their sheets A, B,…<i>Motifs</i>: β beta, turn γ gamma turn, and beta hairpin (red arch). Disulfide bond (1–1, 2–2, 3–3). b, Electrostatic potential distribution on the peptide surfaces (b1-b4). Positive potential is shown in blue, and negative potential is in red; the contouring value of the potential is in kT/e; the bar at the bottom (-5 to +5). The images were drawn using PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA).</p