Research ArticleORGANISMAL BIOLOGY

A comprehensive portrait of the venom of the giant red bull ant, Myrmecia gulosa, reveals a hyperdiverse hymenopteran toxin gene family

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Science Advances  12 Sep 2018:
Vol. 4, no. 9, eaau4640
DOI: 10.1126/sciadv.aau4640
  • Fig. 1 The polypeptidic venom composition of M. gulosa.

    (A) M. gulosa. (B) Total ion chromatogram of M. gulosa venom. Venom (2 μg) was separated by C18 ultrahigh-performance liquid chromatography (uHPLC), using a gradient of 1 to 40% solvent B [90% acetonitrile (ACN) and 0.1% FA] over 45 min and analyzed using MS/MS. Peaks corresponding to identified venom peptides are labeled. Peaks identified as corresponding to partial fragments of larger peptides are labeled with an asterisk. (C) Venom apparatus of M. gulosa. (D) Venom component-encoding transcripts (that is, those encoding peptides detected in the venom itself) comprised 15.9% of total expression. Of these, transcripts encoding aculeatoxin peptides, MIITX2-Mg1a, MIITX3-Mg1a, and venom proteins comprised 93.0, 3.4, 3.3, and 0.3%, respectively, of venom component expression. EGF, epidermal growth factor. (E) Venom component-encoding transcripts (highlighted in red) are found exclusively in the highly expressed portion of the venom apparatus transcriptome, where they constitute most of the most highly expressed transcripts. Inset: Zoom showing the top 50 transcripts (with a linear scale on the y axis). The most highly expressed nonvenom component-encoding transcripts are labeled [cytochrome c oxidase subunits 1 (COI), 2 (COII), and 3 (COIII)].

  • Fig. 2 Venom peptides of the aculeate Hymenoptera.

    Precursor sequences of aculeate Hymenoptera venom peptides (encoding experimentally validated venom peptides) are aligned on the basis of signal and propeptide regions. Mature peptide regions are diverse in sequence (and have not been aligned). Methionine, lysine/arginine, aspartate/glutamate, and cysteine residues are highlighted in purple, blue, red, and yellow, respectively. Posttranslational modifications are not shown. aTorres et al. (22); bKazuma et al. (21); cBouzid et al. (23).

  • Fig. 3 Action of M. gulosa venom peptides on vertebrate sensory neurons.

    (A) Application of whole M. gulosa venom to DRG cells produced a rapid, non–cell-specific increase in [Ca2+]i followed by release of fluorescent dye into the medium. Each trace represents a single cell in the field of view. Snapshots shown of the recording are at 0 s (baseline), 33 s (3 s after addition of venom/fraction), and 150 s. (B) Three different peptides [fractions 8, 24, and 33 (f8, f24, and f33, respectively)] were purified from the whole venom using a single RP-HPLC step. Inset: Reanalysis of purified fractions by RP-HPLC. (C) Fraction 8 (MIITX2-Mg1a) had no effect on [Ca2+]i in sensory neurons, fraction 24 (MIITX1-Mg1a) caused an increase in [Ca2+]i in all sensory neurons, while fraction 33 (MIITX1-Mg2a) induced nonspecific permeabilization of cell membranes.

  • Fig. 4 Defensive and predatory function and mechanism of action of MIITX1-Mg1a.

    (A) Intraplantar injection of MIITX1-Mg1a causes spontaneous dose-dependent nocifensive behavior in mice, lasting for up to 7 min (n = 6 per group). Data are expressed as means ± SEM. Statistical significance compared to vehicle control was determined using two-way analysis of variance (ANOVA) with Sidak’s multiple comparison test. *P < 0.05. (B) Intra-abdominal injection of MIITX1-Mg1a in crickets (A. domesticus) caused dose-dependent, reversible, and nonlethal incapacitation (inability to right after being placed on back). Effects were observed at doses ≥1 μg/g (0.3 nmol/g), and at the maximum dose tested (60 μg/g, 19 nmol/g), incapacitation lasted an average of 53 min. (C) MIITX1-Mg1a caused an increase in [Ca2+]i in DRG cells, which was mostly blocked in the presence of TTX. (D) Representative sodium currents obtained in HEK293 cells heterologously expressing hNaV1.7, in the presence and absence of 3 μM MIITX1-Mg1a. Currents were obtained by a 50-ms voltage pulse to −20 mV from a holding potential of −90 mV. Compared with buffer control, in hNaV1.7-expressing cells, MIITX1-Mg1a did not change peak current or activation/inactivation kinetics, and it did induce persistent currents. (E) In HEK293 cells [independent of hNaV1.7 expression or the presence of TTX (1 μM)], MIITX1-Mg1a alters membrane potential and causes Na+ and Ca2+ influx in a concentration-dependent manner. Concentration-response curves were evaluated by fitting the data with a four-parameter Hill equation with variable slope. FLIPR data are expressed as the means ± SEM and are representative of at least three independent experiments. AUC, area under the curve.

  • Table 1 Polypeptide venom components of M. gulosa.

    TPM, transcripts per million.

    Mass (Da)TPMSequenceFeatures
    MIITX1-Mg1a3144.8530120GLGRLIGKIAKKGAKIAAEAAANAAAEKAAEAL*
    MIITX1-Mg2a8482.7829318LLSKDQALKHVWGVLKKLGKAAMEYVIQQICAKYNKK1 Cys (homodimer)
    MIITX1-Mg3a4325.3216248KNEETMEEALKGLNELKERLKKQGIDTAALNLDEKLLT
    MIITX-Mg4a9220.8511836SLVGCPRPDFLPSWNRCKSCVCKNNKLKCPKILKGSLLKTAA5 Cys (homodimer)
    MIITX-Mg4b8876.522518SLVGCPRPNFLPSWNRCKCICKNNKPMCRKLPNLLKTTA5 Cys (homodimer?)
    MIITX1-Mg5a2516.409654SINVKNLMDMIREQITSRLKK
    MIITX1-Mg5b2515.423556SINVKNLMNMIREQITSRLKK
    MIITX1-Mg6a1509.809484FRGPCLKIKGYKC2 Cys
    MIITX1-Mg7a7110.848100KRSKSSSKTKPKKPKKPKKKIKIPDWVKSGGKMVGEAVAGAVADAAVSAVMDAAVGTTAEPEQ3 HexNAc
    MIITX1-Mg7b5596.047808KRRRGLKKIIGKVIKGTGKVAGEAAASAVADAAVSAAIDAVVGTTEEPEQ3 HexNAc
    MIITX1-Mg7c5736.174343KRRRRLRKIIRKVIKGTGKVAGEAAASAVAGAAVSAAIDAAVGTTEEPEQ3 HexNAc?
    MIITX1-Mg8a7632.291027WLGALFSFIRFIAPYVIRAVRVLIQVVSKVVKPAKVAMKYAKKIATNVAKDVAKDMATDIAIDTITGGDE
    MIITX1-Mg8b7373.97773WLGALFSFIRFIAPYVIKAIRFVVQVASKLVKPAKAAIKFAKNIAKDIAEDMAMDFAMDVITGGDDE
    MIITX1-Mg9a3246.96861NIKWSKYAKKVGKVIVKHGIPLAASIALSQ*
    MIITX2-Mg1a5787.375354DISDYGDPCSDDLKDYCIHGDCFFLKELNQPACRCYTGYYGSRCEHIDHN6 Cys
    MIITX1-Mg10a?11791
    MIITX3-Mg1a~19,4005208
    CAP~24,200378
    Phosphatase~43,70090
    Esterase~61,80059
    Hyaluronidase~38,70026
    DPP-4~86,500233

    *C-terminal amidation.

    The TPM value is the sum of more than one assembled transcript encoding the same peptide.

    Predicted mass (complete primary structure not confirmed by MS/MS).

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaau4640/DC1

      Supplementary Materials and Methods

      Results

      Fig. S1. MALDI-MS analysis of M. gulosa venom apparatus.

      Fig. S2. Coelution of native (purified from venom) and synthetic MIITX1-Mg1a.

      Fig. S3. MIITX1-Mg1a–induced changes in paw withdrawal.

      Table S1. Venom- or toxin-associated annotation of nonvenom component transcripts.

      Table S2. Assessment of antimicrobial, cytotoxic, and hemolytic activity of MIITX1-Mg1a.

      Movie S1. Collection of venom from M. gulosa.

    • Supplementary Materials

      The PDF file includes:

      • Supplementary Materials and Methods
      • Results
      • Fig. S1. MALDI-MS analysis of M. gulosa venom apparatus.
      • Fig. S2. Coelution of native (purified from venom) and synthetic MIITX1-Mg1a.
      • Fig. S3. MIITX1-Mg1a–induced changes in paw withdrawal.
      • Table S1. Venom- or toxin-associated annotation of nonvenom component transcripts.
      • Table S2. Assessment of antimicrobial, cytotoxic, and hemolytic activity of MIITX1-Mg1a.

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      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.mov format). Collection of venom from M. gulosa.

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