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. 2016 Jul 7:6:29538.
doi: 10.1038/srep29538.

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami

Volker Herzig  1 , Maria Ikonomopoulou  1 , Jennifer J Smith  1 , Sławomir Dziemborowicz  2 , John Gilchrist  3 , Lucia Kuhn-Nentwig  4 , Fernanda Oliveira Rezende  5 , Luciano Andrade Moreira  5 , Graham M Nicholson  2 , Frank Bosmans  3 , Glenn F King  1
Affiliations

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami

Volker Herzig et al. Sci Rep. .

Abstract

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

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Figures

Figure 1
Figure 1. Isolation of Ae1a.
Chromatogram resulting from fractionation of crude Augacephalus ezendami venom using C18 RP-HPLC. The dashed line indicates the gradient of solvent B (90% acetonitrile/0.05% TFA). The shaded peak containing the active peptide Ae1a was lethal when injected into Drosophila melanogaster. The inset shows the MALDI MS spectrum of purified Ae1a.
Figure 2
Figure 2. Production of recombinant Ae1a.
RP-HPLC chromatogram showing final purification of rAe1a (shaded peak). The dashed line indicates the gradient of solvent B (90% acetonitrile/0.043% TFA). The inset is an SDS-PAGE gel showing cleavage of the MBP-Ae1a fusion protein with TEV protease. Lanes 1 and 2 correspond to pre- and post-cleavage samples, while lane M contains molecular weight markers (masses indicated in kDa). The bands corresponding to uncleaved and cleaved fusion protein are indicated. Note that this image is cropped from the original gel scan, which is shown in its entirety as Supplementary Figure 1, but is otherwise unmodified.
Figure 3
Figure 3. Insecticidal effects of Ae1a.
(A) rAe1a was injected into blowflies (Lucilia cuprina) and paralytic effects were measured 0.5 and 1 h after injection. (B) rAe1a was injected into fruit flies (Drosophila melanogaster) and paralytic and lethal effects were measured at 3 h and 24 h post injection. (C) rAe1a and Hv1a were injected into the triatomine bug Rhodnius prolixus and lethality was measured 24 h after injection. PD50 and LD50 values were calculated as described previously.
Figure 4
Figure 4. Effect of rAe1a on BgNav1 and hNaV1.5.
Left panels: Representative traces showing the effect of rAe1a (200 nM) on sodium currents mediated by (A) BgNaV1, (C) mutant BgNaV1 (H805Y/D812E), and (E) hNaV1.5 heterologously expressed in Xenopus oocytes. Currents were evoked by a depolarization to −15 mV, with black and red traces corresponding to the current before and after toxin application, respectively. Right panels: Effect of 200 nM rAe1a on normalized conductance-voltage (G–V) relationships (G/Gmax) and steady-state inactivation (SSI) relationships (I/Imax) for (B) BgNaV1, (D) mutant BgNaV1, and (F) hNaV1.5. G/Gmax and I/Imax are shown by closed and open circles respectively, before (black) and after (red) toxin addition. Normalization was performed relative to the peak current before toxin addition. Oocytes were depolarized by steps of 5 mV from a holding potential of −90 mV up to 5 mV for 50 ms, followed by a depolarizing pulse to −15 mV for 50 ms. Peak current from the initial step series was converted to conductance and normalized to obtain the G-V relationship while peak current from the following −15 mV voltage depolarization step was normalized to yield the SSI relationship. rAe1a inhibited the mutant BgNaV1 channel to a lesser extent than wild-type BgNaV1, and it had no effect on the human NaV1.5 channel. Data points are mean ± SEM, and n = 3–5 for all experiments shown.
Figure 5
Figure 5. Effect of rAe1a on NaV channel currents in P. americana DUM neurons.
(A) Representative traces showing the effect of 1 μM rAe1a on INa mediated by PaNaV1 in P. americana DUM neurons. Currents were evoked by depolarizations to −10 mV, from a holding potential of −90 mV (panel B), with black and red traces corresponding to the current before, and 5 min after, toxin application, respectively. (C) Time course of rAe1a actions on PaNaV1. Peak INa were recorded at a rate of 0.1 Hz before (black circles), and for 5 min during, application of 1 μM rAe1a (red circles) and normalized against maximal peak INa (−1/Imax; n = 3). (D) GNa-V relationships in P. americana DUM neurons. A voltage protocol with depolarisation steps from −90 mV to +40 mV in 10 mV increments (lower panel B) was used to generate families of INa. The normalized conductance-voltage (GNa-V) relationship (G/Gmax) is shown before (black circles) and after (red circles) a 5 min perfusion with 1 μM rAe1a (n = 4). Conductance was normalized relative to the peak current before toxin addition.
Figure 6
Figure 6. Alignment of the DII S1–S2 region of insect NaV channels and human NaV1.5.
Regions corresponding to the S1 and S2 transmembrane helices are shaded grey, and the numbering above the sequences corresponds to Blattella germanica BgNaV1. Sequence changes relative to the BgNaV1 sequence shown at the top of the alignment are highlighted in blue. The resistance-conferring Tyr at position 805 is highlighted in red when present. Note the vastly different S1–S2 loop sequence in the human NaV1.5 channel (bottom sequence).
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