Bioinsecticides for the Control of Human Disease Vectors Niraj S Bende B

Bioinsecticides for the control of human disease vectors Niraj S Bende B. Pharm, MRes. Bioinformatics

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience

Abstract

Many human diseases such as malaria, Chagas disease, chikunguniya and dengue fever are transmitted via insect vectors. Control of human disease vectors is a major worldwide health issue. After decades of persistent use of a limited number of chemical insecticides, vector species have developed resistance to virtually all classes of insecticides. Moreover, considering the hazardous effects of some chemical insecticides to environment and the scarce introduction of new insecticides over the last 20 years, there is an urgent need for the discovery of safe, potent, and eco-friendly bioinsecticides.

To this end, the entomopathogenic fungus Metarhizium anisopliae is a promising candidate. For this approach to become viable, however, limitations such as slow onset of death and high cost of currently required spore doses must be addressed. Genetic engineering of Metarhizium to express insecticidal toxins has been shown to increase the potency and decrease the required spore dose. Thus, the primary aim of my thesis was to engineer transgenes encoding highly potent insecticidal spider toxins into Metarhizium in order to enhance its efficacy in controlling vectors of human disease, specifically mosquitoes and triatomine bugs. As a prelude to the genetic engineering studies, I surveyed 14 insecticidal spider venom peptides (ISVPs) in order to compare their potency against key disease vectors (mosquitoes and triatomine bugs)

In this thesis, we present the structural and functional analysis of key ISVPs and describe the engineering of Metarhizium strains to express most potent ISVPs. To facilitate this, an E. coli expression system was developed to produce sufficient amounts of each ISVP for functional and structural studies.

The five most potent ISVPs, namely U2-CUTX-As1a (As1a), ω/κ-hexatoxin-Hv1a (Hybrid), µ-

DGTX-Dc1a (Dc1a), U1-AGTX-Ta1a (Ta1a), and ω-hexatoxin-Hv1a (Hv1a) were successfully expressed in Metarhizium anisopliae-1630 (a mosquito specialist strain) and M. anisopliae-1548 (a bug specialist strain). Mosquitocidal assay of recombinant M. anisopliae-1630 strains revealed a significant improvement in virulence compare to wild type strain, with the engineered strains requiring less time to kill mosquitoes.

This work described in this thesis represents the first step towards the development of engineered Metarhizium strains with the potential to control vectors of human disease.

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

Publications during candidature

A) Peer-reviewed journal articles: - • Bende, N.S., Kang, E., Herzig V., Bosmans, F., Nicholson G.M., Mobli, M. & King,G.F. The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels. Biochemical Pharmacology 85, 1542- 1554 (2013).

• Klint, J.K., Senff, S., Saez, N.J., Seshadri, R., Lau, H.Y., Bende, N.S., Undheim, E. A. B., Rash, L. D., Mobli, M. & King, G.F. (2013). Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E.coli. PLoS ONE 8, e63865 (2013).

• Bende, N.S., Dziemborowicz, S., Mobli, M., Herzig, V., Gilchrist, J., Wagner, J., Nicholson G.M., King,G.F & Bosmans, F. A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nat Commun 5, 4350 (2014).

• Bende, N.S., Dziemborowicz, S., Herzig, V., Brown, G.W., Bosmans, F., Nicholson G.M., King,G.F &. Mobli, M. The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels. FEBS J, 13189 (2015).

B) Book Chapters: - • Herzig, V., Bende, N.S., Alam, M.S., Tedford, H.W., Kennedy, R.M. and King, G.F. (2014) Methods for deployment of spider-venom peptides as bioinsecticides. In: Advances in Insect Physiology: Insect Midgut and Insecticidal Proteins for Insect Control (Dhadialla, T.S. & Gill, S.S., eds), Chapter 5.

III Publications included in this thesis

1) Bende, N.S., Kang, E., Herzig V., Bosmans, F., Nicholson G.M., Mobli, M. & King,G.F. The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage- gated sodium channels. Biochemical Pharmacology 85, 1542- 1554 (2013).

Incorporated as Chapter 3.

Contributor Statement of contribution Bende, N.S. (My self) Designed and performed experiments (60%) Wrote the manuscript (45%) Kang, E. Performed experiments (15%) Herzig, V. Performed experiments (10%) Wrote manuscript (5%) Bosmans, F. Performed experiments (5%) Wrote manuscript (5%) Nicholson G.M. Wrote and edited manuscript (15%)

Mobli, M. Performed experiments (10%) Wrote manuscript (5%) King,G.F. Designed the research (40%) Wrote and edited manuscript (25%)

IV 2)

Bende, N.S., Dziemborowicz, S., Herzig, V., Brown, G.W., Bosmans, F., Nicholson G.M., King,G.F &. Mobli, M. The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels. FEBS J, 13189 (2015)

Incorporated as Chapter 4.

Contributor Statement of contribution Bende, N.S. (My self) Designed and performed experiments (50%) Wrote the manuscript (45%) Dziemborowicz, S. Performed experiments (15%) Wrote the manuscript (5%) Herzig, V. Performed experiments (10%) Brown, G.W. Performed experiments (5%)

Bosmans, F. Performed experiments (5%)

Nicholson G.M. Wrote and edited the manuscript (15%) Designed experiments (10%) King,G.F. Designed research (25%) Wrote and edited the manuscript (15%) Mobli, M. Designed and performed experiments (15%) Wrote and edited the manuscript (20%)

3) Bende, N.S., Dziemborowicz, S., Mobli, M., Herzig, V., Gilchrist, J., Wagner, J., Nicholson G.M., King,G.F & Bosmans, F. A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nat Commun 5, 4350 (2014)

Incorporated as Chapter 5.

Contributor Statement of contribution Bende, N.S. (My self) Designed and performed experiments (45%) Wrote the manuscript (40%) Dziemborowicz, S. Performed experiments (10%) Herzig, V. Performed experiments (5%) Gilchrist, J. Performed experiments (5%)

Wagner, J. Performed experiments (5%)

Nicholson G.M. Wrote and edited the manuscript (10%)

Mobli, M. Performed experiments (5%)

King,G.F. Designed research (30%) Wrote and edited manuscript (20%) Bosmans, F. Designed and performed experiments (25%) Wrote and edited the manuscript (30%)

VI Contributions by others to the thesis Chapter 6: Metarhizium genetic engineering Contributor Statement of contribution Bende, N.S. (My self) Designed experiments (70%) Performed experiments (100%) Wrote the chapter (100%) Dr Hsiao-Ling Lu Designed experiments (30%) Prof. Raymond St Leger Supervised Research (50%) King G.F. Supervised Research (50%)

Dr Luciaono Andrade Moreira contributed in bioassays of mosquitoes and triatomine bugs described in Chapter 2.

Statement of parts of the thesis submitted to qualify for the award of another degree

Portions of Chapter 2 were used in an MSc thesis submitted by Christopher Weir (2013).

VII Acknowledgements Completing this PhD has been a life changing experience for me. It would not have been possible without support and guidance I received from many people.

First and foremost, I wish to extend my heartfelt and sincere gratitude to my advisor Professor Glenn King, Institute for Molecular Bioscience, University of Queensland. Glenn is person you instantly love and never forget once you meet him. He is lively, enthusiastic, energetic and supportive and has provided me the freedom to do my research without objection. His expert supervision, guidance and encouragement made possible to complete this PhD. I am also very grateful to my associate advisor, Dr Mehdi Mobli for his scientific advice and many insightful discussions. With his mentoring efforts, I could solve NMR structures.

Special thanks to my thesis committee – Prof. Richard Lewis, Associate Prof. Bryan Fry and Associate Prof. Sassan Asgari for their guidance and helpful suggestions to shape-up this thesis.

I would like to thank my all wonderful collaborators – Prof. Raymond St. Leger, Dr Hsiao-ling Lu at University of Maryland; Prof. Frank Bosmans at John Hopkins University; Prof Graham Nicholson and Slawomir Dziemborowicz at University of Technology, Sydney; Luciaono Moreira at Fiocruz Institute, Brazil. It has been great pleasure in collaborating with you all.

Big thanks to all members of King group for creating such friendly and co-operative atmosphere. In particular, I would like to thank Dr Nausad Shaikh, Dr Julie Klint, Dr Sandy Pineda, Dr Maria Ikonomopoulou, Dr Margaret Hardy, Dr Brit Winnen, Dr David Morgenstern, Dr Raveendra Anangi, Dr Volker Herzig, Mr Carus Lau, Mr Sebastian Senff and Ms Jessie Er for insightful discussion and help in laboratory.

I would like to acknowledge all support staff of the Queensland Bioscience Precinct and Institute for Molecular Bioscience. Big thanks to Dr Amanda Carozzi and Cody Mudgway for their help in PhD scholarship application and admission. My all PhD milestones and candidature would have not been possible without your help Amanda. Also big thanks to our floor manager Mikiko Miyagi.

I am grateful to the Institute for Molecular Bioscience, UQ for providing world-class infrastructure and facilities to conduct this research and also University of Queensland for funding via UQ International Research Tuition Award (UQIRTA) and UQ Research Scholarship (UQRS).

My heartfelt thanks to my parents - Aai & Baba and my brother Dhiraj for always believing in me and encouraging me to follow my dreams. My in-laws – Mummy & Pappa for helping me in whatever way they could during this challenging period.

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Finally, I would like to dedicate this PhD to my beloved wife Teju, who has supported my decision to start this PhD, has been there always for me throughout this journey and without whom I would not have had completed this journey. My darling son Yash, who brought happiness in our lives and inspired me to complete this PhD.

IX Keywords bioinsecticides, disease vectors , insecticidal spider venom peptides, metarhizium genetic engineering, NMR spectroscopy , voltage-gated sodium channel, recombinant peptide, transgenic entomopathogens.

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060112, Structural biology, 40% ANZSRC code: 100202, Biological control, 30% ANZSRC code: 060505, Mycology, 30%

Fields of Research (FoR) Classification

FoR code: 0604, Genetics, 20% FoR code: 0601, Biochemistry and Cell Biology, 50% FoR code: 1002, Environmental Biotechnology, 30%

Table of Content

1 Introduction………………………………………………………………………………………………….1 1.1 Vector-borne disease ...... ……………………………………1 1.2 Vector control ...... 2 1.2.1 Importance of vector control ...... 3 1.3 Malaria ...... 5 1.3.1 Malaria management ...... 7 1.3.2 Why has malaria control failed? ...... 8 1.4 Chagas Disease ...... 9 1.4.1 Chagas Disease Management…………………………………………………………………………….9 1.4.2 Why has Chagas disease control failed? ...... 10 1.5 The problem of insecticide resistance ...... 11 1.6 How to manage insecticide resistance?...... 12 1.7 Entomopathogens as a biological control agent ...... 13 1.7.1 Metarhizium—mode of infection and pathogensis ...... 15 1.7.2 Metarhizium as a bioinsecticide ...... 16 1.7.3 Commercial development of entomopathogens as bioinsecticides ...... 17 1.7.4 Obstacles using Metarhizium as a bioinsecticide………………………………………………17 1.7.5 Genetic engineering of Metarhizium to improve virulence and tolerance ...... 18 1.7.6 Spiders: source of insecticidal toxins for Metarhizium genetic engineering……….18 1.8 Aim & objectives ...... 19 1.9 Thesis Outline ...... 20

2 Recombinant expression and assay of insecticidal spider venom peptides (ISVPs)………………………………………………………………………………………………………..21 2.1 Introduction ...... 21 2.2 Methods ...... 21 2.2.1 Periplasmic expression of ISVPs in E. coli ...... 21 2.2.2 Blowfly toxicity assay ...... 23 2.2.3 Mosquito and triatomine bug toxicity assay ...... 24 2.3 Results and Discussion ...... 25 2.3.1 Production of recombinant ISVPs ...... 25 2.3.2 ISVPs are paralytic and lethal to sheep blowflies ...... 26 2.3.3 ISVPs are lethal to mosquito and triatomine bugs ...... 28 2.3.4 ISVPs for Metarhizium genetic engineering ...... 29

3 The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels………………………………...... 30

4 The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels…………………………………………………….31

5 A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a……………………………………………………………………………….32

6 Metarhizium Genetic Engineering……………………………………………………………….33 6.1 Metarhizium Strain selection ...... 34 6.2 ISVP selection for Metrhizium genetic engineering ...... 34 6.3 Material and Methods ...... 35 6.3.1 Vector construction ...... 35 6.3.2 Metarhizium transformation ...... 36 XI

6.3.3 Transgenic Metarhizium genomic DNA extraction ...... 37 6.3.4 Characterization of expression of ISVP’s in Metarhizium ...... 38 6.3.5 Mosquitocidal Bioassay ...... 38 6.4 Results and Discussion ...... 41 6.4.1 Genetic transformation of Metarhizium ...... 41 6.4.2 ISVP gene expression is induced by insect hemolymph ...... 42 6.4.3 Transgenic M. anisopliae-1630 is more virulent to mosquitos ...... 42 6.4.4 Transgenic M. anisopliae -1548 ...... 46 6.4.5 Recombinant Metarhizium as a bioinsecticide ...... 47

7 Summarizing Discussion…………………………………………………………………………….48 7.1 Research findings ...... 48 7.1.1 Recombinant ISVP production ...... 48 7.1.2 Insect bioassay of ISVPs ...... 49 7.1.3 Mechanism of action of ISVPs ...... 50 7.1.4 Structure determination of ISVPs ...... 50 7.1.5 Metarhizium genetic engineering ...... 52 7.2 Future perspectives ...... 53 7.2.1 Combining GM Metarhizium & insecticides ...... 53 7.2.2 GM Metarhizium expressing multiple ISVPs ...... 53 7.2.3 GM Metarhizium to control agriculture pest ...... 54 7.2.4 Optimizing fungal production and efficacy ...... 54 7.2.5 Field trials of GM Metarhizium ...... 55 7.3 Conclusion ...... 56

List of References: ...... 57

Appendices………………………………………………………………………………………………………...66 Appendix A: Stock Solution and Media Plates………………………………………………………66 Appendix B: Methods for deployment of spider venom peptides as bioinsecticide……...69 Appendix C: Supplementary data- A distinct sodium channel voltage-°©-sensor locus determines insect selectivity of the spider toxin Dc1a…………………………………………………..70

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List of Figures

Figure 1.1:Global distribution of Malaria………………………………………………………………………..5

Figure 1.2: Life cycle of malaria parasite illustrating the different life stages of the parasite during infection of mosquitoes and humans……………………………………………………….6

Figure 1.3: Mechanisms of mosquito resistance to the four classes of WHO-approved public health insecticides………………………………………………………………………………………………12

Figure 1.4: Mechanism of pathogenesis of entomopathogenic Metarhizium…………………….16

Figure 2.1: Production of ISVPs……………………………………………………………………………………..23

Figure 2.2: Acute toxicity of ISVPs in sheep blowflies (L. cuprina)…………………………………...27

Figure 2.3: Acute toxicity of ISVPs in mosquitoes (A. aegypti)…………………………………...... 28

Figure 2.4: Acute toxicity of ISVPs in triatomine bugs (R. prolixus)...... 29

Figure 6.1: Map of the vector pPK2-bar-GFP-pMCL1-Hybrid…………………………………………...35

Figure 6.2: Growth and sporulation of wild type and transgenic M. anisopliae-1630 strains expressing selected ISVPs………………………………………………………………………………………………39

Figure 6.3: Mosquito bioassay……………………………………………………………………………………….40

Figure 6.4: Agarose gel showing PCR-based detection of ISVP transgenes in transformed M. anisopliae-1630…………………………………………………………………………………………………………41

Figure 6.5: Quantification of ISVPs in transgenic Metarhizium………………………………………...42

Figure 6.6: Effect of transgenic Metarhizium on mosquitoes…………………………………………...43

Figure 6.7: Bioassay of transgenic Metarhizium against mosquitoes (A. gambiae). ………….44

Figure 6.8: Combined effects on blood feeding efficiency and killing potency of transgenic and wild type Metarhizium strains at day 4……………………………………………………………………..45

Figure 6.9: Combined effect of M1630-Dc1a and M1630-Hybrid……………………………………..45

Figure 6.10: Growth and sporulation of transgenic M. anisopliae-1548 strains expressing selected ISVPs………………………………………………………………………………………………………………..46

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List of Tables

Table 1.1: List of WHO recognized vector-borne diseases…………………………………………………2

Table 1.2: Insecticides used for malaria vector control……………………………………………………..4

Table 1.3: List of anti-malarial drugs and their parasite life-cycle specificities…………………..7

Table 1.4: Pyrethroids used for Chagas disease vector control………………………………………..10

Table 1.5: Molecular targets of insecticides……………………………………………………………………11

Table 2.1: Selected insecticidal spider toxins…………………………………………………………………22

Table 2.2: Production of recombinant spider venom peptides………………………………………..26

Table 6.1: Primers used in this study and their respective usage…………………………………….38

Table 6.2: Bioassay of transgenic M. anisopliae strains against A. gambiae………………………43

Table 7.1: Summary of the insect bioassays of selected ISVPs…………………………………………49

Table 7.2: Summary of the outcomes of ISVP characterisation in this thesis…………………….51

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1 Introduction

1.1 Vector-borne disease

In the context of human disease, vectors are living organisms that serve as vehicles to transmit a pathogen (a disease-causing agent such as a virus or parasite) from a host to a human. Some vectors actively transmit pathogens, whereas others are passive transmitters1. Mosquitoes are the most important vector of human disease as they actively transmit diseases such as malaria and dengue2. Houseflies in comparison passively transmit diseases. Ticks are a typical example of vectors that can transmit diseases to humans as well as both domestic and farm animals (e.g., dogs or cattle). Birds and mammals can also be vectors (e.g. rats and mice, which can carry and transmit diseases)3.

According to the World Health Organization (WHO), a vector-borne disease is a disease in which the pathogenic microorganism is transmitted from an infected individual to another individual by an arthropod or other agent, sometimes with other animals serving as intermediary hosts (see Table 1.1 for examples). Vector-borne diseases have a direct and significant negative impact on human and animal health and wellbeing as well as an indirect impact by affecting the livelihood of people, resulting in a huge economic burden on the global economy. Although vector-borne diseases are currently only considered a major health hazard in tropical and subtropical regions, global warming could create appropriate conditions for outbreaks in temperate regions as well4.

1 Table1.1. List of WHO recognized vector-borne diseases5,6.

Disease Manifestation Causative Vector Geographic Treatment/ Agent Distribution Prevention Malaria Severe chills, Plasmodium Anopheline Tropic; Anti-malarial fever, sweating, mosquitoes Subtropics drugs and vector anaemia control Chikungunya Fever, joint pain, Alphavirus of Aedes Africa; Asia; Supportive fatigue, nausea the family mosquitoes Indian treatment and and rash Togaviridae subcontinent vector control Lymphatic Inflammation of Nematodes Culex, Aedes Tropic; Albendazole + filariasis lymphatic system of family and Ano- Subtropics Ivermectin and Filariodidea pheles vector control mosquitoes. Onchocerciasis Blindness, sever Onchocerca Blackflies of Africa; Ivermectine and itching volvulus species Latin America vector control Simulium damnosum Schisto- Haematuria, Schistosoma Water snails Tropic; Anthelmentics somiasis bladder and flatworm Subtropics urethral fibrosis African trypano- Joint pain, Trypano- Tsetse fly Sub-Saharan Various drugs somiasis swollen lymph soma brucei Africa and vector (sleeping nodes, sleep control sickness) disturbances Leishmaniasis Fever, damage to Leishmania Phlebotomine Southern Treatment of spleen and liver (protozoan) sandfly hemisphere; infection and and anaemia Mediterranean vector control Countries Babesiosis Fever and red Babesia Tick South Europe Antibiotics and urine and Africa Vector control Chagas disease Facial oedema, Trypano- Various Central and Antiparasitic (American fever, headache soma cruzi assassin bugs South America drugs, treatment trypano- and, in chronic of subfamily of symptoms, somiasis) phase, Triatominae and vector arrhythmia, control cardiomyopathy, digestive lesions Dengue fever Fever and Flavivirus Aedes Tropics; Supportive arthritis mosquitoes Subtropics; treatment and Southern vector control Europe Yellow Fever Fever, Yellow fever Mosquitoes Africa; South Vaccine, bradycardia, virus America supportive bleeding treatment, and vector control

1.2 Vector control

Vector control refers to any preventive method used to eradicate or control vector populations and thus limit the transmission and spread of vector-borne diseases. Different vector control methods include: (i) Chemical control: use of larvicides, insecticides, and rodenticides to control disease- causing vectors (see Table 1.2 for list of chemical insecticides and larvicides)7; (ii) Personal protection methods: agents that reduce the risk of exposure to disease causing vectors by acting as barriers for vectors (e.g., bed nets, and window and door fly screens)8. Repellent oils and

2 creams, smoldering coils, and liquid vapourizers are popular methods9. Plant oils are being used for repelling mosquitoes in many countries (e.g., turmeric, gingili, mustard, lemon grass and neem oil)10; (iii) Habitat control: methods that reduce the number of places where vectors can breed. (e.g., removal of stagnant water, which serves as a mosquito breeding habitat11); (iv) Biological control: use of predators (natural enemies), bacterial toxins, insect killing viruses, and fungal pathogens. Biological agents that have been used successfully against mosquito larvae include fishes such as Gambusia, Tilapia, and Poeciellia10. Two bacterial species, Bacillus thureingiensis israelensis (Bti) and Bacillus sphaericus (Bs), are also effective against mosquito larvae12. Other promising insecticides include the fungal entomopathogens Metarhizium anisopliae and Beauveria bassiana13.

1.2.1 Importance of vector control

Prevention of the emergence and spread of disease is always a better alternative to treating the disease. Considering that vector-borne diseases have a devastating impact on humans and animals, vector control plays a vital role in public health and livestock management programs around the world. In the absence of vector control measures, dangerous vector-borne diseases would proliferate and spread globally. It is thus crucial for reducing disease incidence, especially for diseases where there are no effective treatment or preventive medical options available (e.g., dengue fever, Chikungunya, and Chagas disease). Even for vector-borne diseases such as malaria for which effective medical treatment is available, other issues such as cost, diagnosis and drug resistance makes the use of drug treatment alone a fragile option. Both vector control measures and medical treatment are necessary for effective disease management. The focus of this thesis is primarily on controlling spread of two of the most devastating vector- borne diseases, malaria and Chagas disease.

3 Table 1.2. Insecticides used for malaria vector control. Insecticides are categorized

according to type of application and their chemical nature1,8,14.

Type of application Class of insecticides Insecticidal compounds Indoor residual spraying Organochlorines DDT (IRS) Organophosphates Chlorpyrifos Chlorpyrifos-methyl Fenthion Fenitrothion Malathion Pirimiphos-methyl Temephos Carbamates Bendiocarb Propoxur Pyrethroids Cypermethrin Bifenthrin Cyfluthrin Deltamethrin Etofenprox Lambda-cyhalothrin Permethrin Treatment of mosquito nets Pyrethroids Alpha-cypermethrin Cyfluthrin Delatmethrin Etofenprox Lambda-cyhalothrin Permethrin Larviciding Organophosphates Chlorpyrifos Chlorpyrifos-methyl Fenthion Fenitrothion Malathion Space spraying Organophosphates Clhlorpyrifos, Fenthion, Fenitothrion, Temephos. Pyrethroids Cypermethrin, Bifenthrin, Deltamethrin, Permethrin.

4 1.3 Malaria

Malaria is the most common vector-borne disease in many tropical and subtropical regions, specifically Africa, Asia, and some parts of America. Despite more than 120 years of research since discovery of the malaria parasite in 1880 by Charles Laveran, it continues to be major health problem15. This life-threatening disease is caused by one of four plasmodial species Plasmodium falciparum, P. ovale, P. vivax and P. malariae16. Mosquito vectors are solely responsible for transmitting this disease, which results from multiplication of malaria parasites within red blood cells. Symptoms include severe chills, fever, and headache, with severe cases progressing to coma and death.

Currently, about 3.3 billion people, almost half of the world’s population, is at risk of contracting malaria (see Figure 1.1). In 2010, there were 219 million cases of malaria and 660,000 deaths. Though malaria is a serious problem across the world, it is predominantly a problem in Africa. In Africa, a child under the age of 5 dies every minute from malaria, accounting for 20% of overall childhood deaths. The prevalence of malaria has a significant negative impact on the economy of countries with high levels of transmission, accounting for a decrease in the gross domestic product (GDP) by as much as 1.3%17. In some heavily malarious countries, the disease accounts for up to 40% of all public health budgets17.

Figure 1.1: Global distribution of Malaria. Malaria is prevalent in 108 tropical and semitropical countries. The most severely affected areas are Sub-Saharan Africa, south Asia (India, Nepal, Bhutan, Bangladesh, Srilanka and Indonesia), and east and south-east Asia (Vietnam, Malaysia, Myanmar, Cambodia, Singapore, Brunei, Philippines). Taken from WHO world malaria report, 201217.

5 The complicated life cycle of the plasmodium parasite, which involves several different life stages, is a major obstacle for malaria management (see Figure 1.2). Plasmodium are transmitted through the bite of female Anopheles mosquitoes (sporozoites from the parasite are transmitted from the mosquito into the human bloodstream). Within 30 min, sporozoites enter liver cells. They multiply for 5–7 days and form thousands of merozoites infecting liver cells. The merozoites burst out of the liver into the bloodstream and invade red blood cells. Within the red blood cell, the parasite multiplies to form new merozoites. This takes about 2 days. The infected erythrocyte then bursts, releasing merozoites, which infect new cells. Few merozoites invade the red blood cells and develop into gametocytes, which is the sexual stage of the parasite. The mosquito, while taking a blood meal, also takes up these gametocytes. In the mosquito gut the gametocytes develop into gametes and fuse to form a zygote. After fertilization, the zygote transforms into a motile ookinete. The ookinete becomes an oocyst, which divides to produce about 1000 sporozoites. After 5–7 days, the sporozoites move to the salivary glands, ready to infect another human.13,18-20.

Figure 1.2. Life cycle of malaria parasite illustrating the different life stages of the parasite during infection of mosquitoes and humans. (Taken from Su et al, 2007).20

1.3.1 Malaria management

There are two main approaches to malaria management, prevention (vector control and vaccines) and medical treatment (anti-plasmodial drugs).

Malaria drug treatment

There are several different generations of drugs for the treatment of malaria. These drugs act on specific life stages of the malaria parasite21 (see Table 1.3). Regrettably, malaria parasites have developed resistance to most anti-malarial drugs, including the aminoquinolines and their analogs (chloroquine, mefloquine, halofantrine and quinine), guanidine, and the antifolates22. Artemisinin derivatives offer a promising alternative approach, as these are effective against P. falciparum, which is resistant to the older generation of drugs23. Many questions regarding their neurologic toxicity, however, remain unresolved24,25. Furthermore, artemisinin treatment is expensive and beyond the reach of many patients in poor countries26. Thus, effective treatment of infected patients remains under threat of widespread drug resistance across all malaria endemic regions27. More recently, resistance of Plasmodium falciparum to artemisinin has emerged in Thailand and along the Cambodian border28.

Table 1.3. List of anti-malarial drugs and their parasite life-cycle specificities22,29.

Drugs Life stage of malaria parasite

Chloroquine, amodiaquine, mefloquine Schizonts, blood stage

Primaquine Hypnozoite stage, hepatic stage schizonts

Atovaquone-proguanil Hepatic stage schizonts

Sulfadoxine-pyrimethamine Gametocyte stage

Artemisinin derivatives Gametocyte, tropozoites, other blood stages

Malaria vaccine

Research scientists have been trying to develop an effective malaria vaccine for more than five decades but there are still no approved vaccines for this disease30. There have been many challenges in developing malaria vaccines, including: (i) malaria parasites have a complex life cycle with different life stages; (ii) four known species of malaria parasites infect people and each of these species are genetically different and have different life stages; (iii) currently we do not fully understand the complex immune responses that protect against the disease31. Therefore, a viable malaria vaccine must address the genetic diversity of the parasites and the host and the need to

7 provide immunity against the different life cycle stages of the parasite32,33.

Malaria Vector control

Because of increasing drug resistance in the malaria parasite and challenges in malaria vaccine development, considerable emphasis is placed on vector control by world health authorities. Insecticides remain the primary vector control tool throughout the world34. Dichlorodiphenyltrichloroethane (DDT) was the first synthetic organic insecticide used successfully for malaria vector control. However, in 1972, due to ecological toxicity concerns, the Environmental Protection Agency (EPA) in the United States of America announced a ban on DDT. This ban, however, excludes uses in public health emergencies, which includes indoor residual spraying (IRS) in sub-Saharan Africa. Other classes of insecticides (see Table 3) used for malaria vector control are pyrethroids, carbamates, and organophosphates7. IRS and more recently long-lasting insecticide-treated nets (LLINs) are the mainstream of insecticide-based malarria vector control. Organochlorines (DDT), organophosphates, carbamates, and pyrethroids are used for IRS (see Table 3). Currently, pyrethroids are the only class of insecticides approved by the World Health Organization Pesticides Evaluation Scheme (WHOPES) for use in LLINs.

1.3.2 Why has malaria control failed?

There are many reasons that explain the failure of efforts to control malaria: (i) The malaria parasite exists in two hosts, humans and female anopheline mosquitoes. Hence, controlling malaria involves three living beings, the malaria parasite itself, the human and the mosquito host. Humans and mosquitoes keep moving and travelling, spreading disease from place to place, making it difficult to track these parasite carriers; (ii) malaria parasites have a great ability to escape the human immune system. They can survive within human hosts for years without causing harm but nevertheless facilitating the spread of the disease through mosquito vectors35; (iii) malaria parasites are highly adaptive and can develop resistance against drugs; (iv) malaria vectors (mosquitoes) have developed resistance to most available insecticides17; (v) malaria is predominantly present in poor countries, where infrastructure, treatment options, and monitoring of treatment success is often not available36.

8 1.4 Chagas Disease

Chagas disease is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. It is a vector-borne disease predominant in Latin America, where it is mostly transmitted to humans by the faeces of triatomine bugs. It can also be transmitted through blood transfusions and organ transplantation. Triatomine bugs occur in both forested and dry areas in the Americas. A number of species have adapted to living in and near houses, where there is a presence of domesticated animals such as chickens, cattle, goats, cats and dogs37. They normally hide in dark crevices during the day staying close to their host. Their bite is not usually painful but in some cases severe itching and other skin problems occurs. Triatomine bugs prefers to feed on blood from the face and they defecate straight after feeding, so bites are often contaminated with faecal matter containing the trypanosome that cause the disease38. These bugs can also cause anemia in extreme circumstances. Common vectors are Triatoma infestans, T. dimidiata, Rhodinus prolixus, T. sorida, and T. sanguisuga39. There is geographical variation in the predominance of Chagas disease vectors. T. infestans is the most important vector in southern parts of South America whereas R. prolixus is typically more important in northern South America and Central America. T. dimidiata occupies Central and South America and extends up to Mexico40. Approximately 8 million people are infected with T. cruzi causing Chagas disease and more than 25 million people are at risk. In 2008, Chagas disease killed more than 15,000 people but actual figures could be substantially higher because of under-reporting and difficulties in diagnosis of the disease41. Almost 30% of chronically infected people develop cardiac alterations and 10% develop digestive, neurological and mixed alterations6,42.

1.4.1 Chagas disease management

Vector control (primary), blood transfusion screening, and medical care of the patient (secondary) are the basic strategies of Chagas disease management40,43.

Vector control Methods of Chagas disease vector control include IRS to eliminate domestic and peridomestic populations and housing improvements to eliminate cracks in walls and ceilings where triatomine bugs seek shelter during the day43. In the past, organophosphates were used for Chagas vector control but in recent times pyrethroids (see Table 1.4) have become the favoured insecticides for IRS because of their better safety profile and effectiveness. Peridomestic and domestic populations are controlled in the same way and should be treated at the same time, as the bugs may walk or fly from peridomestic habitats to domestic habitats. The most common peridomestic locations are chicken coops, corrals, woodpiles etc44. Another way to control triatomine bugs is to improve the building quality of houses with crack-free, smooth walls and ceilings to limit available

9 shelters for the bugs. This is an expensive option, but in the long-term it can help to eradicate domestic bug populations43,45.

Medical Care

The overall aim of the medical care approach is to eliminate the parasites (T. cruzi) with specific drug treatments, and to manage the symptoms and health conditions associated with the disease. Two antiprotozoal agents, benznidazole and nifurtimox are available for specific treatment of T. cruzi infections. These drugs are effective only in acute parasitemia and not in chronically infected patients. In general, the younger the patient and the closer to the time of parasite acquisition, the more chances of parasitologic cure with benznidazole and nifurtimox46,47. The chronic phase of this disease is incurable and treatment decisions are made on case-by-case basis after thorough assessment and continuous monitoring of cardiac and gastrointestinal symptoms of the patient43,48,49.

Table 1.4. Pyrethroids used for Chagas disease vector control. Formulation and required dose for insecticidal activity is given44,50,51.

Pyrethroids Formulation Target dose (mg a.i./m²)3 Deltamethrin 2.5% SC1 or 5% WP2 25 Cyfluthrin 10% WP 50 Beta-cyfluthrin 12.5% SC 25 Cypermethrin 20-40% WP 125 Lambda-cyhalothrin 10% WP 30

1SC, suspension concentrate; 2WP, wettable powder; 3a.i./m2, amount of active ingredient applied per surface area of house (m2) in a 1 litre spray solution.

1.4.2 Why has Chagas disease control failed?

There are many reasons to explain the failure to control Chagas disease, including: (i) there is no vaccine available; (ii) effective diagnostics are not available to identify disease at an early stage, and thus patients may remain unaware of the infection for several years52; (iii) the antiprotozoal drugs benznidazole and nifurtimox both have severe side effects, require long courses of treatment, and are effective only in the acute phase of the disease53; (iv) due to environmental and human health hazards, many chemical insecticides are restricted for IRS use50; (v) vector control in the peridomestic population is rarely satisfactory and peridomestic bugs migrate to residential areas; (vi) more recently, triatomine bugs have developed resistance to nearly all insecticides including pyrethroids50, and no new insecticides for IRS have been introduced in the last 20 years54; (vii) no novel drugs have been introduced for decades; (viii) lack of research and political support to take up the challenges of developing new insecticides and drug leads54,55.

10 1.5 The problem of insecticide resistance

The effectiveness and sustainability of insecticide-based vector control relies on the continuing susceptibility of the vectors to available chemicals. Although there appears to be a vast array of commercially available chemical insecticides, they mostly act on one of only five nervous system targets (see Table 1.5) (i.e., acetylcholinesterase, voltage-gated sodium (Nav) channels, nicotinic acetylcholine receptors, and γ-aminobutyric acid (GABA)- and glutamate-gated chloride channels)56. This reliance on just a handful of targets has promoted the evolution of insecticide resistance. Widespread and long-term use of limited insecticides in public health situations has caused the selection and spread of resistance in disease-causing vectors14,57. An increasing incidence of insecticide resistance has been reported in the major African malaria vectors Anopheles gambiae58,59, A. funestus60 and A. arabiensis61,62. Mosquitoes can become resistant to 63 both DDT and pyrethroids through a single point-mutation in the Nav channel gene . This so-called target-site resistance prevents mosquito paralysis and hence it is known as knockdown resistance16,64,65. Enzymes that metabolically degrade insecticides can also cause resistance. Mosquitoes with elevated levels of monooxygenases, esterases or glutathione S-transferases have shown resistance to insecticides (see Figure 3)16,66,67. In some cases, resistant insects show resistance to other compounds in the same insecticide class (e.g., resistance against one pyrethroid usually results in resistance against the whole group of pyrethroids). This is known as cross-resistance. Cross-resistance can even arise between different classes of insecticides (e.g., organophosphates and carbamates16). Insecticide resistance in anopheline populations in several parts of Africa and triatomine populations in Latin America has led to a significant reduction in the effectiveness of insecticide-based vector control.

Table 1.5. Molecular targets of insecticides68.

Nervous System Targets Insecticides

Nav channels DDT, pyrethroids Nicotinic acetylcholine receptors Imidacloprid, spinosad Acetylcholinesterase Organophosphates and carbamates GABA-gated chloride channels Fipronil Glutamate-gated chloride channels Avermectin

11 1.6 How to manage insecticide resistance?

The management of insecticide resistance, or more precisely, the management of vector susceptibility, is crucial. It is considered one of the most challenging issues in modern applied entomology. Currently there are only four WHO-approved chemical classes of insecticides (organochlorines, organophosphates, carbamates and pyrethroids) addressing just two different insect target sites. Hence, approaches to manage insecticide resistance include: (i) rotation of different insecticides with different modes of action ; and (ii) most importantly, the development of new insecticides with a different mode of action compared to current classes of chemical insecticides14,69-71. Hence, in order to control both malaria and Chagas disease vectors, a novel, cost effective, potent and environment-friendly insecticide is urgently required.

Fig. 1.3. Mechanisms of mosquito resistance to the four classes of WHO-approved public health insecticides. The two major target-site mechanisms are kdr (knock-down resistance), resulting from a mutation in the NaV channel (pink), and MACE (modified acetyl-cholinesterase) (green). Large spots indicate the most important mechanisms (from Nauen, 2007)14.

12 1.7 Entomopathogens as a biological control agent

Entomopathogen are pathogens that kill or seriously disables insects. There is a wide range of entomopathogens—bacterial, viral and fungal, —which have been studied and used as bioinsecticides. Examples include the soli bacterium Bacillus thuringiensis, baculoviruses, and entomopathogenic fungi such as Metarhizium anisopliae (See Appendix B). Entomopathogens plays a vital role in natural regulation of insect pests that vector diseases and destroy crops72.

Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a naturally occurring spore forming bacterium from the family Bacillaceae. It is widespread in soil and lethal to a range of insects. Sporulation results in formation of insecticidal crystal protein (ICP) to form protoxin crystals (Bt crystals). Bt crystals contain Cry toxins73. Cry toxins are pore-forming toxins responsible for the insecticidal activity of Bt. Cry toxins are toxic to specific species of insects, especially larval stages, and they have proved to be safe to humans and other vertebrates. Bt crystals have to be ingested to cause mortality in insects. After ingestion, Bt crystals cleaved within the insect gut and form the active toxin (Cry toxin). The Cry toxin binds to receptors in the midgut to form pores, which results in insect paralysis and/or bacterial septicaemia73,74. Most of the Bt species are active against Lepidoptera, Diptera and Coleoptera. The sub-species Bt israelensis is effective against the larval stage of mosquitoes75. Despite their potential, limited Bt products have been used as sprayable insecticides. The revolutionary transgenic Bt crops expressing Cry toxin were introduced in 1996. Within 3 years of its introduction, 29 million acres of Bt cotton, potato and corn were grown worldwide76. By 2010, Bt crops had been planted in 29 countries covering 148 million hectares of cropland74. Recently, resistance to Bt toxins has been reported, especially in diamondback moths77,78, triggering the need to investigate novel toxins to replace or augment Cry toxins in transgenic insect-resistant crops.

Baculoviruses

Baculoviruses are one of a diverse group of viruses only pathogenic to insects, mostly of the order Lepidoptera, Hymenoptera and Coleoptera79. The most widely studied baculovirus is Autographa californica nuclepolyhedrovirus (AcNPV)80. Baculoviruses occurs in nature on plants, plant wreckage, and in the soil in the form of virions. Virons are responsible for the insecticidal activity of baculovirus. They are occluded within proteinaceous crystals called polyhedra. Similar to Bt, baculovirus have to be eaten by insect to cause mortality to insects. The infection cycle starts when insects ingest polyhedra, which results in the release of hundreds of virons in the gut. Virions then pass through the peritrophic matrix to enter midgut cells. The virus then replicates in the nuclei of epithelial cells of the midgut, producing a large number of virions. Infected insects continue to feed up to 5–14 days following the infection, until they die79.Baculoviruses have been

13 registered and successfully used for decades as safe and effective bioinsecticides against crop and forest pests. Natural baculovirus-based products are used in North America for forest pest control81. They are used in Brazil on over a million hectares of farmland to control the velvet bean caterpillar Anticarsia gemmatalis82, the major pest of soybean. Although baculoviruses have been used successfully for soybean and forest pest control, they have not been able to compete successfully with chemical insecticides. Many crops can tolerate only minimal fruit or foliar damage. Natural baculovirus takes 5–14 days to kill the insect pest and during this time the insect continues to feed, whereas chemical insecticides usually kill within a day. In the field, several baculovirus species may need to be applied in combination to control multiple pests because of the narrow host specificity. Furthermore, baculovirus is effective only at the larval stage of the pest. Some of these issues have been addressed using genetic engineering technology to produce transgenic nucleopolyhedroviruses that express insecticidal venom peptides from venomous animals such as scorpion or spiders83. The introduction of venom-peptide transgenes reduced the time to kill and the time taken until insects stopped feeding84. Though natural baculovirus have their own limitations, transgenic baculovirus have great potential for development as bioinsecticides considering their safety profile, specificity and efficacy.

Entomopathogenic fungi

Entomopathogenic fungi act as parasites of insects and kill or seriously disable them85,86. Almost 400–500 species of fungi are known to have insect pathogenic properties, and they have been used for biocontrol of pest insects for ~150 years. All five sub-divisions of fungi (Mastigomycotina, Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina) contain entomopathogenic species85. Compared to other microorganisms, fungi infect a broader range of insects including Lepidoptera, Homoptera, Hymenoptera, Coleoptera and Diptera87. Therefore entomopathogenic fungi can be found to infect hosts in a wide variety of ecological niches88. Many fungi have been shown to be highly virulent towards a number of economically and medically important arthropods87. Fungi can be applied as conidia or blastospores. Both spore types are robust and have evolved to withstand a range of environmental conditions. Most other insect pathogens require living insects or complex culture systems for inoculum production. On contrary, fungal spores have the capacity for mass production and conidia can be mass-produced on simple artificial media such as rice89. Fungal spores can also be applied in a range of formulation types, which could be water-based, oil-based, powders or granules90. Mass produced conidia can be dried and stored at low temperatures either as a powder or formulated in oil for comparatively long periods of time with minimal loss of viability91,92. Over the past three decades there has been much research on developing fungal bioinsecticides to control arthropod pests, mostly insect pests of crops and pastures. Most research has been directed to the genera Beauveria and Metarhizium whose distribution is cosmopolitan. B. bassiana and B. brongniartii are the most widely used and

14 studied Beauveria species while M. anisopliae is the most widely used Metarhizium species throughout the world. It was recently found that pyrethroid resistance in the malaria vector A. gambiae and the Chagas disease vector T. infestans leads to increased susceptibility to the entomopathogenic fungi M. anisopliae and B. bassiana93,94.

Most importantly, the ability of entomopathogenic fungi to directly parasitise arthropods via penetration of the host integument (e.g., cuticle or shell) sets them apart from bacteria and viruses, which must be ingested to gain host entry86,95. Hence the focus of thesis is to explore selected Metarhizium strains engineered to express insecticidal venom peptides for development as bioinsecticides to control malaria and Chagas disease vectors.

1.7.1 Metarhizium—mode of infection and pathogensis

Metarhizium spores are small (2–6 µm) hydrophobic propagules that can attach to the insect cuticle through hydrophobic interactions96. Under suitable (moist) conditions they can germinate and produce germ tubes (see Figure 1.4). After germination the germ tube may swell to form an appressorium as a prerequisite for host penetration. An appressorium is a “cushion” of cells produced on an invasion surface. Roles attributed to the appressorium include: anchorage for penetration, softening of the cuticle through enzyme production, and concentration of components involved in penetration97. A range of cuticle degrading enzymes are produced during penetration; the three most important classes are lipases, proteases, and chitinases. There is immense variation in the type and quantity of enzymes produced by different species and even different strains of the same species98-100. Once the pathogen has penetrated the host cuticle it is believed to proliferate within the haemocoel as hyphal bodies. This growth form helps the fungus in overcoming insect defence mechanisms101. Hyphal bodies are quickly disseminated throughout insect fluids, thus evading host immune cells. During this stage a number of toxic metabolites may be produced by the pathogen. For M. anisopliae, the primary toxins is destruxin, a cyclic depsipeptide toxin that appears to have various effects on the host, usually leading to paralysis102. Insect death may result from a combination of actions, including depletion of nutrients, physical obstruction, or invasion of organs or toxicosis103. This infection process takes several days, with the overall time to death depending mostly on fungal dose and virulence of the fungal isolate. When humidity is high enough, the fungus can grow out of the dead insect and produce conidia that can infect new insects98,104.

15 Figure 1.4. Mechanism of pathogenesis of entomopathogenic Metarhizium103. Infection steps are: (1) Conidia adherence to the host cuticle through hydrophobic interactions and thin mucilaginous material; (2) Conidia germination and development; (3) Germ-tube differentiation into apressoria; (4) Cuticle penetration; (5) Hyphae differentiation into blastospores; (6) Host colonization; (7) Extrusion to the host cadaver surface; (8) Conidiophore formation and conidia production (from Acevedo, 2007).

1.7.2 Metarhizium as a bioinsecticide

The fungus Entomopthora anisopliae, later renamed Metarhizium anisopliae Sorokin, was used for the first time in the 1880s to infect and kill healthy larvae of the scarab beetle Anisoplia austriaca105. A few years later in 1884 Isaak Krassilstschik mass produced Metarhizium spores and applied them to field plots to control larvae of the root weevil (Cleonus spp.)105. Studies using insects as bait for entomopathogenic fungi suggest that Metarhizium is a part of the soil flora worldwide106. It is believed that Metarhizium has co-evolved in association with insects. Because of the worldwide distribution of this fungus, local isolates can be easily obtained from the soil or dead mycosed insects107. In Australia, there are currently four fungal bioinsecticides produced from endemic isolates of Metarhizium by Becker Underwood and CSIRO Entomology in conjunction with the Australian Plague Locust Commission. These products are registered for the control of locusts (Green Guard ULV; Green Guard SC), greyback cane grub (BioCane), and scarab beetle larvae (Chafer Guard).

16 Metarhizium is an anamorphic fungus producing asexual conidia (spores) from hyphae108. The conidia are produced on conidiophores in a candelabrum-like arrangement109. The compact conidiophores form on a cushion-like hymenial layer or sporodochium (see Figure 1.4). Metarhizium species are easily identified by the green cylindrical conidia produced in chains, which form a dense compact layer of dry powdery spores110. A revision of the genus Metarhizium based on a phylogenetic analysis of rDNA sequence recognised three species: M. anisopliae, M. flavoviride and M. album111.

1.7.3 Commercial development of entomopathogens as bioinsecticides

More than 100 fungus-based insect control products have been registered for commercial use by the USA EPA since 1995 and undergone the required stringent environmental safety tests112,113. Mycotrol, for example, is a Beauveria-based mycoinsecticide that is commercially produced in the USA. Mycotrol formulations can be used on crops for control of grasshoppers, whiteflies, thrips and aphids, and conidia can remain infective for 12 months when stored at 25°C113. GreenMuscle is a mycoinsecticide that is commercially produced in Africa; it contains spores of M. anisopliae var. acridum, which are selectively pathogenic to locusts and grasshoppers. Green Muscle was developed during a 12 year research programme called LUBILOSA114, in which substantial advances were made in mass-production, formulation, and application of Metarhizium115. The product can be sprayed with ultra-low-volume sprayers, and it can remain infective for >12 months when stored at 30°C115,116. Green Muscle showed no detrimental effects on non-target organisms and is now registered and recommended for locust and grasshopper control by the United Nations116. These and several other environmental risk studies demonstrate that fungal biopesticides are relatively safe for the environment, especially when compared to chemical pesticides106.

1.7.4 Obstacles using Metarhizium as a bioinsecticide

Metarhizium may take 1–3 weeks to kill its host because of low virulence. This slow kill time, along with its inconsistency in performance, is a major obstacle for widespread use of Metarhizium to control vectors of human disease. To some extent, field conditions such as UV and high temperature affect the performance of Metarhizium. Moreover, Metarhizium is more expensive than chemical insecticides. Thus, to improve the insecticidal market share of fungal pathogens, genetic modification is likely to be required.

1.7.5 Genetic engineering of Metarhizium to improve virulence and tolerance

With advances in recombinant DNA techniques, it is possible to significantly improve the

17 insecticidal potency of Metarhizium and its tolerance to UV radiation. For example, genetic engineering of Metarhizium to express insecticidal toxins can improve its pathogenicity. Addition of a transgene encoding the insecticidal scorpion toxin AaIT1 in M. anisopliae strain 549 enabled the same mortality rates in the tobacco hornworm Manduca sexta to be obtained at a 22-fold lower spore dose (LC50 – spore concentration required to kill 50% of test population) compared to the wild-type strain. Similar results were obtained with mosquitoes, where the LC50 was reduced by 9- fold117,118.

Another possibility for increasing the ability of the fungus to reduce the spread of malaria would be to engineer it to express a transgene with anti-plasmodial properties in addition to an insecticidal toxin transgene. This approach would provide a dual mechanism to control the disease. Recently it was also demonstrated that the overexpression of CPD (cyclobutane pyrimidine dimer) photolyases increases the tolerance of Metarhizium robertsii to UV radiation. Thus, genetic engineering provides opportunities for improving the bioinsecticidal properties of Metarhizium.

1.7.6 Spiders: source of insecticidal toxins for Metarhizium genetic engineering

Spiders are the most successful insect predators on earth. There are more than 3,900 genera and 44,000 extant species of spiders119,120. They use their venom to paralyse or kill their prey. Only five genera of spiders are potentially lethal to humans121 and their venom is mostly toxic only to their targeted prey (i.e., insects). The major constituents of spider venoms are small disulfide-rich peptides. Many of these contain an inhibitor cystine knot (ICK) motif. This ICK motif provides resistance to extremes of pH, high temperature, and proteolytic enzymes122,123. A large number of spider toxins have been identified and studied for their insecticidal activity against different insect targets. Currently the ArachnoServer database (a manually curated database of spider toxins which provides information on their sequence, structure and biological activity) holds 1078 spider toxin records of which ~70% have characterised insecticidal activity. The molecular target for 162 spider toxins has been experimentally determined119,120 . Thus, spider toxins have a well- established efficacy against insects and represent promising candidates for Metarhizium genetic engineering.

18 1.8 Aim & objectives

The work presented in this thesis is aimed to develop environmentally friendly bioinsecticides for the control of vectors of human diseases (specifically mosquitoes and triatomine bugs). The first part of the research was focused on production, characterisation, and prioritisation of potent insecticidal venom peptides that could be used for genetic engineering of fungal entomopathogens. The second part of the thesis is focused on genetic engineering and testing of fungal entomopathogens that express insecticidal venom-peptide trangenes. The specific research objectives were as follows:

Aim 1: Compare the insecticidal potency of selected insecticidal spider-venom toxins against mosquitoes and triatomid bugs. Aim 2: Determine the structure and mode of action of the most promising toxins. Aim 3: Engineer transgenes encoding the most potent insecticidal spider toxins into Metarhizium anisopliae. Aim 4: Compare the insecticidal potency of the recombinant and wild-type fungi against mosquitoes and triatomine bugs.

1.9 Thesis Outline

Chapter 2 provides details of recombinant production of the selected insecticidal spider-venom peptides (ISVPs) in E. coli as well as assessment of their insecticidal activity. This chapter describes an in-house insecticidal bioassay on sheep blowflies (L. cuprina). It also describes the insecticidal bioassay of these ISVPs in mosquitos (A. gambiae) and triatomine bugs (R. prolixus) which were performed in collaboration with Dr Luciano Moreira at Fiocruz, Brazil.

Chapter 3 is a manuscript published in Biochemical Pharmacology that describes the recombinant production, 3D structure, and molecular target of an ISVP known as Aps III.

Chapter 4 is a manuscript submitted to FEBS Journal that describes the recombinant production, 3D structure, molecular target determination and insecticidal activity of an ISVP known as Sf1a.

Chapter 5 is a manuscript accepted in Nature Communications and currently in press that describes the recombinant production, 3D structure, and molecular target determination of an IVSP known as Dc1a.

Chapter 6 describes genetic engineering of Metarhizium to express the most promising ISVPs and comparison of the insecticidal efficacy of the parental wild-type strain and the recombinant Metarhizium strains against mosquitoes.

Chapter 7 provides a summary of the research findings and discusses future prospects for use of fungal bioinsecticides for controlling vectors of human diseases and agricultural pests.

2 Recombinant expression and assay of insecticidal spider venom peptides (ISVPs)

2.1 Introduction

Recombinant production of venom peptides is often challenging due to the presence of multiple disulfide bonds, which cannot be formed in the cytoplasm of most prokaryotic and eukaryotic cells due to the reducing intracellular environment. An approach that has proved successful for expression of disulfide-rich spider toxins is production in the periplasm of E. coli, where the enzymes involved in disulfide-bond formation are located124. Eleven of the most potent insecticidal spider-venom peptides (ISVPs) described in the literature were selected from a curated database of spider toxins (ArachnoServer)120 (see Table 2.1). In order to obtain sufficient quantities of these peptides for functional and structural studies, they were produced recombinantly using an E. coli periplasmic expression system optimised for production of disulfide-rich peptides124. After production of the ISVPs, insecticidal assays were performed on sheep blowflies (Lucilia cuprina), mosquitoes (Aedes aegypti) and triatomine bugs (Rhodnius prolixus). On the basis of these insecticidal assays, a panel of the five most potent ISVPs were selected for Metarhizium transformation.

2.2 Methods

2.2.1 Periplasmic expression of ISVPs in E. coli

Synthetic genes for all ISVPs were ordered from GeneArt (Invitrogen, Regensburg, Germany).

21 Genes encoding the peptide of interest were codon optimised for E. coli expression and inserted into the specially designed pLIC-MBP expression vector. This vector (pLIC-NSB) contains the LacI promoter, which can be induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), and it encodes ampicillin resistance. The plasmid encodes the ISVP as a fusion protein (see Figure 2.1A) containing a MalE signal sequence for export to the periplasm, MBP (maltose binding protein) to increase protein solubility, His6 tag for nickel affinity purification, plus a TEV (tobacco etch virus) protease recognition site between the MBP and toxin-encoding regions to facilitate cleavage of the ISVP from the fusion protein125,126.

Table 2.1. Selected insecticidal spider toxins.

LD50, Lethal Dose, 50%; PD50, Paralysing Dose, 50%; DSBs, disulfide bonds Data from ArachnoServer (www.arachnoserver.org)120.

Spider toxin name Number of residues and LD50 /PD50 in insects disulfide bonds ω/κ -hexatoxin-Hv1a 39 residues; 3 DSBs –

ω-hexatoxin-Hv1a 37 residues; 3 DSBs LD50: Acheta domesticus 89 pmol/g; Musca domestica 77 pmol/g; PD50: Heliothis virescens 250 pmol/g

κ-hexatoxin-Hv1c 37 residues; 4 DSBs LD50: Spodoptera frugiperda 3070 pmol/g; A. domesticus 167 pmol/g; M. domestica 91 pmol/g; H. virescens 3195 pmol/g

U1-AGTX-Ta1a 50 residues; 3 DSBs PD50: 780–900 pmol/g in lepidopterans;

µ-DGTX-Dc1a 56 residues; 4 DSBs PD50: H. virescens 380 pmol/g;

U2-CUTX-As1a 37 residues; 4 DSBs LD50: M. sexta 133 pmol/g

U2-SGTX-Sf1a 46 residues; 4 DSBs LD50: H. virescens 2011 pmol/g;

U1-PLTX-Pt1a 46 residues; 5 DSBs LD50: M. sexta 13.8 pmol/g

U1-CUTX-As1c 76 residues; 4 DSBs LD50: M. sexta 2.4 pmol/g

δ-CNTX-Pn1a 48 residues; 5 DSBs LD50: M. domestica 36 pmol/g

µ-AGTX-Aa1d 37 residues; 4 DSBs LD50: M. domestica 30 pmol/g

The MBP-toxin fusion protein can be obtained from the periplasm by osmotic shock, sonication, or whole cell lysis. The fusion protein is then purified using nickel affinity chromatography and cleaved by recombinant His6-taggedTEV protease to yield the mature ISVP. The cleaved His6-

MBP and His6-TEV are removed by one of three methods: (i) passing the cleaved fusion protein over a charged Ni-NTA Superflow resin (Qiagen, Chadstone, Australia);,(ii) passing the solution

22 through a Solid-Phase Extraction (SPE) column; (iii) precipitating MBP and TEV with 1% trifluoroacetic acid (TFA) .The pure peptide is then recovered using reverse-phase HPLC (RP- HPLC).

Figure 2.1. Production of ISVPs. (A) Schematic representation of the pLicC-NSB vector used for periplasmic expression of ISVPs. The coding region includes a MalE signal sequence (MalESS) for periplasmic export, a His6 affinity tag, an MBP fusion tag, and a codon-optimized gene encoding the ISVP, with a TEV protease recognition site inserted between the MBP and toxin coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS). (B) E. coli periplasmic expression system. Upon induction with IPTG, the MBP-toxin fusion protein is expressed and transported to the periplasm via the SecYEG translocase system. The DsbA machinery handles oxidative folding of the ISVP in the periplasm (Figure 2.1B from Klint et al., 2013)124

2.2.2 Blowfly toxicity assay

To determine the toxicity of ISVPs in sheep blowfly (Lucilia cuprina), the method described in Bende et al. 2013127 was used. In short, ISVPs were dissolved in insect saline (composition: 200 mM NaCl, 3.1 mM KCl, 5.4 mM CaCl2, 4.0 mM MgCl2, 2.0 mM NaHCO3 and 0.1 mM Na2HPO4; pH adjusted to 7.2) and injected into the ventro-lateral thoracic region of sheep blowflies with mass of 19.5–23.3 mg. A 1.0 ml Terumo Insulin syringe (B-D Ultra-Fine, Terumo Medical Corporation, Maryland, USA) with a fixed 29 G needle fitted to an Arnold hand micro-applicator (Burkard Manufacturing Co. Ltd., England) was used to inject a maximum volume of 2 µl per fly. Three 23 separate experiments were run with various doses of each ISVP injected into 10 flies. After ISVP injection, each blowfly was individually housed in a plastic tube and provided with a 20% sugar solution. Insect saline was used as a control. Paralytic and lethal effects were measured 24 h post- injection. The insect is declared as paralysed with the only sign of life being minor movements of the proboscis. All PD50 and LD50 values were calculated separately for each experiment by plotting dose (in pmol.g-1) vs. percentage of deaths at 24 h post injection. Then log dose–response curves were fitted using following equation128.

Where y is the percentage of deaths in the sample population at 24 h postinjection, x is the toxin dose in pmol.g-1, n is the variable slope factor, a is the maximum response and b is the minimum response.

2.2.3 Mosquito and triatomine bug toxicity assay

Mosquito and triatomine bug assays were performed in the lab of Dr Luciano Moreira at Fiocruz Institute, Brazil. The method used was adopted from Luna et al. (2007)129 with some modifications. Due to time constraints, toxicity assays were performed using ω/κ-hexatoxin-Hv1a ("hybrid" toxin) and ω-hexatoxin-Hv1a ("omega" toxin). ISVPs (Hybrid, Omega) were dissolved in insect saline and injected into the lateral thoracic region of female adult mosquitoes (Aedes aegypti) with an average mass of 2.5 mg. Microinjection needles were made by pulling borosilicate glass capillaries with a needle puller (Sutter Instrument CO, CA, USA) and fitted on a Nanoject II auto injector

(Drummond Scientific Company, Broomall, PA, USA). Mosquitoes were anesthetized with CO2/dry ice and placed on a pre-cooled petri dish for injections. A maximum volume of 69 nl was injected per mosquito. Experiments were performed in triplicate using various doses of ISVP, with each dose injected into 10 mosquitoes. The control group was injected with insect saline. After injections, mosquitoes were transferred into plastic cages and kept at 27ºC with 80% relative humidity. Cotton balls soaked in 80% sugar solution were provided. Lethal effects were measured 130 24 h post-injection. LD50 values were calculated as described in Herzig & Hodgson (2008) and averaged to produce a final LD50 for each IVSP.

ISVPs toxicity was also tested in the triatomine bug Rhodnius prolixus, the second most important vector of Chagas disease, using same experimental set-up with minor changes. Second instar nymphs of R. prolixus with an average mass of 1.4 mg were injected with various doses of ISVPs in the lateral thoracic region. Three separate sets of experiments were performed for each ISVP dose, with 7 bugs injected per dose. The control group was injected with insect saline. After injection, the bugs were transferred to plastic cages and maintained at 26ºC with 50% relative

24 humidity. As a food source, 80% sugar solution was provided. Lethal effects were measured 24 h 130 post injection and LD50 values were calculated as described previously .

2.3 Results and Discussion

2.3.1 Production of recombinant ISVPs

E. coli is the most widely used recombinant protein expression system as it offers high yields and involves simple plasmid constructs. The proteins are expressed in the cytoplasm of the bacterium where the intracellular thioredoxin and glutaredoxin disulfide-reducing pathways keep cytoplasmic cysteine residues in the reduced state131,132. This problem can be overcome by transport of the expressed protein by SecYEG translocase system to the periplasmic space, where the endogenous bacterial machinery for disulfide bond formation is located133,134 (see Figure 2.1B). Bacterial expression further facilitates facile and high yield 15N/13C isotopic labelling of proteins for subsequent structural studies using nuclear magnetic resonance (NMR) spectroscopy.

An efficient E. coli expression system was established for the production of selected ISVPs. Cell growth and purification conditions were optimized for individual toxins. The outcome of recombinant toxin production is outlined in Table 2.2. In summary, seven peptides were produced in sufficient quantities for insecticidal bioassays against blowflies, mosquitoes and triatomine bugs, namely ω/κ -hexatoxin-Hv1a (Hybrid), ω-hexatoxin-Hv1a (Omega), κ-hexatoxin-Hv1c (Kappa), U1-

AGTX-Ta1a (Ta1a), µ-DGTX-Dc1a (Dc1a), U2-CUTX-As1a (As1a), and U2-SGTX-Sf1a (Sf1a). Sufficient amounts of Hybrid, Omega and Kappa were obtained in crude form from Vestaron Corporation, USA (www.vestaron.com) and re-purified to >95% purity using RP-HPLC. Details on production of As1a, Sf1a and Dc1a are covered in Chapter 3, Chapter 4 and Chapter 5 respectively. Throughout the course of this project extensive efforts were made, without success, to optimize E. coli expression of U1-CUTX-As1c (As1c), U1-PLTX-Pt1a (Pt1a) and µ-AGTX-Aa1d (Aa1d). Further studies using these toxins may be possible using other recombinant expression techniques or via chemical synthesis, but these approaches were not explored here.

Table 2.2: Production of recombinant spider venom peptides

Toxin Plasmid vector E. coli expression Yield of Expression of pure toxin 13C/15N-labelled toxin ω/κ -hexatoxin- Yes Yes +++ Yes Hv1a

ω-hexatoxin-Hv1a Yes Yes +++ Yes

κ-hexatoxin-Hv1c Yes Yes +++ Yes

U -AGTX-Ta1a Yes Yes +++ Yes 1

µ-DGTX-Dc1a Yes Yes ++ Yes

U2-CUTX-As1a Yes Yes +++ Yes

U2-SGTX-Sf1a Yes Yes +++ Yes

µ-AGTX-Aa1d Yes Yes (Unfolded) - No

U1-PLTX-Pt1a Yes Yes (Unfolded) - No

U1-CUTX-As1c Yes Yes (Insoluble) - No

δ-CNTX-Pn1a Yes Yes (Unfolded) - No

‘++’ = > 1mg expression of soluble peptide per litre; ‘+++’ = > 3mg expression of soluble peptide per litre

2.3.2 ISVPs are paralytic and lethal to sheep blowflies

As previously stated, seven ISVPs were produced in sufficient amounts for use in insect toxicity assays. In the first instance, sheep blowflies (L. cuprina) were used to assess the relative insecticidal toxicity of the ISVPs. At 24 h post-injection, six ISVPs (Ta1a, Hybrid, Omega, Kappa, Dc1a, and Sf1a) caused a high level of blowfly mortality. As1a induced flaccid paralysis and flies remained paralysed after 24 h; the PD50 measured 24 h after injection was 700 ± 35 pmol/g (see Figure 2.2 G). The assay used does not allow measurement of toxic effects for periods extending beyond 24 h as the survival rate in control blowflies begins to decrease. However, flies paralysed by a high dose of As1a did not recover but died two days post-injection. We conclude that As1a produces an irreversible paralysis in blowflies that eventually results in death. More details about As1a is covered in Chapter 3.

26 LD50 values were calculated from dose-response curves for the remaining six ISVPs (Figure 2.2).

Ta1a stands out as the most potent ISVP with LD50 of 198 ± 33 pmol/g (see Figure 2.2D) followed by Dc1a with LD50 of 231 ± 32 pmol/g (see Figure 2.2E). Hybrid and Kappa were found to be almost equipotent with LD50 of 259 ± 17 pmol/g and 278 ± 7 pmol/g respectively (see Figure

2.2A,B). Sf1a was found to have very weak insecticidal activity with LD50 of 32,0000 ± 8,600 pmol/g (see Figure 2.2F). More details about Sf1a and Dc1a are covered in Chapters 4 and 5, respectively.

27 Figure 2.2. Acute toxicity of ISVPs in sheep blowflies (L. cuprina). Dose-response curves for the lethal or paralytic effects of ISVPs determined 24 h after injection. The ISVPs exmained were (A) ω/κ -hexatoxin-Hv1a (Hybrid); (B) ω- hexatoxin-Hv1a (Omega); (C) κ-hexatoxin-Hv1c (Kappa); (D) U1-AGTX-Ta1a (Ta1a); (E) µ-DGTX-Dc1a (Dc1a); (F) U2- CUTX-As1a (As1a); and (G) U2-SGTX-Sf1a (Sf1a).

2.3.3 ISVPs are lethal to mosquito and triatomine bugs

Initial toxicity screens revealed that all chosen ISVPs except Sf1a were potent against sheep blowflies. Mosquitoes (dipterans) and triatomine bugs (hemipterans) are not closely related, with their last common ancestor dating back more than 300 million years135, and therefore we wondered whether these dipteran-toxic IVSPs would also be toxic to triatomines. We therefore compared the toxicity of the Hybrid and Omega IVSPs against mosquitoes (Aedes aegypti) and triatomine bugs

(R. prolixus). LD50 values were calculated 24 h post-injection. In mosquitoes, the Hybrid toxin (LD50 of 37.4 ± 9.7 pmol/g) was found to be approximately 2-fold more potent than the Omega toxin

(LD50 of 64.4 ± 3.5 pmol/g; Figure 2.3). Remarkably, both toxins were even more toxic to triatomine bugs, with the Hybrid toxin (LD50 of 15.8 ± 4.5 pmol/g) again being more potent than the Omega toxin (LD50 of 47.4 ± 14.5 pmol/g) (see Figure 2.4).

Figure 2.3. Acute toxicity of ISVPs in mosquitoes, A. aegypti. Dose-response curves for lethal effects of (A) ω/κ- hexatoxin-Hv1a (Hybrid) and (B) ω-hexatoxin-Hv1a (Omega) determined 24 h after injection into mosquitoes.

Figure 2.4 Acute toxicity of ISVPs in triatomine bugs, R. prolixus. Dose-response curves for lethal effects of (A) ω/κ- hexatoxin-Hv1a (Hybrid) and (B) ω-hexatoxin-Hv1a (Omega) determined 24 h after injection into triatomine bugs.

2.3.4 ISVPs for Metarhizium genetic engineering

There are no standard assays or target species for comparison of the insecticidal potency of venom peptides. Several experimental factors could affect the measurement of insecticidal potency, including the route of toxin application, site of injection, dose of injection, injection volume, type of injector used, the time intervals between toxin injection, development stages of the insect, the temperature, and relative humidity at which test insects are kept post-injection. Hence, the insecticidal values reported in the literature for different toxin peptides cannot be directly compared136. Here, for the first time, we produced ISVPs and performed toxicity assay against blowflies, mosquitoes and triatomine bugs under identical experimental conditions. These assays allowed a more accurate comparison of the relative potency (LD50 and PD50) of the ISVPs, which in turn allowed us to select a panel of the five most potent ISVPs for engineering toxin-encoding transgenes into Metarhizium. The selected ISVPs cover a range of pharmacologies including block 137 of NaV channels (As1a; see Chapter 3), block of both KCa and CaV channels (Hybrid ); activation 138 of NaV channels (Dc1a; see Chapter 5); selective block of CaV channels (Hv1a ), and Ta1a, for which the molecular target remains to be determined.

The selected ISVPs all have high potency, environmental stability, insect specificity and in some cases novel molecular targets, all ideal attributes for development as bioinsecticides139.

3 The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels.

Published manuscript in Biochemical Pharmacology, 2013

30 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights

Author's personal copy

Biochemical Pharmacology 85 (2013) 1542–1554

Contents lists available at SciVerse ScienceDirect

Biochemical Pharmacology

jo urnal homepage: www.elsevier.com/locate/biochempharm

The insecticidal neurotoxin Aps III is an atypical knottin peptide that

potently blocks insect voltage-gated sodium channels

a b a c,d b

Niraj S. Bende , Eunji Kang , Volker Herzig , Frank Bosmans , Graham M. Nicholson ,

a a,

Mehdi Mobli , Glenn F. King *

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia

School of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway, NSW 2007, Australia

Department of Physiology, Johns Hopkins University–School of Medicine, Baltimore, MD 21205, USA

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University–School of Medicine, Baltimore, MD 21205, USA

A R T I C L E I N F O A B S T R A C T

Article history: One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the

Received 31 January 2013

trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a

Accepted 27 February 2013

promising candidate for bioinsecticide development. However, its disulﬁde-connectivity, three-

Available online 6 March 2013

dimensional structure, and mode of action have not been determined. Here we show that recombinant

Aps III (rAps III) is an atypical knottin peptide; three of the disulﬁde bridges form a classical inhibitor

Keywords:

cystine knot motif while the fourth disulﬁde acts as a molecular staple that restricts the ﬂexibility of an

Voltage-gated sodium channel

unusually large b hairpin loop that often houses the pharmacophore in this class of toxins. We

Neurotoxin

demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of

Spider-venom peptide

insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it

Pore blocker

occurs without signiﬁcant alteration in the voltage-dependence of channel activation or steady-state

Gating modiﬁer

Inhibitor cystine knot inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-

gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium

channel modulators isolated from spider venoms that act as gating modiﬁers by interacting with one or

more of the four voltage-sensing domains of the channel.

1. Introduction resistant to one or more classes of chemical insecticides [6]. In

addition, key classes of insecticides have been withdrawn from

Insects serve as vectors for a wide range of debilitating and sale or their use has been restricted by regulatory authorities due

potentially lethal human diseases such as malaria, dengue, Chagas to growing environmental and human health concerns [7]. Thus,

disease, and yellow fever [1]. About 3.3 billion people, almost half there is an urgent need to develop novel classes of insecticides or

of the world’s population, are at risk of contracting vector-borne alternative methods of insect pest control.

disease [2]. Moreover, despite intensive control measures, insect A promising approach in the agricultural sector is to engineer

pests reduce world crop yields by 10–14% annually [3,4]. crops to produce insecticidal toxins. By 2010, 148 million hectares

Despite the widespread introduction of insect-resistant trans- of genetically modiﬁed (GM) crops had been planted in 29

genic crops, chemical insecticides remain the dominant method for countries, representing 10% of all cropland [8]. While the

controlling insect pests in both the agricultural and public health introduction of GM crops that express Bacillus thuringiensis (Bt)

arenas. These chemicals target a very small number of molecular toxins has provided an alternative and potentially safer method of

targets in the insect nervous system [5]. As a result, their insect control than chemical insecticides, alternative insect-toxin

widespread use over several decades has promoted the evolution transgenes are urgently needed as constitutive expression of Bt

of resistant insect populations, with >600 insects and mites now toxin in transgenic plants is likely to expedite resistance

development [9].

There are very few well characterised toxins that could be

considered as alternatives or adjuncts to Bt. However, some of the

* Corresponding author at: Institute for Molecular Bioscience, The University of

most promising candidates are novel insecticidal peptides that

Queensland, 306 Carmody Road, St Lucia, Queensland 4072, Australia.

have been isolated from the venom of spiders [7,10–12], the most

Tel.: +61 7 3346 2025; fax: +61 7 3346 2021.

E-mail address: [email protected] (G.F. King). successful insect predators on the planet. Most of these peptides

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N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554 1543

are highly stable because they contain an inhibitor cystine knot passing the extract (buffered in 40 mM Tris, 500 mM NaCl, pH 8.0)

(ICK) motif [13,14] that provides them with resistance to over Ni-NTA Superﬂow resin (Qiagen). Proteins bound non-

extremes of pH, high temperatures, and proteolytic enzymes speciﬁcally were removed by washing with 10 mM imidazole

[7,15]. One of the ﬁrst insecticidal spider-venom peptides to be then the fusion protein was eluted with 500 mM imidazole. The

reported was Aps III from the venom of the trapdoor spider eluted fusion protein was concentrated to 10 ml and the buffer was

Apomastus schlingeri [16]. With a reported LD50 of 133 pmol/g exchanged to remove imidazole. Reduced and oxidised glutathione

against the tobacco hornworm Manduca sexta, this peptide is one were then added to 0.6 mM and 0.4 mM, respectively, to maintain

of the most potent insect toxins described to date according to TEV protease activity and promote folding of the protein.

ArachnoServer [17,18]. Aps III comprises 37-residues with four Approximately 100 mg of His6-tagged TEV protease was added

disulﬁde-bonds, but its three-dimensional structure and mode of per mg of rAps III, then the cleavage reaction was allowed to

action are unknown. proceed at room temperature for 12 h. The cleaved His6-MBP and

Here we describe the development of an efﬁcient E. coli His6-TEV were removed by passing the solution over Ni-NTA

expression system that was used to produce recombinant Aps III Superﬂow resin, while the eluate containing rAps III was collected

(rAps III) for functional and structural studies. The 3D solution for further puriﬁcation using reverse-phase HPLC (RP-HPLC). RP-

structure of rAps III determined using NMR spectroscopy revealed HPLC was performed on a Vydac C18 column (250 4.6 mm,

an inhibitor cystine knot motif that is commonly found in spider- particle size 5 mm) using a ﬂow rate of 1 ml/min and a gradient of

venom peptides. However, rAps III contains an additional disulﬁde 20–40% Solvent B (0.043% triﬂuoroacetic acid (TFA) in 90%

bridge that is employed as a molecular staple to tie together the acetonitrile) in Solvent A (0.05% TFA in water) over 20 min. rAps

ends of a very large b-hairpin loop. We demonstrate that the III contains a non-native N-terminal serine residue (a vestige of the

insecticidal activity of rAps III results from a potent block of insect TEV protease cleavage site), making it one-residue longer than

voltage-gated sodium (Nav) channels in combination with a native Aps III (Fig. 1B).

weaker block of insect voltage-gated calcium (Cav) channels.

However, in striking contrast to previously characterised Nav 2.3. Mass spectrometry

channel blockers from spiders, all of which are gating modiﬁers

[19], rAps III appears to be a pore blocker that plugs the outer Toxin masses were conﬁrmed by matrix assisted laser

vestibule of insect Nav channels. desorption ionisation–time of ﬂight mass spectrometry (MALDI-

TOF MS) using a Model 4700 Proteomics Bioanalyser (Applied

2. Material and methods Biosystems, CA, USA). RP-HPLC fractions were mixed (1:1 v:v) with

a-cyano-4-hydroxy-cinnamic acid matrix (5 mg/ml in 50/50

2.1. Chemicals acetonitrile/H2O) and MALDI-TOF spectra were acquired in

positive reﬂector mode. All reported masses are for monoisotopic

All chemicals were purchased from Sigma–Aldrich Australia [M+H] ions.

(Castle Hill, NSW, Australia), Sigma–Aldrich USA (St Louis, MO,

USA), or Merck Chemicals (Kilsyth, Victoria, Australia) with the 2.4. Insecticidal assays

exception of isopropyl-b-D-thiogalactopyranoside (IPTG) and

streptomycin (Life Technologies, Victoria, Australia), tetrodotoxin rAps III dissolved in insect-saline [25] was injected into the

(Alomone Labs, Israel), and HPLC-grade acetonitrile (RCI Labscan, ventro-lateral thoracic region of sheep blowﬂies (Lucilia cuprina;

13 15

Bangkok, Thailand). C6-glucose and NH4Cl were from Sigma– mass 19.7–23.9 mg) using a 1.0 ml Terumo Insulin syringe (B-D

Aldrich Australia. Recombinant His6-TEV protease (EC 3.4.22.44) Ultra-Fine, Terumo Medical Corporation, MD, USA) with a ﬁxed 29

was produced in-house used a published protocol [20]. gauge needle ﬁtted to an Arnold hand micro-applicator (Burkard

Manufacturing Co. Ltd., England). A maximum volume of 2 ml was

2.2. Production of recombinant Aps III injected per ﬂy. Thereafter, ﬂies were individually housed in 2 ml

tubes and the paralytic activity was determined after 24 h. A total

A synthetic gene encoding Aps III, with codons optimised for of three tests were carried out and for each test seven doses of rAps

expression in Escherichia coli, was produced and cloned into a III (n = 10 ﬂies per dose) and the appropriate control (insect saline;

variant of the pLIC-MBP expression vector [21] by GeneArt n = 30 ﬂies each) were used. PD50 values were calculated as

(Invitrogen, Regensburg, Germany). This vector (pLIC-NSB1) described previously [26].

encodes a MalE signal sequence for periplasmic export [22], a

His6 tag for afﬁnity puriﬁcation, a maltose binding protein (MBP) 2.5. Electrophysiological measurements

fusion tag to aid solubility [23], and a tobacco etch virus (TEV)

protease recognition site directly preceding the codon-optimised 2.5.1. Primary cell culture

Aps III gene (Fig. 1A). Dorsal unpaired median (DUM) neurons were isolated from

The plasmid encoding Aps III was transformed into E. coli strain unsexed adult American cockroaches (Periplaneta americana) as

BL21(lDE3) for recombinant toxin production. Protein expression described previously [27,28]. Brieﬂy, terminal abdominal ganglia

and puriﬁcation were performed as described previously [24] with were removed and placed in normal insect saline (NIS) containing

minor modifications. Briefly, cultures were grown in Terrific Broth (in mM): NaCl 180, KCl 3.1, N-hydroxyethylpiperazine-N-ethane-

at 37 8C with shaking at 120 rpm. Toxin gene expression was sulfonic acid (HEPES) 10 and D-glucose 20. Ganglia were then

induced with 1 mM IPTG at an OD600 of 1.1–1.2, then cells were incubated in 1 mg/ml collagenase (type IA) (EC 3.4.24.3) for 40 min

grown at 18 8C for a further 12 h before harvesting by centrifuga- at 29 8C. Following enzymatic treatment, ganglia were washed

13 15

tion for 15 min at 8000 rpm. For production of uniformly C/ N- three times in NIS and triturated through a ﬁre-polished Pasteur

labelled rAps III, cultures were grown in minimal medium pipette. The resultant cell suspension was then distributed onto

13 15

supplemented with C6-glucose and NH4Cl as the sole carbon 12-mm diameter glass coverslips pre-coated with 2 mg/ml

and nitrogen sources, respectively. concanavalin A (type IV). DUM neurons were maintained in NIS

The His6-MBP-toxin fusion protein was extracted from the supplemented with 5 mM CaCl2, 4 mM MgCl2, 5% foetal bovine

bacterial periplasm by cell disruption at 26 kPa (TS Series Cell serum and 1% penicillin and streptomycin, and maintained at

Disrupter, Constant Systems Ltd, Northants, UK), then captured by 29 8C, 100% humidity.

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1544 N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554

Fig. 1. Production and functional analysis of recombinant Aps III. (A) Schematic representation of the pLicC-NSB1 vector used for periplasmic expression of Aps III. The coding

region includes a MalE signal sequence (MalESS) for periplasmic export, a His6 afﬁnity tag, an MBP fusion tag, and a codon-optimised gene encoding Aps III, with a TEV protease

recognition site inserted between the MBP and toxin coding regions. The locations of key elements of the vector are shown, including the ribosome binding site (RBS). (B)

Primary structure of rAps III. The non-native N-terminal Ser residue is highlighted in grey and the triglycine sequence is underlined. The disulﬁde framework of rAps III as

determined in the current study is shown above the amino acid sequence. (C) SDS-PAGE gels illustrating different steps in the puriﬁcation of rAps III. Lanes are as follows: M,

molecular weight markers; lane 1, E. coli cell extract prior to IPTG induction; lane 2, E. coli cell extract after IPTG induction; lane 3, lysate resulting from cell disruption; lane 4,

soluble periplasmic extract; lane 5, Ni-NTA beads after loading the cell lysate (the His6-MBP-Aps III fusion protein is evident at 49 kDa); lane 6, eluate from washing Ni-NTA

resin with loading buffer; lane 7, eluate from washing Ni-NTA resin with 10 mM imidazole; lane 8, eluate from washing Ni-NTA resin with 500 mM imidazole; lane 9, puriﬁed

fusion protein before TEV cleavage; lane 10, fusion protein sample after TEV protease cleavage, showing complete cleavage of fusion protein to His6-MBP. (D) RP-HPLC

chromatogram showing the ﬁnal step in the puriﬁcation of rAps III. The asterisk denotes the peak corresponding to correctly folded rAps III. Inset is a MALDI-TOF MS spectrum

showing the [M+H] ion for the puriﬁed recombinant toxin (obs. = 3846.58 Da; calc. = 3846.49 Da). (E) Dose–response curve for the paralytic effects of rAps III determined

24 h after injection into sheep blowﬂies (L. cuprina).

2.5.2. Patch-clamp electrophysiology the inﬂuence of fast time-dependent shifts in steady-state

Ionic currents were recorded in voltage-clamp mode using the inactivation resulting in current rundown, particularly when

whole-cell patch-clamp technique employing version 10.2 of the recording NaV channel currents (INa). Time-dependent shifts in

pCLAMP data acquisition system (Molecular Devices, Sunnyvale, steady-state NaV channel inactivation are typically of the order of

CA). Data were filtered at 5–10 kHz with a low-pass Bessel filter 2–3 mV beyond this period and do not significantly influence

with leakage and capacitative currents subtracted using P-P/4 current amplitude. Experiments were performed with a single

procedures. Digital sampling rates were set between 15 and 25 kHz concentration of toxin tested on one cell, which was subsequently

depending of the length of the protocol. Single-use 0.8–2.5 MV repeated in separate experiments using unexposed cells. The

electrodes were pulled from borosilicate glass and ﬁre-polished number of independent recordings for each type of experiment is

prior to current recordings. Liquid junction potentials were provided in the relevant sections of the results. All experiments

calculated using JPCALC [29], and all data were compensated for were performed at ambient room temperature (20–23 8C).

these values. Cells were bathed in external solution through a To record INa, the external bath solution contained (in mM):

continuous pressurised perfusion system at 1 ml/min, while toxin NaCl 80, CsCl 5, CaCl2 1.8, tetraethylammonium chloride (TEA-Cl)

solutions were introduced via direct pressurised application via a 50, 4-aminopyridine (4-AP) 5, HEPES 10, NiCl2 0.1, and CdCl2 1,

perfusion needle at 50 ml/min (Automate Scientiﬁc, San Fran- adjusted to pH 7.4 with 1 M NaOH. The pipette solution contained

cisco, CA) to a bath volume of 300 ml. To avoid issues of (in mM): NaCl 34, CsF 135, MgCl2 1, HEPES 10, ethylene glycol-

0 0

desensitisation or rundown of currents, particularly with CaV bis(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA) 5, and

channel currents, recording periods were kept as short as possible ATP-Na2 3, adjusted to pH 7.4 with 1 M CsOH. Due to the reported

with the effect of toxin recorded within 5 min of control current rundown with calcium as a charge carrier [30], BaCl2

recordings. Control data was not acquired until at least 20 min replaced CaCl2 in all experiments on voltage-activated calcium

after whole-cell conﬁguration was achieved. This was to eliminate (CaV) channels.

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N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554 1545

The external bath solution for barium current (IBa) recordings currents via two-electrode voltage-clamp recording techniques

contained (in mM): Na acetate 140, TEA-Br 30, BaCl2 3 and HEPES using an OC-725C Oocyte Clamp Ampliﬁer (Warner Instruments,

10, adjusted to pH 7.4 with 1 M TEA-OH. The external solution also CT, USA) with a 150-ml recording chamber. Data were ﬁltered at

contained 300 nM tetrodotoxin (TTX) to block NaV channels. 4 kHz and digitised at 50 kHz using pCLAMP 10. Microelectrode

Pipette solutions contained (in mM): Na acetate 10, CsCl 110, TEA- resistances were 0.1–1 MV when ﬁlled with 3 M KCl. The external

Br 50, ATP-Na2 2, CaCl2 0.5, EGTA 10 and HEPES 10, adjusted to pH recording solution contained (in mM): 96 NaCl, 2 KCl, 5 HEPES, 1

7.4 with 1 M CsOH. The external bath solution for recording global MgCl2 and 1.8 CaCl2, pH 7.6. Experiments were performed at

voltage-activated potassium (KV) channel currents (IK) contained ambient temperature (22 8C) and leak and background conduc-

(in mM): NaCl 200, K gluconate 50, CaCl2 5, MgCl2 4, TTX 0.3, HEPES tance was subtracted by blocking the residual sodium current

10 and D-glucose 10, adjusted to pH 7.4 with 1 M NaOH. The with TTX.

pipette solution consisted of (in mM): K gluconate 135, KF 25, NaCl Voltage–activation relationships were obtained by measuring

9, CaCl2 0.1, MgCl2 1, EGTA 1, HEPES 10 and ATP-Na2 3, adjusted to steady-state currents elicited by stepwise depolarisations of 5 mV

pH 7.4 with 1 M KOH. from a holding potential of–90 mV and calculating conductance (G)

To eliminate any inﬂuence of differences in osmotic pressure, using G = I/(Vm Erev) in which G is conductance, I is peak inward

all internal and external solutions were adjusted to 400 5 mOs- current, Vm is the test potential, and Erev is the reversal potential.

mOsmol/l with sucrose. Experiments were rejected if there were large Reversal potentials were individually estimated for each data set

leak currents or currents showed signs of poor space clamping. [33]. After addition of the toxin to the recording chamber (150 ml),

the equilibration between the toxin and the channel was

2.5.3. Curve-ﬁtting and statistical analyses monitored using weak depolarizations (50-ms test pulse to a

Data were analysed using AXOGRAPH X version 1.3 (Molecular voltage near the foot of the G-V curve, 30 mV) elicited at

Devices). Curve-ﬁtting of I-V data was performed using GraphPad intervals of 5 s. We recorded voltage–activation relationships in

Prism version 5.00d for Macintosh (GraphPad Software, San Diego). the absence and presence of toxin. Off-line data analysis was

Comparisons of two sample means were made using a paired performed using Clampﬁt 10 (Molecular Devices, USA) and Origin 8

Student’s t-test and differences were considered to be signiﬁcant if (OriginLab, MA, USA).

p < 0.05. All data are presented as mean standard error of the

mean (SEM) of n independent experiments. 2.6. Structure determination

Concentration–response curves were ﬁtted using the following

15 13

Logistic equation: Recombinant N/ C-labelled Aps III was dissolved in 20 mM

sodium phosphate, pH 6.0 to a ﬁnal concentration of 450 mM. 5%

1 2

y ¼ (1) H2O was added, then the sample was ﬁltered using a low-protein-

1 þ ð½x=IC50Þ

binding Ultrafree-MC centrifugal ﬁlter (0.22 mm pore size;

Millipore, MA, USA) and 300 mL was added to a susceptibility

where x is the toxin dose, nH is the Hill coefﬁcient (slope

matched 5 mm outer-diameter microtube (Shigemi Inc., Japan).

parameter), and IC50 is the median inhibitory concentration to

NMR data were acquired at 25 8C using a 900 MHz NMR

block channel currents.

spectrometer (Bruker BioSpin, Germany) equipped with a cryo-

The following equation was employed to ﬁt current-voltage (I-

genically cooled probe. 3D and 4D data used for resonance

V) curves:

assignments were acquired using non-uniform sampling (NUS).

1 Sampling schedules that approximated the signal decay in each

I ¼ gmax 1 ðV V revÞ (2)

1 þ exp½ðV V 1=2Þ=s indirect dimension were generated using sched3D [34]. NUS data

were processed using the Rowland NMR toolkit (www.rowlan-

where I is the amplitude of the current at a given test potential V,

d.org/rnmrtk/toolkit.html) and maximum entropy parameters

gmax is the maximal conductance, V1/2 is the voltage at half- 13

were automatically selected as previously described [35]. C-

maximal activation, s is the slope factor, and Vrev is the reversal 15

and N-edited HSQC-NOESY (mixing time of 200 ms) experiments

potential.

were acquired using uniform sampling. All experiments were

The voltage dependence of steady-state NaV channel inactiva- 13

acquired in H2O except for the C-edited HSQC-NOESY, which was

tion (h1/V) data were normalised to the maximum peak current in

acquired in D2O.

the control or maximum peak current and ﬁtted using the

Dihedral angles (29 f, 30 c) were derived from TALOS+

following Boltzmann equation:

chemical shift analysis [36] and the restraint range for structure

A calculations was set to twice the estimated standard deviation. The

h ¼ (3)

1 Thr6–Pro7 peptide bond was determined to be in the trans

1 þ exp½ðV V 1=2Þ=k

conformation on the basis of characteristic NOEs and the Ca and Cb

where A is the fraction of control maximal peak INa (value of 1.0 chemical shifts of the Pro residue.

under control conditions), V1/2 is the midpoint of inactivation, k is Six backbone amide protons were identiﬁed as being involved

1 15

the slope factor, and V is the prepulse voltage. in hydrogen-bonds by comparison of 2D H- N HSQC spectra

acquired either in H2O or 60 min after reconstitution of lyophilised

2.5.4. Two-electrode voltage-clamp recordings from Xenopus oocytes protein in D2O. The presence of intense NOESY crosspeaks for the

To prepare cRNA for oocyte injection, separate plasmids hydroxyl proton of Thr 4 indicated that it is also engaged in a

encoding the a-subunit of the NaV1 channel from the German hydrogen bond. Hydrogen-bond acceptors and disulﬁde-bond

cockroach Blatella germanica (BgNaV1; [31]) and the Drosophila partners were identiﬁed from preliminary structure calculations,

NaV1 auxiliary subunit TipE [32] were linearised with NotI, and hydrogen-bond and disulﬁde-bond restraints were applied in

followed by in vitro transcription using T7 polymerase (mMES- subsequent structure calculations as described previously [37].

SAGE mMACHINE kit, Life Technologies, CA, USA). After Xenopus NOESY spectra were manually peak picked and integrated, then

oocytes were co-injected at 1:5 molar ratio with cRNA encoding peaklists were automatically assigned, distance restraints

BgNaV1 and TipE, they were incubated for 2–3 days at 17 8C (in extracted, and an ensemble of structures calculated using the

96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl2, 1.8 mM CaCl2, torsion angle dynamics package CYANA 3.0 [38]. The tolerances

50 mg/ml gentamycin, pH 7.6) prior to recording BgNaV1-mediated used for CYANA 3.0 were 0.025 ppm in the direct H dimension,

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1546 N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554

0.03 ppm in the indirect H dimension, and 0.3 ppm for the the absence of any signiﬁcant changes in the time to peak, time

13 15

heteronucleus ( C/ N). During the automated NOESY assign- course of inactivation (decay) kinetics, or time course of tail

ment/structure calculation process, CYANA assigned 86% of all currents at the end of the depolarising test pulse, as shown in the

NOESY crosspeaks (1127 out of 1313). representative current traces in Fig. 2A. At a concentration of

30 nM, rAps III reduced peak INa by 29 6% (n = 5 cells, p < 0.05). In

3. Results separate experiments, higher concentrations of 300 nM and 1 mM

rAps III produced a concentration-dependent reduction in peak INa by

3.1. Production of recombinant Aps III 43 5% (n = 9 cells, p < 0.001) and 53 5% (n = 5 cells, p < 0.01),

respectively.

Recombinant production of venom toxins is often challenging The half-maximal inhibitory concentration (IC50) for rAps III on

due to the presence of multiple disulﬁde bonds, which cannot be DUM neuron INa was estimated to be 540 nM (Fig. 2C). However,

formed in the cytoplasm of most prokaryotic and eukaryotic cells the Hill coefﬁcient was signiﬁcantly less than unity (shallower

because of the reducing intracellular environment. An alternative slope), which may reﬂect incomplete block at higher concentra-

approach that has proved successful for expression of disulﬁde- tions, as has been observed with certain m-conotoxin derivatives

rich spider toxins [24,39,40] is production in the periplasm of [47].

E. coli, where the enzymes involved in disulﬁde-bond formation are

located [41]. Thus, we attempted to produce rAps III using an IPTG- 3.4. rAps III is a classical pore blocker

inducible construct (Fig. 1A) that allowed export of a His6-MBP-

toxin fusion protein to the E. coli periplasm. To determine whether toxin inhibition of peak INa was due to a

Using this expression system, a signiﬁcant amount of His6- depolarising shift in the voltage dependence of activation, families

MBP-toxin fusion protein was recovered in the soluble cell fraction of INa (Fig. 3A,B) were elicited using a test pulse that depolarised

following IPTG induction (Fig. 1C, lanes 1–4). The fusion protein the cell from Vh of 90 mV to +70 mV for 50 ms in 10-mV

was subsequently puriﬁed using nickel afﬁnity chromatography increments (Fig. 3F). Peak INa were then normalised against the

(Fig. 1C, lanes 5–8) then eluted from the column and cleaved with maximum peak INa in the control and plotted against membrane

His6-tagged TEV protease (Fig. 1C, lanes 9–10). The His6-tagged potential (V) to establish an INa-V curve. Peak INa was then ﬁtted to

MBP and TEV protease were removed by passage over a nickel Equation 2 (Materials and Methods) using non-linear regression

column, then the eluted toxin was further puriﬁed using RP-HPLC analysis. In the absence of toxin, INa activated around 60 mV. This

(Fig. 1D). rAps III eluted as a single major disulﬁde-bond isomer threshold did not shift in the presence of any concentration of rAps

with a retention time of 25 min under the chosen experimental III tested as shown by the superimposed control and toxin curves

conditions. The purity of recombinant rAps III following RP-HPLC around 60 mV (Fig. 3C,D). The voltage at half maximum NaV

was >98% as assessed by SDS-PAGE and MALDI-TOF mass channel activation (V1/2) in control cells was only marginally

spectrometry (Fig. 1D, inset), and the ﬁnal yield was 1.5 mg of shifted (4 mV) in the hyperpolarising direction in the presence

toxin per litre of culture. of 1 mM rAps III (control V1/2 = 35 1 mV versus toxin V1/2 =

31 3 mV; n = 4 cells, p < 0.005). This is more clearly observed

3.2. rAps III induces irreversible paralysis in insects as a lack of any signiﬁcant shift in the voltage dependence of

activation when currents recorded in the presence of toxin were

The insecticidal activity of rAps III was tested using the blowﬂy normalised to the peak inward control current (Fig. 3D). No

Lucilia cuprina. This dipteran pest is the causative agent of ﬂystrike, signiﬁcant shifts in V1/2 were observed with either 30 nM or

which results in annual economic losses in Australia of $280 300 nM rAps III. Considering the time-dependent hyperpolarising

million [42]. rAps III induced ﬂaccid paralysis in adult L. cuprina, shifts in V1/2 of around 5 mV over a 10–15 min period that occur in

and the PD50 measured 24 h after injection was 700 35 pmol/g whole-cell patch clamp conﬁgurations, this would indicate that the

(Fig. 1E). The toxicity assay used does not allow measurement of toxic toxin does not alter the voltage-dependence of activation. Impor-

effects for periods extending beyond 24 h as the survival rate in tantly, only a depolarising shift in the voltage-dependence of NaV

control cohorts begins to decrease, possibly due to conﬁnement of the channel activation would reduce INa.

ﬂies in small tubes. However, ﬂies paralysed by a high dose of rAps III To determine whether toxin-induced block of INa was voltage-

did not recover but died two days post-injection. We conclude that dependent, peak INa in the presence of toxin was calculated as a

rAps III produces an irreversible paralysis in blowﬂies and would fraction of the corresponding control INa from the INa-V relation-

most likely produce similar effects in related dipterans such as ships. Data were then ﬁtted by linear regression and the slope

mosquitoes and tsetse ﬂies that vector human diseases. coefﬁcient determined. This revealed that the inhibition of NaV

channels by 1 mM rAps III was voltage-independent and the

3.3. rAps III is a potent blocker of insect NaV channels binding of the toxin to the channel was not relieved at increasing

membrane potentials (Fig. 3E). The slope of the line did not

The majority of insecticidal spider toxins that have been signiﬁcantly deviate from zero (p > 0.05, n = 4 cells).

isolated to date modulate the activity of voltage-gated ion In separate experiments, the effects of rAps III on the voltage-

channels [7,11,28,43,44]. We therefore used patch-clamp electro- dependence of steady-state inactivation (h1/V) were examined to

physiology to examine the ability of rAps III to modulate the determine whether the reduction of peak INa was due to

activity of a variety of ion channels in cockroach DUM neurons. stabilisation of the inactivated (closed) state of the channel, as

NaV channel currents (INa) in DUM neurons were elicited using opposed to a pore blocking mechanism. Accordingly, experiments

50-ms depolarising test pulses from a holding potential (Vh) of– were conducted using a two-pulse protocol consisting of a 1 s

90 mV to 10 mV every 10 s (0.1 Hz) (Fig. 2D). This elicited a conditioning pre-pulse (Vprepulse) followed by a 50 ms test pulse

rapidly activating and inactivating ionic current characteristic of (Vtest) to 10 mV. The conditioning prepulse clamped the

classical INa observed previously in DUM neurons (Fig. 2A,B) membrane potential from 120 mV to 0 mV in 10-mV increments

[28,45,46]. This current was conﬁrmed to be mediated by NaV (see inset to Fig. 4A,B). Due to increasing levels of depolarisation

channels following complete INa inhibition with 300 nM TTX during the conditioning prepulse, channels eventually accrue in

(Fig. 2B). In separate experiments, perfusion with rAps III produced the inactivated state and have insufﬁcient time to recover from

a concentration-dependent inhibition of peak INa. This occurred in inactivation before the test pulse. Thus, INa amplitude decreases

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Fig. 2. Effects of rAps III on NaV channels in cockroach DUM neurons. (Aa-c) Typical effects of increasing concentrations of rAps III on INa. Traces show superimposed control

(black) and toxin (grey and shaded) current traces elicited by a 50-ms depolarising test pulse (Vtest) shown in panel D. The reduction in INa is highlighted in the inset of panels

Aa-c. (B) Complete block of INa by 300 nM TTX, conﬁrming that ionic currents are solely mediated through NaV channels. Dotted lines represent zero current. (C)

Concentration–response relationship of rAps III to inhibit peak INa. The percentage block at increasing concentrations of rAps III were ﬁtted with a logistic function (Eq. 1; see

Materials and Methods). The median inhibitory concentration (IC50) was determined to be 540 nM. Data points are the mean SEM of 5–9 cells.

with increasing prepulse potential (Fig. 4A,C). In the presence of M-LVA and HVA CaV channel currents. This occurred in the

30 nM rAps III, INa was inhibited to 79 3% (n = 3 cells) of control absence of changes in M-LVA and HVA IBa activation and

amplitude (parameter ‘A’ in Equation 3). Normalisation of the toxin inactivation kinetics, with no alteration in the time to reach peak

data to the maximum peak INa during the test pulse revealed that the or timecourse of current decay (Fig. 5A). The M-LVA CaV channel

curves almost completely overlap (Fig. 4D) with an insigniﬁcant 2-mV currents were reduced by 28 5% (p < 0.01, n = 5 cells) and HVA

hyperpolarising shift in h1/V from 56 1 mV in controls to CaV channel currents by 31 7% (p < 0.01, n = 6 cells). The small

58 1 mV in the presence of rAps III (p > 0.05, n = 3 cells; difference in the block between inhibition of M-LVA and HVA current

Fig. 3D). Thus the 21% reduction in NaV channel current does not was statistically insigniﬁcant (unpaired Student’s t-test, p > 0.05).

appear to be the result of a reduction in channel availability due to Given the weak effects on CaV channel currents at 1 mM rAps III,

stabilisation of the channels in the inactivated state. Therefore rAps III experiments were not conducted at 30 nM or 300 nM concentra-

appears to be a classical pore blocker. tions. In an additional smaller number of cells there was a partial

recovery from CaV channel current inhibition. In these cells initial

3.5. rAps III is a weak blocker of insect CaV channels rapid inhibition was followed by partial recovery to a steady-state

level. This resulted in a statistically insigniﬁcant block of both M-LVA

We next tested the ability of rAps III to modulate the activity of CaV channel currents (18 6% block, p > 0.05, n = 4 cells) and HVA

insect CaV channels as numerous spider-venom peptides have CaV channel currents (19 12%, p > 0.05, n = 3 cells).

been demonstrated to inhibit this channel [48,49]. Two distinct To investigate whether the weak block of CaV channels by rAps

CaV channel subtypes have been previously observed in DUM III was due to a shift in the threshold of CaV channel activation, the

neurons: mid to low-voltage-activated (M-LVA) and high-voltage IBa-V relationship was examined. The IBa-V relationships were

activated (HVA) CaV channels [46]. M-LVA and HVA CaV channel established from families of IBa generated by 100-ms depolarising

barium currents (IBa) were elicited using alternating 100-ms test potentials from Vh of 90 mV to +40 mV, at 5-mV increments

depolarising test pulses to 30 mV (M-LVA IBa) and +20 mV (HVA every 7 s. Families of peak inward IBa were normalised against

IBa) from a Vh of 90 mV every 7 s. These elicited inward currents the maximum control inward peak IBa and plotted against the

characteristic of classical IBa observed previously in DUM neurons membrane potential (Fig. 5Ba). CaV channels activated around

[46]. Perfusion with 1 mM rAps III caused weak inhibition of both 60 mV, and this threshold was not altered in the presence of

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1548 N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554

Fig. 3. Effects of rAps III on the voltage-dependence of NaV channel activation in cockroach DUM neurons. Typical families of INa recorded prior to (A), and following (B),

application of 1 mM rAps III. Nav channel currents were elicited by the test pulse protocol shown in panel F. (C-D) Normalised peak INa-V relationships. Currents recorded in

the presence of 1 mM rAps III were normalised to the maximum inward INa in controls (C), or maximum inward INa (D). Data shows INa before (closed symbols), and after (open

symbols and shaded), application of 1 mM rAps III. Data were ﬁtted with Eq. (2) (see Materials and Methods). As can be seen in panel (D), no signiﬁcant shifts in the voltage

dependence of NaV channel activation were observed. (E) Fractional block of INa by 1 mM rAps III showing lack of voltage dependence. Normalised peak INa in the presence of

toxin were calculated as a fraction of peak control INa and plotted against the test potential. Data were taken from panel C and ﬁtted using linear regression. All data are

expressed as the mean SEM of 4 cells.

1 mM rAps III. Additionally, there were no signiﬁcant shifts in the non-inactivating delayed-rectiﬁer [IK(DR)], transient ‘‘A-type’’

V1/2 of channel activation (p > 0.05, n = 4 cells) and currents were [IK(A)], and large-conductance Ca -activated [IBK(Ca)] KV channel

essentially superimposable when normalised to the peak IBa currents [28,50]. To determine the effects of 1 mM rAps III on KV

(Fig. 5Bb). The partial block of CaV channels by rAps III was also channels, global KV channel currents (IK) were generated by 100-

voltage-independent (p > 0.05, n = 4 cells, data not shown). ms depolarising test pulses to +25 mV from a Vh of 80 mV, every

5 s (0.2 Hz). This generated a large outward IK that displayed fast

3.6. rAps III does not modulate the activity of insect KV channels activation and partial inactivation, consistent with global IK

previously observed in cockroach DUM neurons [27,28]. Global IK

The major outward KV channel current subtypes present were measured at the peak and at the end of the test pulse

in cockroach DUM neurons include a slowly activating, (100 ms). The early peak global IK results mainly from the

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Fig. 4. Effects of rAps III on steady-state NaV channel inactivation (h1). Steady-state inactivation was determined using a two-pulse protocol (see inset). (A and B) Typical peak

INa recorded during the test pulse (Vtest) are shown following 1-s prepulse potentials (Vprepulse) to 120 mV, 60 mV and 40 mV recorded before (left-hand traces), and

following (right-hand shaded traces), perfusion with 30 nM rAps III. Dotted lines represent zero current. (C and D) Peak INa, recorded during Vtest, were expressed as a fraction

of maximum control INa (C), or normalised to peak INa amplitude (D), and plotted against prepulse potential. Panels show the proportion of INa that is available for activation

under control conditions (closed circles), and during perfusion with 30 nM rAps III (open circles and shaded). The h1/V curves were ﬁtted with Eq. 3 (see Materials and

Methods). All data are expressed as the mean SEM of 3 cells.

contribution of rapidly activating IK(A) and IK(Ca), while the late cloned BgNaV1 channel [31]. At 1 mM concentration, rAps III

global IK results from the slowly activating IK(DR) and slow strongly inhibited BgNaV1-mediated sodium currents over a wide

inactivating component of the IK(Ca). voltage range (Fig. 6A,B). Boltzmann ﬁts of conductance-voltage

Application of 1 mM rAps III caused minimal inhibition of global relationships before (V1/2 = 27 1 mV; slope factor = 4.1 0.2)

IK. rAps III reduced the peak global IK by only 2 2% (p > 0.05, n = 4 and after (V1/2 = –27 1 mV; slope factor = 4.6 0.3; n = 3 cells)

cells; Fig. 5C) while late global IK were inhibited only slightly by toxin addition as well as the steady-state inactivation relationships

5 2% (p < 0.05, n = 4 cells; Fig. 5C). This lack of overt activity was (control: V1/2 = 53 1 mV; slope factor = 4.3 0.1 and toxin: V1/

mirrored by the lack of effect on the voltage-dependence of global IK 2 = 55 1 mV; slope factor = 4.3 0.2; n = 3 cells) revealed no

activation. Global IK-V relationships for early and late currents were signiﬁcant changes in the midpoints or slope factors (Fig. 6C). The

not signiﬁcantly altered with no marked differences in V1/2 values onset of rAps III action is rapid, and there is a fast and complete

between the IK-V for controls (V1/2 = 3 mV for early and 4 mV for late recovery of channel current upon toxin washout (Fig. 6D). In contrast

IK) versus toxin (V1/2 = 4 mV for early and 9 mV for late IK) to gating-modiﬁer toxins that inhibit channel opening by interacting

(Fig. 5D). Due to the lack of any overt activity of 1 mM rAps III on with one or more of the four NaV channel voltage sensors [52], rAps III

global KV channel currents, the effect of rAps III on individual KV decreased INa without shifting the midpoint of activation to more

channel sub-types was not pursued. We conclude that rAps III does depolarised voltages (Fig. 6B). Together with the experiments

not modulate the activity of delayed-rectiﬁer, ‘A-type’ or BKCa reported above on DUM neurons (Fig. 3), this is consistent with a

potassium channels that are the major contributors to the global pore-blocking activity.

outward Kv channel current in DUM neurons [51].

3.8. High-resolution solution structure of rAps III

3.7. rAps III is a potent blocker of cloned cockroach NaV channels

The development of an efﬁcient bacterial expression

13 15

To further investigate the ability of rAps III to inhibit insect NaV system allowed us to produce uniformly C/ N-labelled

channels, we applied the toxin to Xenopus oocytes expressing the rAps III for structure determination using heteronuclear NMR.

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Fig. 5. Effect of rAps III on KV and CaV channel currents in cockroach DUM neurons. (A) Whole-cell M-LVA and HVA IBa in the absence (black traces), and presence (dark grey

and shaded traces), of 1 mM rAps III. M-LVA and HVA IBa were activated by a 100 ms Vtest to 30 mV and +20 mV, respectively, as shown in the inset in panel C. Perfusion with

1 mM rAps III partially blocked M-LVA (Aa) and HVA (Ab) CaV channel currents. Dotted lines represent zero current. (B) Effects of 1 mM rAps III on the voltage-dependence of

CaV channel activation. Families of CaV channel currents were generated by the Vtest protocol shown in the inset of panel C. Currents recorded in the presence of 1 mM rAps III

were normalised to the maximum inward IBa in control (Ba) or maximum inward IBa in toxin (Bb). IBa-V relationships show current recorded before (closed circles), and after

(open circles and shaded), perfusion with 1 mM rAps III. Normalised I-V relationships were ﬁtted using Eq. 2. (C) Typical superimposed global IK recorded prior to (black

traces), and following (dark grey and shaded traces), application of 1 mM rAps III. Currents were generated by 100-ms depolarising test pulses (Vtest) as shown in the as shown

in the inset in panel C. (D) Effects of rAps III on the voltage-dependence of global KV channel activation. Families of outward IK were recorded before (closed circles), and after

(open circles and shaded), application of 1 mM rAps III. Families of IK were generated by the Vtest protocol shown in the inset of panel C. Global IK-V relationships show effects

of the toxin on peak (Da) and late (Db) global IK. Late currents were measured at 100 ms. All data are expressed as the mean SEM of 4 cells.

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Table 1

Structural statistics for the ensemble of rAps III structures.

Experimental restraints

Interproton distance restraints

Intraresidue 111

Sequential 159

Medium range (i–j < 5) 93

Long range (i–j 5) 165 3

Hydrogen-bond restraints 14

Disulﬁde-bond restraints 12

Dihedral-angle restraints (f, c) 59

Total number of restraints per residue 16.1

R.m.s. deviation from mean

coordinate structure (A˚ )

All backbone atoms (residues 1–38) 0.33 0.08

All heavy atoms (residues 1–38) 0.54 0.07

Backbone atoms (residues 2–27, 32–38) 0.13 0.03

Heavy atoms (residues 2–27, 32–38) 0.43 0.06 4

Stereochemical quality

Residues in most favoured 84.5 2.8

Ramachandran region (%)

Ramachandran outliers (%) 2.4 1.4

Unfavourable sidechain rotamers (%) 5.0 2.1

Clashscore, all atoms 0.0 0.0

Overall MolProbity score 1.67 0.15

Fig. 6. rAps III inhibits BgNa 1-mediated sodium currents. (A) Inhibition of BgNa 1-

V V 1

All statistics are given as mean S.D.

mediated sodium currents by 1 M rAps III at a depolarization to 20 mV from a

m 2

Only structurally relevant restraints, as deﬁned by CYANA, are included.

holding voltage of 90 mV. (B) Representative I -V relationship for BgNa 1 before

Na V 3

Two restraints were used per hydrogen bond.

(black) and after (grey) addition of 1 M rAps III. Currents were elicited by 5-mV

m 4

According to MolProbity (http://molprobity.biochem.duke.edu).

step depolarizations from a holding voltage of 90 mV. (C) Comparison of the

Deﬁned as the number of steric overlaps >0.4 A˚ per thousand atoms.

gating properties of BgNaV1 before (black) and after (grey) addition of 1 mM rAps III.

Shown are the deduced conductance (G)–voltage (ﬁlled circles) and steady-state

inactivation (open circles) relationships. Error bars denote SEM, with n = 3 cells. (D)

Onset of BgNaV1-mediated INa inhibition by 1 mM rAps III at depolarizations to

20 mV (holding voltage was 90 mV) followed by a complete recovery after toxin Fig. 7A shows a backbone overlay of the ensemble of 20 rAps III

washout.

structures, while a schematic of the top-ranked structure

highlighting key secondary structure elements is shown in

1 15 13 13 13 0

HN, N, Ca, Cb, and C resonance assignments for the toxin Fig. 7B. Three of the four disulﬁde bonds in rAps III form a

were obtained from analysis of amide-proton strips in 3D classical ICK motif in which the Cys2–16 and Cys9–20 disulﬁde

1 13

HNCACB, CBCA(CO)NH, and HNCO spectra. Sidechain H and C bonds and the intervening sections of polypeptide backbone form a

chemical shifts were obtained using a 4D HCC(CO)NH-TOCSY 13-residue ring that is pierced by the Cys15–Cys36 disulﬁde bond

experiment, which has the advantage of providing sidechain (Fig. 7A). This region forms a highly structured, disulﬁde-rich core

1 13

H– C connectivities [34]. Complete chemical shift assignments from which emerges an unusual b-hairpin with a very large

have been deposited in BioMagResBank (Accession Number hairpin loop (Fig. 7B). The two b-strands of the hairpin are formed

18946). by residues 19–21 (b2) and 34–37 (b3), while the loop comprises

CYANA was used for automated NOESY assignment and residues 22–33. Residues 7–9 form a third b-strand (b1) and

structure calculation [38]. The disulﬁde-bond pattern (1–4, 2–5, residues 11–14 form a single turn of 310-helix (a1) (Fig. 7B). A

3–8, 6–7; see Fig. 1B) was unambiguously determined from summary of the secondary structure of rAps III as judged by

preliminary structures calculated without disulﬁde-bond PDBsum [56] is shown in Fig. 7C.

restraints [53]; this disulﬁde framework is notably different from While the compact ICK region of the rAps III structure is similar

the 1–4, 2–5, 3–6, 7–8 framework predicted in the UniProt entry to previously determined structures of knottin peptides [57], the

for rAps III (P49268). Disulﬁde-bond and hydrogen-bond restraints protruding b-hairpin loop, which often houses the pharmacophore

were used in the ﬁnal round of structure calculations. 200 in this class of spider toxins [15], is highly unusual. First, this

structures were calculated from random starting conformations, hairpin is unusually large, comprising 13 residues (Fig. 7A,B). In

then the 20 conformers with highest stereochemical quality as comparison, the average size of the b-hairpin loop in the 35 spider-

judged by MolProbity [54] were selected to represent the solution venom ICK toxins listed in ArachnoServer [17,18] is 4.8 1.8

structure of rAps III. Coordinates for the ﬁnal ensemble of residues. Second, the hairpin loop contains a glycine triplet (Gly29–

structures are available from the Protein Data Bank (Accession Gly30–Gly31; underlined in Fig. 1B) that is rare in this class of toxins.

Number 2M36). Third, the glycine-rich portion of the loop is poorly deﬁned in the

Statistics highlighting the high precision and stereochemical ensemble of rAps II structures (Fig. 7A). Gly30 and to a lesser extent

quality of the ensemble of rAps III structures are shown in Table 1. Gly32 consistently fall into unfavourable regions of the Ramachan-

The average MolProbity score of 1.67 places the ensemble in the dran plot, suggesting that this region is highly dynamic in solution.

90th percentile relative to all other structures ranked by Nevertheless, the dynamics of the triglycine loop is likely to be limited

MolProbity. The high stereochemical quality of the ensemble by the Cys27–Cys32 disulﬁde bond (Fig. 7A,B) which forms a

stems from a complete absence of bad close contacts, very few molecular staple that serves to isolate this loop from the remainder

unfavourable sidechain rotamers (5%), and reasonably high of the b-hairpin, which in contrast is well structured.

Ramachandran plot quality (85% of residues in the most favoured It should be noted that the disulﬁde framework has not been

region). The structural ensemble is also highly precise with experimentally established for native Aps III. However, the fact

backbone and heavy-atom RMSD values over all residues of that rAps III is insecticidal and that it contains an ICK motif that is

0.32 0.09 A˚ and 0.54 0.07 A˚ , respectively. The ensemble of rAps the deﬁning structural characteristic of this class of toxins suggests

III structures ranks as ‘‘high resolution’’ based on these measures of that the recombinant peptide has the same disulﬁde framework

precision and stereochemical quality [55]. and 3D fold as the native peptide.

Author's personal copy

1552 N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554

Fig. 7. Three-dimensional structure of rAps III. (A) Stereoview of an overlay of the ensemble of 20 rAps III structures. Disulﬁde bonds are highlighted in red and the N- and C-

termini are labelled. The structures are overlaid over the backbone atoms of residues 2–27 and 32–38 in order to highlight the disordered nature of the triglycine loop

(residues 27–32) relative to the well-structured ICK region of the toxin. (B) Ribbon representation of the rAps III structure highlighting the various secondary structure

elements and disulﬁde bonds. The views in panels (B) and (C) are related by a 1808 rotation around the long axis of the molecule. (C) Topology map of the secondary structure

of rAps III. The N- and C-termini are labelled.

4. Discussion voltage-dependence of steady-state NaV channel inactivation.

Thus rAps III does not share a common mode of action with

4.1. rAps III potently inhibits insect NaV channels m-TMTX-Hme1a, despite its ability to inhibit INa.

Interestingly, rAps III displays weak sequence homology with a

In the present study, the effect of recombinantly produced Aps range of spider d-toxins (41% homology). All d-toxins are gating

III was examined on three families of insect voltage-activated ion modiﬁers that delay NaV channel inactivation via an interaction

channels: NaV, CaV and KV channels. The main effect of rAps III was with the voltage sensor of channel domain IV [62–67]. As a result,

to produce a concentration-dependent inhibition of insect NaV cells generate spontaneous and repetitive action potentials at, or

channels with an estimated IC50 of 540 nM. The pharmacological near, the resting membrane potential to induce contractile

properties of rAps III resemble those of ‘pore blocking’ toxins that paralysis [65]. A similar effect has been noted with spider b-

target NaV channels such as the guanidinium compounds TTX and toxins that shift the voltage-dependence of activation in the

saxitoxin as well as m-conotoxins from marine cone snail venoms hyperpolarising direction primarily via an interaction with the

that reduce peak INa [58]. voltage sensor of channel domain II [52,68,69]. However, the lack

rAps III displays similar activity to m-TRTX-Hhn2b (hainan- of any signiﬁcant shifts in the voltage-dependence of NaV channel

toxin-I) and m-TRTX-Hh1a (huwentoxin-III), depressant neuro- activation or slowing of NaV channel inactivation kinetics indicates

toxins from the venom of the Chinese tarantulas Haplopelma that the effect of rAps III on NaV channels is not due a modiﬁcation

hainanum and H. huwenum, respectively. These toxins have been of channel gating. Other toxins, such as the depressant toxin b-

postulated to block insect NaV channels via binding to neurotoxin TRTX-Cm1a (ceratotoxin-1) from the straight-horned tarantula

receptor site-1 near the mouth of the channel [59,60]. Both toxins Ceratogyrus marshalli, can inhibit INa by shifting the voltage

block insect NaV channels more potently than vertebrate NaV dependence of activation in the depolarising direction to produce a

channels. For example, m-TRTX-Hhn2b blocks Drosophila melano- depressant phenotype [70]. In contrast, rAps III inhibits insect NaV

gaster NaV channels expressed in Xenopus oocytes with an IC50 of channel conductance in the absence of any depolarising shift in the

4.5 mM compared with a 15-fold higher IC50 of 68 6 mM for block voltage-dependence of activation. In summary, rAps III does not

of rat NaV1.2 channels [59]. m-TRTX-Hh1a did not block any of the cause a hyperpolarising shift (as seen for excitatory spider b-

vertebrate NaV channel subtypes expressed in rat dorsal root ganglion toxins) or a depolarising shift (as observed for depressant spider b-

neurons but it inhibited insect NaV channel currents in cockroach toxins) in the voltage-dependence of NaV channel activation, nor

DUM neurons with an IC50 of 1.1 mM [60]. Thus, although both m- does it slow the kinetics of NaV channel inactivation as observed for

TRTX-Hhn2b and m-TRTX-Hh1a selectively block insect NaV channels, spider d-toxins.

rAps III targets the insect channel with higher afﬁnity, consistent with Based on the pharmacology described here, Aps III should be

its high level of lethality (LD50 = 133 pmol/g) in larvae of the tobacco renamed m-cyrtautoxin-As1a (m-CUTX-As1a) based on the

hornworm M. sexta [16]. rational nomenclature recently proposed for spider-venom

One mechanism that can lead to inhibition of INa is an increase peptides [71]. This is consistent with its action to induce ﬂaccid

in the number of NaV channels stabilised in the inactivated state. paralysis in M. sexta by inhibiting neuronal excitability [16], in

m-TMTX-Hme1a (Hm-1) from the venom of the spider Heriaeus contrast with the spastic paralysis observed with d-toxins and

melloteei inhibits mammalian NaV channels expressed in Xenopus excitatory b-toxins.

oocytes without an alteration in the activation or inactivation

kinetics of the channel [61]. The reduction in peak INa produced by 4.2. Promiscuous activity of rAps III on insect NaV and CaV channels

m-TMTX-Hme1a results from a shift in steady-state NaV channel

inactivation in the hyperpolarising direction [61]. Thus, the While rAps III inhibits insect NaV channels with higher potency

number of closed channels available for opening is reduced, than other insect-selective spider neurotoxins such as m-TRTX-

resulting in ﬂaccid paralysis. Unlike m-TMTX-Hme1a, neither Hhn2b and m-TRTX-Hh1a, the IC50 value (540 nM) is still relatively

rAps III nor m-TRTX-Hh1a produce a signiﬁcant shift in the high compared to spider toxins that inhibit vertebrate NaV

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N.S. Bende et al. / Biochemical Pharmacology 85 (2013) 1542–1554 1553

Table 2

required to determine the precise mechanism of action and

Block of cockroach DUM neuron CaV channels by spider neurotoxins.

binding site of rAps III on insect NaV channels, the current data

CaV channel subtype suggests that it might bind in the outer vestibule of the channel

rather than interact with one of the voltage-sensor domains. The

Toxin M-LVA IC50 (nM) HVA IC50 (nM) Reference

large b-hairpin loop increases the footprint of this region of the

v-HXTX-Hv1a 279 1080 [46]

toxin compared with other ICK-containing spider-venom toxins,

v-HXTX-Ar1a 692 644 [46]

* and computer modelling (E. Deplazes and G.F. King, unpublished)

v-CNTX-Cs1a 467 274 [79]

* indicates that the b-hairpin loop is an ideal size to ﬁt into the turret

Insecticidal toxin also active on mammalian CaV channels.

region of the recently determined crystal structures of bacterial

NaV channels [78]. Thus, in addition to being a useful bioinsecticide

channels [45]. This suggests that rAps III may also target other lead, rAps III might prove to be a valuable pharmacological tool for

voltage-activated channels. Spider toxins can interact with more the study of invertebrate NaV channels.

than one target often across voltage-activated ion channel families

and/or across channel subtypes to elicit multiple functions. This Conﬂict of interest

non-selective activity is due to the common structural elements

shared between voltage-activated ion channels that are recognised The authors declare that they have no conﬂicts of interest.

by these toxins [72]. NaV and CaV channels, in particular, are closely

related and contain shared structural motifs and functional Acknowledgments

domains. Thus it is not surprising to ﬁnd toxins with promiscuous

activity across these channel families [49,67]. Promiscuous activity We thank Ke Dong (Michigan State University) for the BgNav1

of spider toxins with high afﬁnity for both NaV and KV, or NaV and and TipE clones, Geoff Brown (Department of Agriculture, Fisheries

CaV, channels is not without precedence and has been previously and Forestry, Brisbane) for the supply of blowﬂies, and the

demonstrated by vertebrate active spider toxins including the Queensland NMR Network for access to the 900 MHz NMR

NaV/KV channel toxins m/v-TRTX-Hh1a [73] and b/k-TRTX-Cj1a spectrometer at the University of Queensland. This work was

[74] and the NaV/CaV channel toxin b/v-TRTX-Tp2a (ProTx-II) [75]. supported by grants from the Australian Research Council

Moreover, spider toxins targeting CaV channels with additional low (Discovery Grant DP1095728 to G.F.K.) and the National Institute

afﬁnity for NaV channels have also been described including v- of Neurological Disorders And Stroke of the National Institutes of

TRTX-Hg1a (SNX482) [76], v-agatoxin-Aa4a (v-Aga-IVA) [77] and Health (Award Number R00NS073797 to F.B.).

v-hexa toxin-Ar1a [46].

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4 The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage- gated sodium channels.

Published manuscript FEBS Journal, 2015

31 The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large b-hairpin loop Niraj S. Bende1, Sławomir Dziemborowicz2, Volker Herzig1, Venkatraman Ramanujam3, Geoffrey W. Brown4, Frank Bosmans5,6, Graham M. Nicholson2, Glenn F. King1 and Mehdi Mobli3

1 Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia 2 School of Medical & Molecular Biosciences, University of Technology Sydney, NSW, Australia 3 Centre for Advanced Imaging, The University of Queensland, St. Lucia, Qld, Australia 4 Queensland Department of Agriculture, Fisheries and Forestry, Brisbane, Qld, Australia 5 Department of Physiology, Johns Hopkins University, Baltimore, MA, USA 6 Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MA, USA

Keywords Spider venoms contain a plethora of insecticidal peptides that act on neuro- disulfide-rich peptide; heteronuclear NMR; nal ion channels and receptors. Because of their high specificity, potency and pore blocker; spider toxin; voltage-gated stability, these peptides have attracted much attention as potential environ- sodium channel mentally friendly insecticides. Although many insecticidal spider venom Correspondence peptides have been isolated, the molecular target, mode of action and struc- G. F. King, Institute for Molecular ture of only a small minority have been explored. Sf1a, a 46-residue peptide Bioscience, The University of Queensland, isolated from the venom of the tube-web spider Segesteria florentina, is insec- St. Lucia, QLD 4072, Australia ticidal to a wide range of insects, but nontoxic to vertebrates. In order to Fax: +61 7 33462101 investigate its structure and mode of action, we developed an efficient bacte- Tel: +61 7 33462025 rial expression system for the production of Sf1a. We determined a high-reso- E-mail: [email protected] lution solution structure of Sf1a using multidimensional 3D/4D NMR M. Mobli, Centre for Advanced Imaging, The University of Queensland, St. Lucia, spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually QLD 4072, Australia large b-hairpin loop that accounts for a third of the peptide length. This loop Fax: +61 7 33653833 is delimited by a fourth disulfide bond that is not commonly found in knottin Tel: +61 7 33460352 peptides. We showed, through mutagenesis, that this large loop is function- E-mail: [email protected] ally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its ‘depres- (Received 10 June 2014, revised 15 sant’ paralytic phenotype in insects. However, in contrast to the majority of December 2014, accepted 27 December 2014) spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to doi:10.1111/febs.13189 act as a pore blocker.

Introduction Despite intensive control measures, herbivorous insects dengue and Chagas disease [3]. Half of the world’s reduce world crop yields by 10–14% and damage as population is at risk of contracting insect-borne much as 30% of stored grain [1,2]. Insects also vector diseases [4], with malaria alone causing ~ 3000 deaths a wide range of human diseases including malaria, per day [5].

Abbreviations

CaV, voltage-gated calcium channel; DUM, dorsal unpaired median neurons; HVA, high-voltage-activated channel current; ICK, inhibitor cystine knot motif; KV, voltage-gated potassium channel; MBP, maltose binding protein; M–LVA, mid/low-voltage-activated channel current;

NaV, voltage-gated sodium channel; NIS, normal insect saline; Sf1a, U2-segestritoxin-Sf1a; TEV, tobacco etch virus.

FEBS Journal (2015) ª 2015 FEBS 1 Structure and function of the spider toxin Sf1a N. S. Bende et al.

Chemical insecticides are the primary method of ever, the oral activity of Sf1a can be markedly controlling insect pests [6]. Although there appears to improved by fusion to the plant lectin Galanthus niva- be a vast array of extant chemical insecticides, there lis agglutinin, which ferries attached peptide toxins are, in fact, only a few major classes that act on a very across the gut epithelium via transcytosis and effec- small number of molecular targets in the insect ner- tively delivers them to the hemocoel, Malpighian vous system [4,7,8]. These conditions have promoted tubules, fat bodies, ovarioles and central nervous sys- the evolution of insecticide resistance, with > 1000 tem [18–20]. Using this approach, Sf1a was found to arthropod species now resistant to one or more classes be highly insecticidal when fed to a range of insects, of chemical insecticide [9]. The evolution of insecticide including the tomato moth Lacanobia oleracea [17], resistance has led to the withdrawal of chemical insec- the peach–potato aphid Myzus persicae [21] and the ticides that are no longer considered effective, and rice brown planthopper Nilaparvata lugens [21]. concerns about the adverse impact of certain classes of The structure and mode of action of Sf1a remain chemical insecticides on the environment and human unknown. Here, we describe the development of an effi- health have also led to widespread deregistrations and cient Escherichia coli expression system for the produc- use cancellations by regulatory authorities [8]. These tion of Sf1a for structural and functional studies. We developments, along with more stringent requirements show that recombinant Sf1a is insecticidal to sheep for the registration of new insecticides, have signifi- blowflies (Lucilia cuprina) and we demonstrate that it cantly reduced the number of available insecticides [8]. acts by inhibiting insect voltage-gated sodium (NaV) For example, over the period 2005–2009 the U.S. channels and not voltage-gated calcium (CaV) channels, Environmental Protection Agency (EPA) deregistered as initially suggested based on sequence homology. or limited the use of 169 insecticides; over the same Moreover, we demonstrate that Sf1a inhibits insect NaV period only nine new insecticides were registered [4]. channels by blocking the pore of the channel and not by Thus, we are being forced to combat an increasing the more typical allosteric voltage-dependent mecha- insect-pest problem with a rapidly diminishing chemi- nism reported for other spider toxins that act as gating- cal arsenal, resulting in an urgent need to discover new modifiers [14]. The 3D solution structure of Sf1a that we and safe insecticidal compounds. determined using multidimensional heteronuclear NMR The success of transgenic plants expressing insecti- spectroscopy revealed that it contains an inhibitor cidal Cry toxins from the bacterium Bacillus thuringi- cystine knot (ICK) motif that is commonly observed in ensis has created a renewed interest in protein- and spider-venom peptides [8,22,23]. However, Sf1a con- peptide-based insecticides [10]. Along with predatory tains an unusually large and disordered b-hairpin loop beetles, spiders are the most successful insect predators that is stabilized by an atypical fourth disulfide bond on the planet and their venoms are replete with insecti- that anchors the ends of the loop. We show that this cidal peptide toxins [8,11]. Remarkably, the venom of large b-hairpin loop contains a conserved sequence a single spider can contain as many as 1000 unique pep- motif that is critical for the toxin’s insecticidal activity. tides [12,13]. Although the EPA recently approved a spider-venom peptide for use as an insecticide (http:// Results www.vestaron.com/epa-approval), very few insecticidal spider toxins have been sufficiently characterized to war- Production of recombinant Sf1a rant consideration as bioinsecticides [8]. One promising candidate is SFI1 (U2-segestritoxin-Sf1a, hereafter Recombinant production of venom peptide toxins is Sf1a), a 46-residue peptide isolated from the venom of often challenging because of the presence of multiple the tube-web spider Segestria florentina [14]. When disulfide bonds, which cannot be formed in the cyto- injected into tobacco budworms (larvae of the noctuid plasm of most prokaryotic and eukaryotic cells because moth Heliothis virescens), Sf1a induced flaccid paralysis of the reducing intracellular environment. An approach within 15 min and killed larvae within 24 h, with an that has proved successful for expression of disulfide- 1 1 LD50 of 10 lgg (~ 2 nmolg ) [14]. Moreover, Sf1a rich spider toxins is production in the periplasm of did not induce toxic effects in mice when injected intra- E. coli, where the enzymes involved in disulfide-bond venously at a dose of 1.5 mgkg1 (~ 300 pmolg1). formation are located [24]. Thus, to produce Sf1a, we Because spiders inject venom into prey via hypoder- employed an IPTG-inducible construct (Fig. 1A) that mic-needle-like fangs, there is no evolutionary selection allowed export of a His6–maltose binding protein pressure for spider toxins to be orally active. Although (MBP)–toxin fusion protein to the E. coli periplasm. some insecticidal spider-venom peptides are orally Using this expression system, a significant amount 1 active [15,16] this is not the case for Sf1a [17]. How- of His6–MBP–toxin fusion protein (> 5mgL ) was

2 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

A T7 promoter T7 terminator lac operator His Kpnl Sspl 6 pLIC-NSB2

U -SGTX-Sf1a Maltose binding protein (MBP) 2

RBS MalEss TEV recognation site

SKECMTDGTVCYIHNHNDCCGSCLCSNGPIARPWEMMVGNCMCGPKA 110 20 30 40 47

C M1 2 M3 4567 8 100 100 75 75

50 50

37 37

25 25 20 20

1.0 DE100 nat-Sf1a PD –1

5 80 50 = 2.2±0.2 nmol·g

5 0 5 60 S-Sf1a 0.5 5055 5060 5065 5070 PD = 1.5±0.3 nmol·g–1 Mass (Da) 40 50 20

(arbitrary units) Paralysed %

214 0

A 0.0 0102030 40 10–12 10–11 10–10 10–9 10–8 10–7 –1 Retention time (min) Dose (mol·g )

Fig. 1. Production and functional analysis of rSf1a. (A) Schematic representation of the pLicC–NSB2 vector used for periplasmic expression of rSf1a. The coding region includes a MalE signal sequence (MalESS) for periplasmic export, a His6 affinity tag, an MBP fusion tag and a codon-optimized gene encoding rSf1a, with a TEV protease recognition site inserted between the MBP and toxin-coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS), T7 promoter and lac operator. (B) Primary structure of rSf1a. The non-native N-terminal Ser residue is highlighted in grey. Disulfide bridge connectivities are indicated above the sequence. (C) SDS/PAGE gels illustrating different steps in the purification of rSf1a. Lanes are as follows: M, molecular mass markers; lane 1, E. coli cell extract prior to IPTG induction; lane 2, E. coli cell extract after IPTG induction; lane 3, soluble periplasmic extract (the

His6–MBP–rSf1a fusion protein is evident at ~ 50 kDa); lane 4, Ni-NTA beads after loading the cell lysate; lane 5, eluate resulting from the passage of cell lysate through Ni-NTA resin; lane 6, eluate from washing Ni-NTA resin with 10 mM imidazole; lane 7, eluate from washing Ni-NTA resin with 500 mM imidazole; lane 8, sample after TEV protease cleavage, showing complete cleavage of the MBP–Sf1a fusion protein. (D) RP-HPLC chromatogram showing the final step in the purification of rSf1a. The arrow denotes the peak corresponding to correctly folded recombinant rSf1a. Inset is a MALDI-TOF MS spectrum showing the [M+H]+ ion for the purified recombinant toxin (obs. = 5058.25 Da; calc. = 5056.98 Da). (E) Paralytic effects of two versions of recombinant Sf1a following injection in sheep blowflies (L. cuprina). nat-Sf1a corresponds to the native sequence, whereas Ser-Sf1a is recombinant toxin with an additional non-native N-terminal serine residue. Paralysis was determined 24 h after injection. recovered in the soluble cell fraction following IPTG preferred P10 residues for TEV protease, was added to induction. The native toxin was recovered after cleavage the N terminus of Sf1a (Fig. 1B). This recombinant of the His6–MBP–Sf1a fusion protein using tobacco Sf1a (referred to hereafter as rSf1a) was found to be etch virus (TEV) protease. The initial construct, how- equipotent with the initial construct that has the native ever, yielded a very small amount of cleaved product sequence, and adopted the same tertiary fold as assessed because of the native N-terminal Lys residue, which is by 1D 1H NMR. Thus, this construct was used in all not one of the preferred residues for the P10 position of subsequent experiments. However, the amino acid resi- TEV protease [25]. To improve the yield of cleaved due numbering used throughout this article is for the protein, a non-native Ser residue, one of the two most native sequence.

FEBS Journal (2015) ª 2015 FEBS 3 Structure and function of the spider toxin Sf1a N. S. Bende et al.

ABa

CD Fig. 2. Inhibition of cockroach DUM

neuron NaV channels by rSf1a. (A, C)

Representative superimposed INa traces before (black) and following (grey and shaded), application of 1 lM (A) and 10 lM (C) rSf1a. Dotted lines represent zero current. (Ba) pulse protocol used to

generate INa shown in (A). (D) Incomplete

inhibition of INa by increasing concentrations of rSf1a (grey bars) and 1 lM rSf1a–R31A–W33A (open bar). (E)

EF Typical time course of INa inhibition by 1 lM rSf1a. Data were ﬁtted with Eqn (3). (F) Effects of depolarizing post-pulses on dissociation of rSf1a were assessed using 10-ms post-pulses to +140 mV applied immediately following a test pulse to 10 mV from 90 mV as detailed in (Bb).

INa were normalized to peak control INa before (●) and after (○), application of 1 lM rSf1a.

The His6–MBP–rSf1a fusion protein was purified almost completely paralyzed and unable to make using nickel affinity chromatography (Fig. 1C, lanes whole-body movements, flies still showed minor move- 1–4). There was minimal loss of fusion protein in the ments of the proboscis or extremities, even when flow through from the Ni-NTA beads (Fig. 1C, lanes injected with the highest doses of Sf1a. Lethality at 5,6). The bound fusion protein was eluted from the 24 h remained < 20%. The median paralytic dose column and cleaved with His6-tagged TEV protease (PD50) values for native Sf1a and rSf1a were deter- 1 (Fig. 1C, lanes 7,8). The His6-tagged MBP and TEV mined to be 2.2 0.2 and 1.5 0.3 nmolg , respec- protease were then removed by passage through a SPE tively (Fig. 1D), which were not significantly different column, and rSf1a subsequently purified using RP- (unpaired t-test, n = 3, P > 0.05). HPLC. rSf1a eluted as a single major disulfide-bond isomer with a retention time of ~ 22 min under the rSf1a is a Na channel pore blocker chosen experimental conditions (Fig. 1E). The purity V of rSf1a was assessed to be > 98% as judged by SDS/ We first investigated whether rSf1a modulated the activ-

PAGE and MALDI-TOF MS (Fig. 1D, inset). The ity of insect NaV channels. Whole-cell sodium currents 1 final yield of rSf1a was ~ 2 mg toxinL culture . (INa) were recorded from dorsal unpaired median (DUM) neurons isolated from the American cockroach Periplaneta americana. The voltage-clamp configuration rSf1a is lethal to insects was used and depolarizing test pulses to 10 mV from a Depending on the dose, rSf1a induced uncoordinated holding potential (Vh)of90 mV (Fig. 2Ba) were movements within 5–10 min following injection into employed to elicit fast activating and inactivating sheep blowflies (L. cuprina) and full paralysis within inward INa. During a 5-min perfusion, 1 lM rSf1a 15–60 min. Flies remained paralyzed until 24 h when caused rapid inhibition of peak INa amplitude final paralysis statistics were recorded. Despite being (55.7 6.2% block of control peak INa, P < 0.05,

4 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

Fig. 3. The effects of rSf1a on the voltage-dependence of NaV gating in cockroach DUM neurons. (A, B) Voltage protocols used to generate INa. (C, D)

Typical superimposed families of INa are shown before (C) and after (D) application of 1 lM rSf1a. Currents were generated using the test pulse protocol shown in (A). EF

(E, F) Normalized INa–V relationships before (●), and after (○ and shaded), application of 1 lM rSf1a generated using the pulse protocol in (B). Data were ﬁtted with Eqn (1) (see Materials and methods). Currents were normalized against maximum peak INa in controls (E) and maximum peak INa (F). (G, H) Effects of rSf1a on steady-state NaV channel inactivation (h∞) were determined using a GH two-pulse protocol detailed in (B). Peak INa recorded during the test pulse before (●), and after (○ and shaded), application of 1 lM rSf1a were normalized to maximum inward INa in controls (G) and maximum inward INa (H) and plotted against prepulse potential. The h∞–V curves were ﬁtted with Eqn (2) (see Materials and methods).

n = 4; Fig. 2A,E) that was only partially reversible INa amplitude was tested as a function of membrane (~ 25% recovery) after prolonged washout in toxin-free potential (INa–V). Families of INa were elicited using a solution. Interestingly, even a very high rSf1a concen- test pulse (Vtest) that depolarized the cell from a Vh of tration (10 lM) failed to completely block INa 90 mV to +70 mV for 50 ms in 10-mV increments (60.0 5.0% block of control peak INa, P < 0.05, (Fig. 3A) before (Fig. 3C) and after (Fig. 3D) applica- n = 3; Fig. 2C), whereas 300 nM rSf1a failed to produce tion of 1 lM rSf1a. Peak INa values were then normal- significant inhibition (7.0 0.6% block of control peak ized against the maximum peak INa in the control and INa, P > 0.05, n = 11; Fig. 2D). At both 1 and 10 lM, plotted against membrane potential (Vm) to establish an rSf1a failed to alter the kinetics of NaV channel activa- INa–V curve. Peak INa were then fitted to Eqn (1) using tion or inactivation (Fig. 2A,C). To test whether a series nonlinear regression analysis (Fig. 3E). In the absence of depolarizing pulses could cause dissociation of the of toxin, INa activated at around 50 mV, which did toxin, post-pulses to +140 mV were applied immediately not shift in the presence of rSf1a (Fig. 3E). The voltage following a test pulse to 10 mV from a Vh of 90 mV at half maximum NaV channel activation (V0.5) in con- (Fig. 2Bb). However, the depolarizing post-pulses failed trol cells was only marginally shifted (1.4 mV) in the to cause any significant relief from the fractional block depolarizing direction in the presence of 1 lM rSf1a of INa by 1 lM rSf1a compared with currents recorded (control: V0.5 = 21.4 1.3 mV; slope factor = 5.1 in the absence of rSf1a (P > 0.05, n = 5; Fig. 2F). 1.0 versus toxin: V0.5 = 20.0 1.1 mV; slope fac- To investigate whether rSf1a-induced inhibition of tor = 5.4 0.8; n = 4, P > 0.05; Fig. 3E). This is more peak INa was because of a depolarizing shift in the clearly observed as a lack of any significant shift in voltage dependence of NaV channel activation, peak the voltage dependence of activation when currents

FEBS Journal (2015) ª 2015 FEBS 5 Structure and function of the spider toxin Sf1a N. S. Bende et al.

Fig. 4. Effects of rSf1a on CaV and KV channel currents in cockroach DUM neurons. (A–D) Inhibition of barium currents through cockroach

DUM neuron CaV channels by rSf1a. (A, B) Representative superimposed IBa traces before (black) and following (grey and shaded) application of 1 lM rSf1a to M-LVA (A) and HVA (B) channels. Dotted lines represent zero current. Currents were generated using the test pulse protocol shown in (Ca). (D) Normalized IBa–V relationships before (●) and after (○ and shaded) application of 1 lM rSf1a. Currents were generated using the test pulse protocol shown in (Cb). Normalized IBa–V relationships were ﬁtted using Eqn (2). (E–H) Inhibition of cockroach DUM neuron KV channels by rSf1a. (E) Representative superimposed IK traces before (black) and following (grey and shaded) application of 1 lM rSf1a. Dotted lines represent zero current. Currents were generated using the test pulse protocol shown in (Fa). (G, H)

Normalized global IK–V relationships before (●) and after (○ and shaded), application of 1 lM rSf1a on peak (G) and late (H) IK. Currents were generated using the test pulse protocol shown in (Fb) and measurements for peak and late current were measured at the circle and square

(50 ms) shown in (E), respectively. Normalized IK–V relationships were ﬁtted using Eqn (2). recorded in the presence of toxin were normalized to the mechanism for recording one current in isolation from peak inward INa in the presence of toxin (Fig. 3F). This the other because no peptide or organic blockers are also revealed that the inhibition of NaV channels by available that exclusively block one type of current 1 lM rSf1a was voltage independent and the binding of [27]. As previously described, depolarizing voltage the toxin to the channel was not relieved at increasing command pulses to different levels were used to inves- membrane potentials, supporting the ﬁnding of the tigate actions on M–LVA and HVA CaV channels, experiments using depolarizing post-pulses. respectively [27,30,31]. CaV channel barium currents The effects of rSf1a on the voltage dependence of (IBa) were evoked by 50-ms depolarizing pulses from a steady-state NaV channel inactivation (h∞V) were also Vh of 90 mV at 7-s intervals to 30 mV for genera- examined to determine whether the reduction of peak tion of predominantly M–LVA CaV channel currents INa was due to stabilization of the inactivated (closed) and to +20 mV to evoke mainly HVA CaV channel state of the channel, and hence a reduction in channel currents (Fig. 4Ca) [27,30]. Depolarizations to availability, as opposed to a pore blocking mechanism. 30 mV caused an inward IBa with slow inactivation, Accordingly, experiments were conducted using a two- consistent with a reduction in Ca2+-dependent fast pulse protocol that utilized a 1-s conditioning prepulse inactivation caused by the use of Ba2+ as the charge

(Vprepulse) from 120 to 0 mV in 10-mV increments, fol- carrier [27], whereas depolarizations to +20 mV elicited lowed by a 50-ms test pulse (Vtest)to10 mV (Fig. 3B). a smaller IBa with a faster inactivating component In the presence of 1 lM rSf1a, INa were inhibited to (Fig. 4A,B). Following a 5-min perfusion, 1 lM rSf1a 51 2% (n = 3) of control peak amplitude (parameter caused only weak inhibition of both M–LVA and ‘A’ in Eqn 2; Fig. 3G). Normalization of the toxin data HVA CaV channel currents. The M–LVA CaV channel to the maximum peak INa in the presence of toxin currents were reduced by 31.5 44.0% (P > 0.05, revealed that the curves almost completely overlap n = 5) and HVA CaV channel currents by (Fig. 3H) with no signiﬁcant shift in h∞V (control: 11.5 9.7% (P > 0.05, n = 5). There were also no sig- V0.5 = 33.8 0.3 mV and toxin: V0.5 = 35.5 niﬁcant changes in M–LVA and HVA IBa activation 0.8 mV: P > 0.05, n = 3; Fig. 3H). Thus, the 49% and inactivation kinetics (Fig. 4A,B). reduction in INa does not appear to be the result of a To investigate whether there was any shift in the reduction in NaV channel availability due to stabiliza- threshold of CaV channel activation, IBaV relation- tion of the channels in the closed-inactivated state. ships were established from families of IBa generated by Thus, rSf1a appears to be a NaV channel pore blocker. 100-ms depolarizing test potentials from Vh of 90 to +40 mV, at 10-mV increments every 7 s (Fig. 4Cb).

Families of peak inward IBa were normalized against the rSf1a has no signiﬁcant effect on CaV or KV channels maximum control inward peak IBa and plotted against Given that NaV channel-targeting spider toxins may the membrane potential. In controls, DUM neuron CaV also interact with CaV channels [26], we tested the abil- channels activated between 50 and 40 mV. In the ity of rSf1a to modulate the activity of insect CaV presence of 1 lM rSf1a this threshold was shifted slightly channels. At least two subtypes of CaV channel cur- in the hyperpolarizing direction (Fig. 4D). This may be rents have been demonstrated in cockroach DUM neu- the cause of the large variance in the magnitude of inhi- rons, including high-voltage-activated (HVA) and mid/ bition of M–LVA CaV channel currents, detailed above. low-voltage-activated (M–LVA) CaV channel currents However, there was no signiﬁcant shift in the V0.5 of [27–30]. Unfortunately, despite differences in the channel activation (control: V0.5 = 29 1 mV versus kinetic and pharmacological properties of M–LVA toxin: V0.5 = 27 1 mV; n = 5, P > 0.05) and cur- and HVA CaV channel currents, there remains no rents were essentially superimposable when the toxin

6 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

Ca D

Fa E

FEBS Journal (2015) ª 2015 FEBS 7 Structure and function of the spider toxin Sf1a N. S. Bende et al.

1 15 13 13 13 data was normalized to the maximum peak IBa in the buffer, pH 3.5. HN, N, Ca, Cb and C reso- presence of toxin (dotted line, Fig. 4D). nance assignments were obtained from analysis of For completeness, we also determined the effect of amide-proton strips in 3D HNCACB, CBCA(CO)NH rSf1a on the major outward voltage-gated potassium and HNCO spectra. Side chain 1H and 13C chemical

(KV) channel current subtypes present in cockroach shifts were obtained using a 4D HCC(CO)NH-TOCSY DUM neurons. These include a slowly activating experiment, which has the advantage of providing side 1 13 delayed-rectifier [IK(DR)], transient ‘A-type’ [IK(A)], and chain H– C connectivities [35]. A fully assigned 2+ 1 15 large-conductance Ca -activated [IBK(Ca)]KV channel H– N HSQC spectrum of rSf1a is shown in Fig. 5. currents [32]. To determine the effects of 1 lM rSf1a Complete chemical shift assignments have been depos- on KV channels, global KV channel currents (IK) were ited in BioMagResBank (accession number 19535). generated by 50-ms depolarizing test pulses to +30 mV CYANA was used for automated NOESY assign- from a Vh of 80 mV, every 5 s (Fig. 4Fa). Global IK ment and structure calculation [36]. Disulfide-bond were measured at the peak (circles) and at the end of connectivities were determined from 15N and 13C NO- the test pulse (50 ms, squares). The early peak global ESY spectra and confirmed by performing structure

IK results mainly from the contribution of rapidly acti- calculations without inclusion of disulfide-bond vating IK(A) and IBK(Ca), whereas the late global IK restraints. The following connectivities were unambigu- results from the slowly activating IK(DR) and slow ously established following this procedure: Cys3– inactivating component of the IBK(Ca). Application of Cys19, Cys10–Cys22, Cys18–Cys42 and Cys24–Cys40, 1 lM rSf1a caused a nonsignificant inhibition of out- which correspond to a 1–4, 2–5, 3–8, 6–7 framework, ward global IK reducing the peak global IK by as described previously for Aps III [8]. For structure 18 15% and the late global IK by 18 13% calculations, interproton distance restraints were sup- (P > 0.05 in both cases, n = 3; Fig. 4E). This lack of plemented with 75 dihedral angle restraints (36 /, overt activity was mirrored by the lack of effect on the 39 w) derived from TALOS+ chemical shift analysis [37]. voltage dependence of global IK activation. Global IK– Two hundred structures were calculated from random V relationships generated by the pulse protocol shown starting conformations, and the 20 structures with the in Fig. 4(Fb) were not significantly altered, with no lowest target function values were selected to represent marked differences in V0.5 values between the IK–V for the solution structure of rSf1a. Coordinates for the controls (V0.5 = 5 2 mV for early and 8 4mV final ensemble of structures have been deposited in the for late IK) versus toxin (V0.5 = 7 3 mV for early Protein Data Bank (accession number 2MF3). and 8 4 mV for late IK)(P > 0.05; n = 3; Fig. 4G, The final ensemble of structures revealed that Sf1a H). Currents were essentially superimposable when the contains a canonical ICK motif [38] (Fig. 6A) in which toxin data was normalized to maximum peak global the Cys10–Cys22 and Cys3–Cys19 disulfide bonds and

IK in the presence of toxin (dotted lines, Fig. 4G,H). the intervening section of the peptide backbone form a ring that is pierced by the Cys18–Cys42 disulfide bond (Fig. 6B). The Cys24–Cys40 disulfide acts as a ‘molecu- High-resolution solution structure of rSf1a lar staple’ to hold together the ends of the extended Buffer conductivity and pH can have a pronounced b-hairpin loop. This loop was poorly defined in the cal- effect on NMR spectra and thus several conditions culated structures and analysis of random coil indices were screened to optimize NMR data quality, includ- using TALOS+ clearly shows that this loop is disordered 2 ing 20 mM phosphate buffer, pH 6, 20 mM Mes buffer, (S ~ 0.65 compared with average of ~ 0.8 for the core pH 6, 20 mM acetate buffer, pH 5, 20 mM citrate buf- of the molecule). When the disordered hairpin loop and fer, pH 4.0 and 20 mM citrate buffer, pH 3.5. These N-terminal residues are excluded, the ensemble of 20 buffer screens revealed that the side chain of Trp33 structures overlay very well, with backbone and heavy- exists in several stable conformations and that the atom rmsd values of 0.13 and 0.43 A, respectively, over population of the minor conformations was reduced at residues 4–26 and 39–45. Based on precision and Rama- low pH and high temperature. Trp33 is adjacent to chandran plot quality (see Table 1), the structure ranks Pro32 and therefore cis–trans isomerization of the as high resolution [39]. The structure has high stereo- Arg31–Pro32 peptide bond might be responsible for chemical quality with a mean MolProbity [40] score of the observed conformational isomerism [33]. Such con- 1.55 0.18, placing it in the 93rd percentile relative to formational dynamics are known to be both tempera- other protein structures. ture and pH dependent [34]. A search for structural homologs [41] revealed that Consequently, NMR data were collected at 40 °C rSf1a has similar topology to numerous other spider 13 15 using C/ N-labeled rSf1a dissolved in 20 mM citrate toxins containing the ICK motif. However, this search

8 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

G27 G20 110 SC (N 14) G38 G7 SC (N 26) SC (N 39) G43 SC (N 16) 115 T8 T5 H13 N14 W33 M4 Y11 S25 3C H15 N39 R31 M35 N16 C18 V37 N26 E34 120 M41 I29 C40 D17 M36 C10 SC (R 31-Folded) N [ppm]

K1 K45 15 C42 S21 E2 1 –15 δ Fig. 5. 2D H N HSQC NMR spectrum C24 I12 C19 of rSf1a acquired at 900 MHz. Sequence- C22 D6 speciﬁc resonance assignments are 125 indicated. Backbone amide assignments A30 are annotated using the one-letter amino V9 acid code. Assignments are also shown SC (W 33) 130 for the side chain (SC) NH correlations of A46 L23 Trp33, the side chain NH2 groups of the four Asn residues, and the aliased peak from the side chain of Arg31. (Note: 10.0 9.0 8.0 7.0 numbering according to the native δ1H [ppm] sequence).

ABC

Disordered loop

Loop staple C10-C22 C24-40

C ICK motif C18-C42

C3-C19

Fig. 6. 3D structure of rSf1a. (A) Ensemble of 20 NMR-derived structures. b-Strands are green, and the disulfide bonds are shown as yellow sticks. The dominant extended and largely disordered loop is highlighted in red. (Note: numbering according to the native sequence.) (B) The ensemble is rotated such that the ICK motif is clearly visible. The ICK ring formed by two disulfide bonds is shown in magenta and the third ICK disulfide bond piercing this ring is also shown. The remainder of the molecule is made transparent for clarity. (C) Overlay of rSf1a (red) with structural homologs tachystatin B (PDB 2DCV; blue) and Aps III (PDB 2M36; green). The structures are made transparent and the disulfide bonds are shown as solid sticks, illustrating the alignment of the ICK motif. The position of the extra disulfide bond that serves as a molecular staple in Sf1a and Aps III is also shown. also revealed close structural homology to tachysta- venom peptide Aps III (l-CUTX-As1a) [8]. In tin B (PDB 2DCV), an antimicrobial ICK-containing Aps III, this loop is comprised of 14 residues, which is peptide isolated from the Japanese horseshoe crab larger than found in typical ICK peptides, but signifi- Tachypleus tridentatus [42]. Tachystatin B does not cantly shorter than the 19-residue loop present in contain the extended hairpin loop found in rSf1a but rSf1a. An overlay of the structures of rSf1a and the two peptides overlay well over their core ICK Aps III reveals that the placement of the non-ICK regions (Fig. 6C). A similarly large hairpin loop is disulfide bond is very different in the two peptides present in the structure of the insecticidal spider- (Fig. 6C).

FEBS Journal (2015) ª 2015 FEBS 9 Structure and function of the spider toxin Sf1a N. S. Bende et al.

Table 1. Structural statistics for ensemble of 20 NMR structures Discussion of rSf1a. All statistics are given as mean SD. Experimental restraintsa Molecular target of Sf1a Interproton distance restraints Intraresidue 147 We examined the effect of rSf1a on insect NaV,CaV Sequential 225 and KV channels that represent common targets for Medium range (i–j < 5) 58 insecticidal spider toxins. The main effect of rSf1a was Long range (i–j ≥ 5) 134 to produce a rapid, but incomplete, block of insect Disulfide-bond restraints 12 NaV channels that occurred in the absence of: (a) a Dihedral-angle restraints (36 /,39w)75 shift in the voltage dependence of activation; (b) a Total number of restraints per residue 13.9 R.m.s.d from mean coordinate structure (A) shift in the voltage dependence of steady-state inactiva- All backbone atoms (residues 1–45) 0.80 0.18 tion; or (c) depolarization-induced dissociation of All heavy atoms (residues 1–45) 1.24 0.19 binding. Furthermore, Sf1a failed to modulate the gat- – – Backbone atoms (residues 4 26, 39 45) 0.13 0.03 ing or kinetics of NaV channel currents and the inhibi- Heavy atoms (residues 4–26, 39–45) 0.43 0.06 tion of I was not relieved by depolarizing pulses. We b Na Stereochemical quality also showed that rSf1a does not significantly affect the Residues in most favored Ramachandran 82.4 2.4 activity of insect Ca channels or delayed-rectifier, region (%) V Ramachandran outliers (%) 0.4 0.9 ‘A-type’ or BKCa potassium channels that are the Unfavorable side chain rotamers (%) 6.8 3.1 major contributors to the global outward KV channel Clash score, all atomsc 0.0 0.0 current in cockroach DUM neurons. Thus, rSf1a Overall MolProbity score 1.55 0.18 appears to be a channel blocker that selectively plugs the outer vestibule of Na channels in a manner aOnly structurally relevant restraints, as defined by CYANA, are V l included. similar to -conotoxins [44]. The incomplete block of + bAccording to MolProbity (http://molprobity.biochem.duke.edu). Na flux induced by rSf1a is consistent with the toxin cDefined as the number of steric overlaps > 0.4 A per thousand binding to the channel outer vestibule rather than lod- atoms. ging deep into the pore region and causing complete

steric block of the insect NaV channel pore. [43]. Most other Na channel blockers isolated from spider Functional importance of the b-hairpin loop V venoms act as ‘gating-modiﬁers’ that reduce INa by The b-hairpin loop in spider-venom ICK peptides causing a depolarizing shift in the voltage dependence of often houses functionally critical residues [7]. The NaV channel activation, thereby inducing a ﬂaccid or unusually large b-hairpin loop found in Sf1a contains ‘depressant’ phenotype. This results from an interaction – – an Arg Pro Trp (RPW) motif that is strictly con- with one or more of the four NaV channel voltage served in this family of toxins [14] and we therefore sensors [45]. These spider toxins include the depressant wondered whether this motif might be critical for the theraphotoxins b-TRTX-Cm1a, -Cm1b and Cm2a toxin’s insecticidal activity. (ceratotoxin-1, -2 and -3) from Ceratogyrus marshalli We produced a mutant of rSf1a in which residues and b-theraphotoxin-Ps1a from Paraphysa scrofa [46].

R31 and W33 were both replaced with alanine. The By contrast, rSf1a inhibits insect NaV channel conduc- mutant peptide (rSf1a–R31A/W33A) was purified and tance in the absence of any depolarizing shift in the volt- characterized as described above for rSf1a and shown age dependence of activation. Given this distinct lack of to adopt the same fold as the native peptide by com- any voltage-dependent shift in channel activation, Sf1a parison of their 2D 1H–15N HSQC spectra. In striking belongs to a growing group of spider l-toxins that = 1 contrast to rSf1a (PD50 1.5 nmolg ), the R31A/ appear to occlude the outer vestibule of the insect NaV W33A double mutant was not insecticidal to blowflies channel. This includes Aps III (l-CUTX-As1a) from at doses up to 95.4 nmolg1 (i.e. there were no signs the unrelated spider Apomastus schlingeri [8]. Unlike of paralysis or lethality up to 24 h after injection). Aps III, which acts on both NaV and to a lesser extent l – Moreover, at a concentration of 1 M, the rSf1a CaV channels, rSf1a selectively blocks insect NaV chan- R31A/W33A mutant failed to significantly inhibit INa nels. Furthermore, Sf1a is marginally more potent than in DUM neurons (5.9 1.8% block of control peak two other l-theraphotoxins that are pore blockers: INa, P > 0.05, n = 3; Fig. 2D), consistent with its lack l-TRTX-Hhn2b (hainantoxin-I) and l-TRTX-Hh1a of insecticidal activity. We therefore conclude that the (huwentoxin-III) from two different species of Chinese b-hairpin loop, and in particular the conserved RPW Haplopelma tarantulas. These two toxins block insect motif, is critical for the activity of Sf1a. NaV channels presumably via binding to neurotoxin

10 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

receptor site 1 near the mouth of the channel with IC50 Unusual structure of Sf1a concentrations for block of the channel between 1.1 and The ICK/knottin topology is by far the dominant struc- 4.5 lM [47,48]. Although the IC for Sf1a on insect 50 tural scaffold found in spider venoms [8] and it is there- Na channels was not definitively determined in the V fore not surprising to find that rSf1a conforms to this present study, it is < 1 lM because this concentration paradigm. Many spider-venom knottins are elaborated caused a 60% block of cockroach DUM neuron I . Na with a fourth non-ICK disulfide bond such as the d- Thus, although both l-TRTX-Hhn2b and l-TRTX- hexatoxins with 1–4, 2–6, 3–7, 5–8 connectivity [60] and Hh1a selectively inhibit insect Na channels, rSf1a V the j-hexatoxins that contain a rare vicinal disulfide targets the insect Na channel with higher affinity, V bond leading to 1–6, 2–7, 3–4, 5–8 connectivity [61,62]. consistent with its modest, but irreversible, depressant However, the 1–4, 2–5, 6–7, 3–8 disulfide connectivity paralytic activity (PD = 1.5 nmolg1). 50 seen in rSf1a is rare and has only been described for the An additional mechanism that can lead to an inhibi- following spider toxins: Aps III (PDB 2M36) [8], tion of I is a decrease in Na channel availability, as Na V U -hexatoxin-Hi1a (PDB 1KQH/1KQI) [33], x-agatox- observed with l-TMTX-Hme1a from venom of the 2 in IVA (PDB 1IVA) [63], d-palutoxin IT2 (PDB 1V91) spider Heriaeus melloteei [49]. This mechanism of [64] and l-agatoxin I (PDB 1EIT) [65]. In all these action causes an increase in the number of Na chan- V structures except Aps III, the non-ICK disulfide bond nels stabilized in the closed-inactivated state as delineates the boundaries of a 4–6-residue loop, poten- observed by a hyperpolarizing shift in the voltage tially as a mechanism for decoupling the dynamics of dependence of steady-state inactivation. By contrast to the loop from the ICK core. In Aps III, the auxiliary l-TMTX-Hme1a, rSf1a failed to produce any signifi- disulfide bond appears to stabilize a dynamic glycine- cant hyperpolarizing shift in the voltage dependence of rich segment within the loop [8]. Notably, in rSf1a this steady-state Na channel inactivation, lending further V loop comprises 19 residues, a third of the entire peptide support to the notion that the toxin occludes the outer sequence, making it by far the largest such loop vestibule of the channel. described for any ICK peptide in this class. Cis–trans Finally, voltage-dependent dissociation of bound isomerization of the Tyr29–Pro30 peptide bond in the toxins from mammalian and insect Na channels has V loop region of U -hexatoxin-Hi1a leads to equilibrium also previously been demonstrated with a range of spi- 2 between two equally populated conformers. In rSf1a, der, scorpion and sea anemone toxins [50–54]. How- we observed multiple conformations of the tryptophan ever, there was no indication of voltage-dependent residue adjacent to Pro32, but the minor conformers dissociation of rSf1a from DUM neurons using depo- made up a small fraction of the equilibrium population. larizing post-pulse protocols. The unusual b-hairpin loop in rSf1a was found to be In summary, rSf1a inhibits Na channels via partial V functionally critical. Simultaneous mutation of R31 and block of the channel pore. This inhibition occurs in W33 within the conserved RPW motif in the b-hairpin the absence of any alteration to the kinetics of Na V loop led to a mutant toxin that lacked insecticidal activ- channel inactivation as observed for spider d-toxins ity and was able to inhibit insect Na channel currents. [55] and Sf1a fails to produce any hyperpolarizing shift V This is also consistent with previous studies showing (as seen for excitatory b-toxins) [56] or a depolarizing that Trp and Arg are the two residues most commonly shift (as observed for depressant b-toxins) [46] in the found at the interface between interacting proteins [66]. voltage dependence of Na channel activation. Based V The only other peptide in this class of knottins for which on the pharmacology described here, rSf1a should be the pharmacophore has been mapped is d-palutoxin IT2 renamed l-segestritoxin-Sf1a (l-SGTX-Sf1a) based on and in this case the b-hairpin loop was also found to be the rational nomenclature recently proposed for spi- functionally important [64]. der-venom peptides [57]. This is consistent with its Finally, we note that the second closest structural action to induce flaccid paralysis, in contrast with the homolog of rSf1a is tachystatin B, an antimicrobial spastic paralysis observed with spider d-toxins [58] and peptide isolated from the Japanese horseshoe crab excitatory b-toxins [59]. Tachypleus tridentatus [42]. The two peptides overlay The incomplete block of Na channels is unlikely to V very closely over their core ICK regions, but deviate be the sole cause of Sf1a toxicity. Given the weak effects significantly over the unusually large disordered loop of Sf1a on neuronal calcium channels, an additional of rSf1a. The origin of the ICK fold remains under target might be the HVA-like dihydropyridine-sensitive debate; there is conflicting evidence about whether the Ca channels found on the surface of invertebrate V ICK motif evolved from an ancestral two-disulfide, skeletal muscle [28], which are critical for conduction of disulfide-directed b-hairpin fold by addition of a disul- muscle action potentials.

FEBS Journal (2015) ª 2015 FEBS 11 Structure and function of the spider toxin Sf1a N. S. Bende et al.

fide bond [61,67] or vice versa [68]. The presence of Victoria, Australia) and HPLC-grade acetonitrile (RCI 13 15 the ICK fold in an ancient marine arthropod suggests Labscan, Bangkok, Thailand). C6-glucose and NH4Cl that the ICK fold may have already been present prior were purchased from Sigma-Aldrich Australia. Recombi- – to its recruitment into the venom of arthropods. In nant His6 TEV protease (EC 3.4.22.44) was produced in- this scenario, the ICK fold would be the plesiotypic house using a previously described protocol [69]. state with the two-disulfide, disulfide-directed b-hairpin fold subsequently evolving via loss of a disulfide bond. Nomenclature This would provide an alternative explanation for the rarity of two-disulfide, disulfide-directed b-hairpin pep- Based on the rational nomenclature proposed for spider- l tides in arachnid venoms [67]. venom peptides [57], SFI1 should be renamed -segestritoxin-Sf1a (l-SGTX-Sf1a) based on its source and activity on

NaV channels. Sf1a as a bioinsecticide We found that Sf1a caused long-lasting, irreversible Production of recombinant Sf1a paralysis in sheep blowflies that eventually led to death A synthetic gene encoding Sf1a, with codons optimized for by starvation. Because of methodological issues (i.e. expression in E. coli, was synthesized and cloned into the high mortality in controls beyond 24 h), we could not pLIC–MBP expression vector by GeneArt (Invitrogen, determine the PD over longer periods. However, flies 50 Regensburg, Germany). This vector (pLIC–NSB2), which paralyzed by high doses of rSf1a remained paralyzed was designed as described earlier [8], encodes an IPTG- 48 h after injection The observed effects of rSf1a in inducible LacI promoter, a MalE signal sequence for peri- blowflies are similar to those recently reported for the plasmic export, a His6 tag for nickel affinity purification, spider-venom peptide Aps III, although Aps III is MBP fusion tag to enhance solubility, and a TEV protease threefold more potent with a PD50 value of cleavage site between the MBP and Sf1a coding regions 1 700 pmolg [8]. Given the observed insecticidal (Fig. 1A). The plasmid was transformed into E. coli strain effects of rSf1a in blowflies, it seems likely that it BL21(kDE3) for rSf1a production. Cultures were grown in would produce similar effects in dipteran pests such as Terrific Broth (TB) medium using baffled flasks at 37 °C mosquitoes and tsetse flies that vector human diseases. with shaking at 110 rpm. Expression of the toxin gene was

Because rSf1a is also active against lepidopteran induced with 1 mM IPTG at D600 = 0.9–1.0, then cells were [14,17] and hemipteran [21] pests, but inactive against grown at 18 °C for a further 15 h and harvested by centrifu- vertebrates [14], it appears to be a suitable lead for the gation for 12 min at 8967 g. For production of uniformly development of novel bioinsecticides [4]. Although the 13C/15N-labeled Sf1a, cultures were grown in minimal med- 13 15 moderate potency of Sf1a would require large amounts ium supplemented with C6-glucose and NH4Cl as the sole to be applied in the field for efficient pest control, carbon and nitrogen sources, respectively. – – delivery of the toxin to pest insects via genetic engi- The His6 MB rSf1a fusion protein was extracted from neering of crops or via insect-specific pathogens (e.g. the bacterial periplasm by cell disruption at 27 kPa (TS entomopathogenic fungi) might provide efficient deliv- Series Cell Disrupter, Constant Systems Ltd, Daventry, ery of the toxin at effective concentrations. UK), then captured by passing the periplasmic extract (buffered in 40 mM Tris, 450 mM NaCl, pH 8.0) over Ni-NTA In summary, we have shown that Sf1a is a NaV-channel-specific insecticidal toxin that acts via a nonvoltage- Superflow resin (Qiagen) followed by washing with 10 mM imidazole to remove any products that bound nonspecifi- dependent mechanism. This is an atypical mode of cally to the resin. The fusion protein was then eluted with action that has previously only been observed in one 500 mM imidazole. The eluted fusion protein was concen- other spider toxin, namely Aps III. Although less potent trated to 5 mL and the buffer was exchanged to remove than Aps III, the specificity of Sf1a towards Na chan- V imidazole. Reduced and oxidized glutathione were then nels makes it an attractive pharmacological tool. added to 0.6 and 0.4 mM, respectively, to maintain TEV

protease activity. Approximately 100 lg of His6-tagged Materials and methods TEV protease was added per mg of fusion protein, and the cleavage reaction was allowed to proceed at room tempera- – – Chemicals ture for 12 h. The cleaved His6 MBP and His6 TEV were removed by using SPE columns; the mixtures were loaded All chemicals were purchased from Sigma-Aldrich Australia onto the column and the peptide eluted with 40% elution (Castle Hill, NSW, Australia), Sigma-Aldrich USA (St solvent (Solvent B: 0.043% triﬂuoroacetic acid in 90% Louis, MO, USA) or Merck Chemicals (Kilsyth, Victoria, acetonitrile). The eluate was lyophilized and then semi-pure Australia) with the exception of IPTG (Life Technologies, rSf1a was subjected to further puriﬁcation using RP-HPLC.

12 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

RP-HPLC was performed on a Vydac C18 column NOESY spectra were manually peak picked and integrated, (250 9 4.6 mm, particle size 5 lm) at a flow rate of peak lists were then automatically assigned, distance 1mLmin1 and a gradient of 20–40% Solvent B in Sol- restraints extracted, and an ensemble of structures calcu- vent A (0.05% trifluoroacetic acid in water) over 20 min. lated using the torsion angle dynamics package CYANA 3.0 [36]. The tolerances used for CYANA were 0.02 ppm in the direct 1H dimension, 0.04 ppm in the indirect 1H dimen- MALDI-TOF mass spectrometry sion, and 0.4 ppm for the heteronucleus (13C/15N). During Peptide masses were determined using MALDI-TOF MS the automated NOESY assignment/structure calculation employing a Model 4700 Proteomics Bioanalyser (Applied process, CYANA assigned 92% of all NOESY cross-peaks Biosystems, Carlsbad, CA, USA). RP-HPLC fractions were (1447 of 1566). The four disulfide bonds were assigned mixed 1 : 1 (v/v) with a-cyano-4 hydroxycinnamic acid matrix based on preliminary structure calculations; subsequent cal- 1 (5 mgmL in 50 : 50 acetonitrile/H2O) and MALDI-TOF culations included distance restraints for these disulfide spectra were acquired in positive reflector mode. All reported bonds as described previously [72]. masses are for monoisotopic [M+H]+ ions. Electrophysiological measurements on insect Blowfly toxicity assay neurons

Insect toxicity assays were performed as described previ- DUM neurons were isolated from unsexed adult American ously [8] Briefly, rSf1a, nat-Sf1a or rSf1a-R31A/W33A were cockroaches (Periplaneta americana) as described previously injected into the ventro-lateral thoracic region of sheep [8]. Briefly, terminal abdominal ganglia were removed and blowflies (Lucilia cuprina) weighing 19.5–25.9 mg. Three placed in normal insect saline (NIS) containing 180 mM separate experiments were conducted for each Sf1a homo- NaCl, 3.1 mM KCl, 10 mM Hepes and 20 mM D-glucose. log, each comprising 3–10 doses, with each dose injected Ganglia were then incubated in 1 mgmL1 collagenase into 10 flies. Paralytic and lethal effects were measured (type IA) for 40 min at 29 °C. Following enzymatic treat-

24 h post injection. PD50 values were calculated as ment, ganglia were washed twice in NIS and resuspended described previously [8] with statistical analysis and in NIS supplemented with 4 mM MgCl2,5mM CaCl2,5% unpaired Student’s t-tests performed using PRISM 6. fetal bovine serum and 1% penicillin/streptomycin (Life Technologies, Victoria, Australia) (NIS+) and triturated through a fire-polished Pasteur pipette. The resultant cell Structure determination suspension was then distributed onto 12-mm diameter glass NMR spectra were acquired at 40 °C on a 900 MHz NMR coverslips precoated with 2 mgmL1 concanavalin A spectrometer (Bruker BioSpin, Germany) equipped with a (type IV). DUM neurons were maintained in NIS+ at cryogenically cooled probe. 15N/13C-labeled rSf1a was dis- 29 °C and 100% humidity. Ionic currents were recorded in solved in 20 mM citrate, pH 3.5 at a final peptide concentra- voltage-clamp mode using the whole-cell patch-clamp tech- tion of 420 lM, followed by addition of 5% D2O. The sample nique, employing version 10.2 of the pCLAMP data acqui- was filtered using a low-protein-binding Ultrafree-MC cen- sition system (Molecular Devices, Sunnyvale, CA, USA). trifugal filter (0.22 lm pore size; Millipore, Bedford, MA, Data were filtered at 5–10 kHz with a low-pass Bessel filter USA), then 300 lL was added to a susceptibility matched with leakage and capacitive currents subtracted using P–P/4 5 mm outer-diameter microtube (Shigemi Inc., Allison Park, procedures. Digital sampling rates were set between 15 and PA, USA). 25 kHz depending on the length of the protocol. Single-use 3D and 4D spectra used for resonance assignments were 0.8–2.5 MΩ electrodes were pulled from borosilicate glass acquired using nonuniform sampling [70]. Sampling sched- and fire-polished prior to current recordings. Liquid junc- ules that approximated the signal decay in each indirect tion potentials were calculated using JPCALC [73], and all dimension were generated using SCHED3D [35]. Nonuniform data were compensated for these values. Cells were bathed sampling data were processed using the Rowland NMR in external solution through a continuous pressurized per- toolkit (www.rowland.org/rnmrtk/toolkit.html) and maxi- fusion system at 1 mLmin1, and toxin solutions were mum entropy parameters were automatically selected as introduced via direct pressurized application using a perfu- previously described [71]. 13C- and 15N-edited HSQC-NO- sion needle at ~ 50 lLmin1 (Automate Scientific, San ESY spectra (mixing time of 200 ms) were acquired using Francisco, CA, USA). Control data were not acquired uniform sampling. Separate 13C-edit HSQC-NOESY spec- until at least 20 min after whole-cell configuration was tra were acquired for the aliphatic and aromatic regions of achieved to eliminate the influence of fast time-dependent the carbon spectrum. shifts in steady-state inactivation resulting in rundown of

Dihedral angles were derived from TALOS+ chemical shift INa from NaV channels. All experiments were performed at analysis [37] and the restraint range for structure calcula- ambient room temperature (20–23 °C). To record INa, the tions was set to twice the estimated standard deviation. external bath solution contained: 230 mM NaCl, 5 mM

FEBS Journal (2015) ª 2015 FEBS 13 Structure and function of the spider toxin Sf1a N. S. Bende et al.

CsCl, 1.8 mM CaCl2,50mM tetraethylammonium chloride, The data for voltage dependence of NaV channel steady- 5mM 4-aminopyridine, 10 mM Hepes, 0.1 mM NiCl2 and state inactivation (h∞/V) were amplitude inverted and fitted 1mM CdCl2, adjusted to pH 7.4 with 1 M NaOH. The pip- using the following Boltzmann equation: ette solution contained: 34 mM NaCl, 135 mM CsF, 1 mM A M M M MgCl2,10m Hepes, 5 m EGTA and 3 m ATP-Na2, h1 ¼ ð2Þ 1 þ exp½ðV V : Þ=k adjusted to pH 7.4 with 1 M CsOH. To eliminate any influ- 0 5 ence of differences in osmotic pressure, all internal and where A is the fraction of control maximal peak INa, external solutions were adjusted to 400 5 mOsmolL1 V0.5 is the midpoint of inactivation, k is the slope fac- with sucrose. Because of the reported current rundown tor, and V is the conditioning prepulse potential. with calcium as a charge carrier [27], BaCl replaced CaCl 2 2 On-rates were determined by fitting time course data in all experiments on voltage-gated Ca channels. The V with the following single exponential association function: external bath solution for IBa recordings contained: 140 mM Na acetate, 30 mM TEA-Br, 3 mM BaCl and 2 Y ¼ Y0 þðA Y0Þð1 expðk tÞÞ ð3Þ 10 mM Hepes, adjusted to pH 7.4 with 1 M TEA-OH. The external solution also contained 300 nM tetrodotoxin to where Y0 is the maximal peak INa, A is the minimum block NaV channels. Pipette solutions contained: 10 mM peak INa, K is the rate constant and t is time. The on- Na acetate, 110 mM CsCl, 50 mM TEA-Br, 2 mM ATP- rate (son) was subsequently determined from the Na2, 0.5 mM CaCl2,10mM EGTA and 10 mM Hepes, inverse of the rate constant (K). adjusted to pH 7.4 with 1 M CsOH. The external bath solution for recording global K channel currents (I ) V K Acknowledgements contained: 200 mM NaCl, 50 mM K gluconate, 5 mM CaCl2,4mM MgCl2, 0.3 mM tetrodotoxin, 10 mM Hepes We thank the Queensland NMR Network for access to and 10 mMD-glucose, adjusted to pH 7.4 with 1 M NaOH. the 900 MHz NMR spectrometer at The University of The pipette solution consisted of: 135 mM K gluconate, Queensland. This work was supported by the Australian 25 mM KF, 9 mM NaCl, 0.1 mM CaCl2,1mM MgCl2, Research Council through Discovery Grants 1mM EGTA, 10 mM Hepes and 3 mM ATP-Na2, adjusted DP140101098 to MM and DP130103813 to GFK and a to pH 7.4 with 1 M KOH. Future Fellowship to MM (FTl10100925). GFK is sup- Experiments were rejected if there were large leak cur- ported by a Principal Research Fellowship from the rents or currents showed signs of poor space clamping. Australian National Health & Medical Research Coun- cil. NSB received support from University of Queens- Curve fitting and statistical analysis land International Research Tuition Award (UQIRTA) and University of Queensland Research Scholarship Peak current amplitude was analyzed offline using AXOG- (UQRS). RAPH X v. 1.5.3 (Molecular Devices). Current amplitudes were normalized against maximal control current amplitude, or maximal current amplitude in the presence of Author contributions 1 lM rSf1a, for all curve-fitted data. All curve fitting was Conceived and designed the experiments: NSB, SD, performed using PRISM 6 for Windows (GraphPad Soft- ware Inc., San Diego, CA, USA) using nonlinear regres- VH, FB, GMN, GFK, MM. Performed the experi- sion and a least-squares method. Comparisons of two ments: NSB, SD, VH, VR, MM. Analyzed the data: sample means were made using a paired Student’s t-test. NSB, SD, VH, GMN, MM. Contributed materials, A test was considered to be significant when P < 0.05. All experimental/analysis tools: NSB, SD, VH, GWB, data represent the mean SEM of n independent experi- GMN, GFK, MM. Wrote the paper: NSB, SD, VH, ments. FB, GMN, GFK, MM. The data for the voltage dependence of channel activation, for all channel types, were fitted using the following References current–voltage (I–V) formula: 1 Oerke EC (2006) Crop losses to pests. J Agric Sci 144, 1 – I ¼ gmax 1 ð Þ ðV VrevÞð1Þ 31 43. 1 exp V V : =s þ ½ð 0 5Þ 2 Boyer S, Zhang H & Lemperiere G (2012) A review of control methods and resistance mechanisms in stored- where I is the amplitude of the current at a given test product insects. Bull Entomol Res 102, 213–229. potential V, gmax is the maximal conductance, V0.5 is 3 Tedford HW, Sollod BL, Maggio F & King GF (2004) the voltage at half-maximal activation, s is the slope Australian funnel-web spiders: master insecticide factor and Vrev is the apparent reversal potential. chemists. Toxicon 43, 601–618.

14 FEBS Journal (2015) ª 2015 FEBS N. S. Bende et al. Structure and function of the spider toxin Sf1a

4 Windley MJ, Herzig V, Dziemborowicz SA, Hardy MC, venom toxin to insect haemolymph following oral King GF & Nicholson GM (2012) Spider-venom ingestion. J Insect Physiol 50,61–71. peptides as bioinsecticides. Toxins 4, 191–227. 18 Fitches E, Woodhouse SD, Edwards JP & Gatehouse 5 Murray CJ, Rosenfeld LC, Lim SS, Andrews KG, JA (2001) In vitro and in vivo binding of snowdrop Foreman KJ, Haring D, Fullman N, Naghavi M, (Galanthus nivalis agglutinin; GNA) and jackbean Lozano R & Lopez AD (2012) Global malaria (Canavalia ensiformis; Con A) lectins within tomato mortality between 1980 and 2010: a systematic analysis. moth (Lacanobia oleracea) larvae; mechanisms of Lancet 379, 413–431. insecticidal action. J Insect Physiol 47, 777–787. 6 Smith JJ, Herzig V, King GF & Alewood PF (2013) 19 Fitches EC, Pyati P, King GF & Gatehouse JA (2012) The insecticidal potential of venom peptides. Cell Mol Fusion to snowdrop lectin magnifies the oral activity of Life Sci 70, 3665–3693. insecticidal x-hexatoxin-Hv1a peptide by enabling its 7 Tedford HW, Gilles N, Menez A, Doering CJ, delivery to the central nervous system. PLoS One 7, Zamponi GW & King GF (2004) Scanning e39389. mutagenesis of x-atracotoxin-Hv1a reveals a spatially 20 Bonning BC & Chougule NP (2014) Delivery of restricted epitope that confers selective activity against intrahemocoelic peptides for insect pest management. insect calcium channels. J Biol Chem 279, 44133– Trends Biotech 32,91–98. 44140. 21 Down RE, Fitches EC, Wiles DP, Corti P, Bell HA, 8 Bende NS, Kang E, Herzig V, Bosmans F, Nicholson Gatehouse JA & Edwards JP (2006) Insecticidal spider GM, Mobli M & King GF (2013) The insecticidal venom toxin fused to snowdrop lectin is toxic to the neurotoxin Aps III is an atypical knottin peptide that peach-potato aphid, Myzus persicae (Hemiptera: potently blocks insect voltage-gated sodium channels. 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PLoS and their modulation in neurons and skeletal muscles. One 8, e73136. Prog Neurobiol 64, 431–525. 17 Fitches E, Edwards MG, Mee C, Grishin E, Gatehouse 29 Grolleau F & Lapied B (1996) Two distinct low- AM, Edwards JP & Gatehouse JA (2004) Fusion voltage-activated Ca2+ currents contribute to the proteins containing insect-specific toxins as pest control pacemaker mechanism in cockroach dorsal unpaired agents: snowdrop lectin delivers fused insecticidal spider median neurons. J Neurophysiol 76, 963–976.

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30 Chong Y, Hayes JL, Sollod B, Wen S, Wilson DT, horseshoe crab antimicrobial peptide tachystatin B with Hains PG, Hodgson WC, Broady KW, King GF & an inhibitory cystine-knot motif. J Pept Sci 13, 269– Nicholson GM (2007) The x-atracotoxins: selective 279. blockers of insect M-LVA and HVA calcium channels. 43 Hui K, Lipkind G, Fozzard HA & French RJ (2002) Biochem Pharmacol 74, 623–638. Electrostatic and steric contributions to block of the 31 Wicher D, Walther C & Penzlin H (1994) skeletal muscle sodium channel by l-conotoxin. J Gen Neurohormone D induces ionic current changes in Physiol 119,45–54. cockroach central neurones. J Comp Physiol A 174, 44 Shon KJ, Olivera BM, Watkins M, Jacobsen R, Gray 507–515. WR, Floresca CZ, Cruz LJ, Hillyard DR, Brink A, 32 Windley MJ, Escoubas P, Valenzuela SM & Nicholson Terlau H et al. (1998) l-Conotoxin PIIIA, a new GM (2011) A novel family of insect-selective peptide peptide for discriminating among tetrodotoxin-sensitive neurotoxins targeting insect large-conductance calcium- Na channel subtypes. J Neurosci 18, 4473–4481. activated K+ channels isolated from the venom of the 45 Bosmans F & Swartz KJ (2010) Targeting voltage theraphosid spider Eucratoscelus constrictus. Mol sensors in sodium channels with spider toxins. Trends Pharmacol 80,1–13. Pharmacol Sci 31, 175–182. 33 Rosengren KJ, Wilson D, Daly NL, Alewood PF & 46 Bosmans F, Rash L, Zhu S, Diochot S, Lazdunski M, Craik DJ (2002) Solution structures of the cis- and Escoubas P & Tytgat J (2006) Four novel tarantula trans-Pro30 isomers of a novel 38-residue toxin from toxins as selective modulators of voltage-gated sodium the venom of Hadronyche infensa sp. that contains a channel subtypes. Mol Pharmacol 69, 419–429. cystine-knot motif within its four disulﬁde bonds. 47 Li D, Xiao Y, Hu W, Xie J, Bosmans F, Tytgat J & Biochemistry 41, 3294–3301. Liang SP (2003) Function and solution structure of 34 King GF, Middlehurst CR & Kuchel PW (1986) Direct hainantoxin-I, a novel insect sodium channel inhibitor NMR evidence that prolidase is speciﬁc for the trans from the Chinese bird spider Selenocosmia hainana. isomer of imidodipeptide substrates. Biochemistry 25, FEBS Lett 555, 616–622. 1054–1062. 48 R-l Wang, Yi S & Liang S-P (2010) Mechanism of 35 Mobli M, Stern AS, Bermel W, King GF & Hoch JC action of two insect toxins huwentoxin-III and (2010) A non-uniformly sampled 4D HCC(CO)NH- hainantoxin-VI on voltage-gated sodium channels. J TOCSY experiment processed using maximum entropy Zhejiang Univ Sci B 11, 451–457. for rapid protein side chain assignment. J Magn Reson 49 Billen B, Vassilevski AA, Nikolsky A, Tytgat J & 204, 160–164. 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Protein Sci 3, 1833–1839. on rat brain and insect sodium channels highlighted by 39 Kwan AH, Mobli M, Gooley PR, King GF & Mackay binding of a funnel-web spider d-atracotoxin. Eur J JP (2011) Macromolecular NMR spectroscopy for the Biochem 269, 1500–1510. non-spectroscopist. FEBS J 278, 687–703. 52 Strichartz G & Wang G (1986) Rapid voltage- 40 Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral dependent dissociation of scorpion a-toxins coupled to GJ, Wang X, Murray LW, Arendall WB 3rd, Snoeyink Na channel inactivation in amphibian myelinated J, Richardson JS et al. (2007) MolProbity: all-atom nerves. J Gen Physiol 88, 413–435. contacts and structure validation for proteins and 53 Schreibmayer W, Kazerani H & Tritthart HA (1987) A nucleic acids. Nucleic Acids Res 35, 375–383. mechanistic interpretation of the action of toxin II from 41 Holm L & Rosenstrom€ P (2010) Dali server: Anemonia sulcata on the cardiac sodium channel. conservation mapping in 3D. Nucleic Acids Res 38, Biochim Biophys Acta 901, 273–282. W545–W549. 54 Grolleau F, Stankiewicz M, Birinyi-Strachan L, Wang 42 Fujitani N, Kouno T, Nakahara T, Takaya K, Osaki X, Nicholson GM, Pelhate M & Lapied B (2001) T, Kawabata S-i, Mizuguchi M, Aizawa T, Demura M, Electrophysiological analysis of the neurotoxic action of Nishimura S-I et al. (2007) The solution structure of a funnel-web spider toxin, d-atracotoxin-Hv1a, on

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insect voltage-gated Na+ channels. J Exp Biol 204, calcium channel-selective omega-agatoxins. Nat Struct 711–721. Mol Biol 1, 853–856. 55 Yamaji N, Little MJ, Nishio H, Billen B, Villegas E, 64 Ferrat G, Bosmans F, Tytgat J, Pimentel C, Chagot B, Nishiuchi Y, Tytgat J, Nicholson GM & Corzo G Gilles N, Nakajima T, Darbon H & Corzo G (2005) (2009) Synthesis, solution structure, and phylum Solution structure of two insect-specific spider toxins selectivity of a spider d-toxin that slows inactivation of and their pharmacological interaction with the insect specific voltage-gated sodium channel subtypes. J Biol voltage-gated Na+ channel. Proteins 59, 368–379. Chem 284, 24568–24582. 65 Omecinsky DO, Holub KE, Adams ME & Reily MD 56 Corzo G, Sabo JK, Bosmans F, Billen B, Villegas E, (1996) Three-dimensional structure analysis of l- Tytgat J & Norton RS (2007) Solution structure and agatoxins: further evidence for common motifs among alanine scan of a spider toxin that affects the activation neurotoxins with diverse ion channel specificities. of mammalian voltage-gated sodium channels. J Biol Biochemistry 35, 2836–2844. Chem 282, 4643–4652. 66 Bogan AA & Thorn KS (1998) Anatomy of hot spots 57 King GF, Gentz MC, Escoubas P & Nicholson GM in protein interfaces. J Mol Biol 280,1–9. (2008) A rational nomenclature for naming peptide 67 Smith JJ, Hill JM, Little MJ, Nicholson GM, King GF toxins from spiders and other venomous animals. & Alewood PF (2011) Unique scorpion toxin with a Toxicon 52, 264–276. putative ancestral fold provides insight into evolution 58 Little MJ, Zappia C, Gilles N, Connor M, Tyler MI, of the inhibitor cystine knot motif. Proc Natl Acad Sci Martin-Eauclaire M-F, Gordon D & Nicholson GM USA 108, 10478–10483. (1998) d-Atracotoxins from Australian funnel-web 68 Sunagar K, Undheim EA, Chan AH, Koludarov I, spiders compete with scorpion a-toxin binding but Munoz-Gomez SA, Antunes A & Fry BG (2013) differentially modulate alkaloid toxin activation of Evolution stings: the origin and diversification of voltage-gated sodium channels. J Biol Chem 273, scorpion toxin peptide scaffolds. Toxins 5, 2456–2487. 27076–27083. 69 Fang L, Jia K, Tang Y, Ma D, Yu M & Hua Z (2007) 59 Zlotkin E, Rochat H, Kopeyan, Miranda F & Lissitzky An improved strategy for high-level production of TEV S (1971) Purification and properties of the insect toxin protease in Escherichia coli and its purification and from the venom of the scorpion Androctonus australis characterization. Protein Expr Purif 51, 102–109. Hector. Biochimie 53, 1073–1078. 70 Hoch JC, Maciejewski MW, Mobli M, Schuyler AD & 60 Fletcher JI, Chapman BE, Mackay JP, Howden MEH Stern AS (2014) Nonuniform sampling and maximum & King GF (1997) The structure of versutoxin (d- entropy reconstruction in m ultidimensional NMR. Acc atracotoxin-Hv1) provides insights into the binding of Chem Res 47, 708–717. site 3 neurotoxins to the voltage-gated sodium channel. 71 Mobli M, Maciejewski MW, Gryk MR & Hoch JC Structure 5, 1525–1535. (2007) An automated tool for maximum entropy 61 Wang X-H, Connor M, Smith R, Maciejewski MW, reconstruction of biomolecular NMR spectra. Nat Meth Howden MEH, Nicholson GM, Christie MJ & King 4, 467–468. GF (2000) Discovery and characterization of a family 72 Fletcher JI, Smith R, O’Donoghue SI, Nilges M, of insecticidal neurotoxins with a rare vicinal disulfide Connor M, Howden MEH, Christie MJ & King GF bond. Nat Struct Biol 7, 505–513. (1997) The structure of a novel insecticidal neurotoxin, 62 Mobli M, de Araujo AD, Lambert LK, Pierens GK, x-atracotoxin-HV1, from the venom of an Australian Windley MJ, Nicholson GM, Alewood PF & King GF funnel web spider. Nat Struct Biol 4, 559–566. (2009) Direct visualization of disulfide bonds through 73 Barry PH (1994) JPCalc, a software package for diselenide proxies using 77Se NMR spectroscopy. calculating liquid junction potential corrections in Angew Chem Int Ed 48, 9312–9314. patch-clamp, intracellular, epithelial and bilayer 63 Reily MD, Holub KE, Gray WR, Norris TM & Adams measurements and for correcting junction potential ME (1994) Structure-activity relationships for P-type measurements. J Neurosci Meth 51, 107–116.

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5 A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

Published manuscript in Nature Communications, 2014

32 ARTICLE

Received 21 Dec 2013 | Accepted 10 Jun 2014 | Published 11 Jul 2014 DOI: 10.1038/ncomms5350 A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

Niraj S. Bende1, Sławomir Dziemborowicz2, Mehdi Mobli3, Volker Herzig1, John Gilchrist4, Jordan Wagner4, Graham M. Nicholson2, Glenn F. King1 & Frank Bosmans4,5

b-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b–S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we ﬁnd that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by

PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identiﬁed two residues within the BgNav1 domain II S1–S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides.

1 Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland QLD 4072, Australia. 2 School of Medical and Molecular Biosciences, University of Technology, Sydney, New South Wales 2007, Australia. 3 Centre for Advanced Imaging, The University of Queensland, St Lucia, Queensland QLD 4072, Australia. 4 Department of Physiology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA. 5 Solomon H. Snyder Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA. Correspondence and requests for materials should be addressed to G.F.K. (email: [email protected]) or to F.B. (email: [email protected]).

NATURE COMMUNICATIONS | 5:4350 | DOI: 10.1038/ncomms5350 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5350

nsect voltage-gated sodium (Nav) channels share a common Nav channels have been governing electrical excitability in a architecture with their mammalian orthologues1. The pore- wide range of organisms for millions of years, even before Iforming subunit consists of four connected domains (DI–IV), the development of neurons2,11. Thus, it is not surprising that each with six transmembrane segments (S1–S6). These ancient organisms such as spiders developed an arsenal of homologous, but not identical, domains each contain a voltage toxins geared towards incapacitating prey by modulating Nav sensor (S1–S4) and a portion of the pore through which Na þ can channel function12–15. One intriguing specimen is the desert 2 diffuse (S5–S6) . While they have yet to be identified in insect Nav bush spider (Diguetia canities) whose habitat spans the channels, each voltage-sensing domain within mammalian and Southwestern deserts of the United States16,17. The venom of bacterial Nav channels contains an S3b–S4 helix-turn-helix motif, this relatively unexplored primitive weaving spider contains the voltage-sensor paddle, which drives voltage-sensor activat- a 57-residue peptide known as DTX9.2 (or b-diguetoxin-Dc1a18, ion3–6. Aside from its vital role in channel gating, the paddle hereafter Dc1a; Fig. 1a), one of the most potent insect-selective motif is also an important pharmacological target, as peptide neurotoxins found in arthropod venoms19. Although its toxins interact with this region to modify channel opening3,4,7. precise molecular target has yet to be elucidated, neurophysiolo- Insect Nav channels likely possess similar motifs since residue gical studies on housefly larvae revealed excitation of sensory substitutions in homologous regions can abolish channel and neuromuscular preparations on Dc1a application that 1 susceptibility to toxins found in animal venoms . Even though could be blocked by the Nav channel-blocking compound voltage-sensing domains are extensively targeted by naturally tetrodotoxin20. occurring peptides, commercially available insecticides such as Here, we show that Dc1a potently promotes opening of the pyrethroids and oxadiazines typically interact with the channel German cockroach Nav channel BgNav1 (ref. 21) by interacting pore region or intracellular linker between S4 and S5 to disrupt with the paddle motif in domain II. Surprisingly, Dc1a has little 8,9 opening or closing (that is, gating) . However, insects have effect on PaNav1-expressing neurons isolated from the American responded to this threat by mutating residues at strategic cockroach (Periplaneta americana)22, even though the toxin- locations within the channel that result in a reduced sensitivity binding site is identical in these two cockroach channels. By 9 to these compounds . Moreover, the conserved nature of the Nav exploring regions responsible for the remarkable insect-family channel pore throughout the animal kingdom often leads to specificity of Dc1a, we uncovered an important role for the undesired biological activity of insecticides in beneficial insect domain II S1–S2 loop in determining PaNav1 resistance to Dc1a. orders or mammals8–10. Since the amino acid composition of Together with the unique solution structure of Dc1a, our results voltage-sensing domains varies considerably between related Nav provide a proof of concept that toxins—and by extension small channel isoforms, these regions may replace the pore as a target molecules—can selectively target insect Nav channels by for designing insecticides with a higher degree of phyletic interacting with their voltage-sensing domains, thus facilitating selectivity, an intriguing notion that has yet to be fully explored. the development of new insecticides.

a SAKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLDCRCLKSGFFSSKCVCRDV 1102030405057

b T7 promoter T7 terminator lac operator Kpnl Sspl His 6 pLIC-NSB3

Maltose binding protein (MBP) β-DGTX-Dc1a

RBS MalEss TEV protease recognition site

c d 1 M1 23M4 56 78910 100 100 6,484 75 75

50 (a. u.) 6,480 6,485 6,490 50 Mass (Da)

37 37 214

25 25 0 20 20 0 10 20 30 40 Retention time (min)

Figure 1 | Recombinant production of Dc1a. (a) Primary structure of rDc1a. The non-native N-terminal Ser residue that is a vestige of the TEV protease cleavage site used for recombinant toxin production is highlighted in grey. Disulphide bridge connectivity is shown above the sequence. (b) Schematic representation of the pLIC-NSB3 vector used for periplasmic expression of rDc1a. The coding region includes a MalE signal sequence (MalESS) for periplasmic export, a His6 afﬁnity tag, an MBP fusion tag and a codon-optimized gene encoding rDc1a, with a TEV protease recognition site inserted between the MBP and toxin-coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS). (c) SDS-polyacrylamide gel electrophoresis gels illustrating different steps in the puriﬁcation of rDc1a. Lanes contain: M, molecular weight markers; lane 1,

E. coli cell extract before IPTG induction; lane 2, E. coli cell extract after IPTG induction; lane 3, soluble periplasmic extract (the His6-MBP-rDc1a fusion protein is evident at B50 kDa); lane 4, Ni-NTA beads after loading the cell lysate; lane 5, eluate-1 from washing Ni-NTA resin with 600 mM imidazole; lane 6, elute-2 from washing Ni-NTA resin with 600 mM imidazole; lane 7, eluate-3 from washing Ni-NTA resin with 600 mM imidazole; lane 8, Ni-NTA beads after elution-3; lane 9, fusion protein sample before TEV protease cleavage; lane 10, fusion protein sample after TEV protease cleavage showing almost complete cleavage of fusion protein to His6-MBP. (d) RP-HPLC chromatogram showing the final step in the purification of rDc1a. The arrow head denotes the peak corresponding to correctly folded recombinant rDc1a. Inset is a MALDI-TOF MS spectrum showing the [M þ H] þ ion for the purified recombinant toxin (obs. ¼ 6,484 Da; calc. ¼ 6,485.39 Da).

2 NATURE COMMUNICATIONS | 5:4350 | DOI: 10.1038/ncomms5350 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5350 ARTICLE

Results Solution structure of rDc1a. The development of an efficient Production of recombinant Dc1a. We first produced recombi- bacterial expression system allowed us to produce uniformly nant Dc1a peptide (rDc1a) using a novel approach that addresses 13C/15N-labelled rDc1a protein for structure determination using 1 15 13 13 13 0 the challenge of correctly folding disulphide-rich spider toxins heteronuclear NMR. HN, N, Ca, Cb, and C resonance in E. coli23. To this end, we created an isopropyl-b-D- assignments for the toxin were obtained from analysis of thiogalactopyranoside (IPTG)-inducible construct (Fig. 1b) that amide-proton strips in three-dimensional (3D) HNCACB, 1 13 allows for export of a His6-MBP-Dc1a fusion protein to the CBCA(CO)NH, and HNCO spectra. Sidechain H and C E. coli periplasm, where the enzymes involved in disulphide- chemical shifts were obtained using a four-dimensional (4D) bond formation are located23. On recovery from the soluble HCC(CO)NH-TOCSY experiment, which has the advantage of cell fraction, the fusion protein was purified using nickel providing sidechain 1H–13C connectivities24. Complete chemical affinity chromatography (Fig. 1c). Subsequent cleavage and shift assignments have been deposited in BioMagResBank chromatographic purification yielded a single major disulphide- (Accession Number 19666). CYANA was used for automated bond isomer with a purity of 498% as assessed by SDS- NOESY assignment and structure calculation25. The disulphide- polyacrylamide gel electrophoresis and matrix-assisted laser bond pattern (1–4, 2–5, 3–8, 6–7) was unambiguously desorption/ionization time-of-flight (MALDI-TOF) mass determined from preliminary structures calculated without spectrometry (Fig. 1d). Note that, with four disulphide bonds, disulphide-bond restraints26 and is identical to the framework there are 105 possible disulphide-bond isomers of rDc1a. Overall, predicted in the UniProtKB entry for Dc1a (P49126). Of the 200 the final yield was B1.1 mg of toxin per litre of culture. Since structures that were calculated from random starting native Dc1a was unavailable to us, we confirmed the insecticidal conformations, 20 conformers with high stereochemical quality activity of rDc1a by injection into the blowfly Lucilia cuprina and (as judged by MolProbity27) were selected to represent the the housefly Musca domestica, which yielded LD50 values of solution structure of rDc1a (Fig. 2a). Atomic coordinates for the 231±32 pmol g 1 and 493±52 pmol g 1, respectively (n ¼ 3) final ensemble of 20 structures are available from the Protein (Supplementary Fig. 1). We therefore conclude that rDc1a Data Bank (PDB; Accession Number 2MI5). has a similar lethality to agricultural pests when compared Statistics highlighting the high precision and stereochemical with native toxin19. quality of the ensemble of rDc1a structures are shown in ab

Disordered hairpin loop loop loop -hairpin -hairpin β β β5 C42 Hairpin staple β C52 CC4 C C40 -sheet 2 -sheet 2 β β C20 C54 β3 Cystine C25 knot C26 β2 C13 -sheet 1 -sheet 1 β β N-terminal β1 extension NNN

Core Core ICK ICK C region C region

C C

NN β-sheet unique β-sheet unique N to Dc1a N to Dc1a

Figure 2 | NMR solution structure of rDc1a. (a) Stereo view of the ensemble of 20 rDc1a structures. The structures are overlaid over the backbone atoms of residues 2–42 and 52–56 in order to highlight the disordered nature of the hairpin loop (residues 43–51, purple) relative to the well-structured ICK region of the toxin. The N and C termini are labelled and the b-strands that form the N- and C-terminal b-sheets are coloured blue and green, respectively. (b) Ribbon representation of rDc1a highlighting the ﬁve b- strands (b1–b5) and four disulphide bonds. The three disulphide bonds that form the ICK motif are shown in red while the fourth disulphide that staples the base of the disordered b4–b5 hairpin loop is highlighted in orange. The b-strands and disordered hairpin loop are coloured as in a.(c) Stereo view of the structures of As1a, a typical knottin peptide30, overlaid with rDc1a. The two structures were overlaid for optimal superposition over their core ICK regions (28 residues; region highlighted in cyan) using the CLICK server70. Note that the N-terminal b-sheet in rDc1a is quite distinct from the region of structural overlap. (d) Electrostatic surface potential of rDc1a with positive and negative charges shown in blue and red, respectively. The molecular orientation is the same as in panels a and c.

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Supplementary Table 1. The average MolProbity score of 1.24 ac1 μM rDc1a washout places the ensemble in the 99th percentile relative to all other 1 1 1 5 s structures ranked by MolProbity. The high stereochemical quality 50 s of the ensemble stems from a complete absence of bad close max max max I I contacts and an excellent Ramachandran plot quality (499% of G / / / I I residues in the most favoured region). The structural ensemble is G also highly precise with backbone and heavy-atom root-mean- square deviation (r.m.s.d.) values over all the structural ordered 0 0 0 regions (residues 3–42, 52–56) of 0.27±0.07 Å and 0.70±0.11 Å, –100 –50 0 0 100 200 respectively. The ensemble of rDc1a structures ranks as ‘very high V (mV) Time (s) resolution’ based on these measures of precision and stereochemical quality28. b 1 d –25 Three of the four disulphide bonds in rDc1a form a classical –30 inhibitor cysteine knot (ICK) motif in which the Cys13–Cys26 –35 max (mV) I and Cys20–Cys40 disulphide bonds and the intervening sections / I

1/2 –40 of the polypeptide backbone form a 23-residue ring that is pierced V –45 by the Cys25–Cys54 disulphide bond (Fig. 2b). This ICK motif is commonly found in spider toxins, and this particular scaffold 0 –50 provides these peptides (so-called ‘knottins’) with an unusually 02550 1 10 100 1,000 high degree of chemical, thermal and biological stability15. Time (ms) rDc1a (nM) However, the structure of rDc1a differs markedly from other Figure 3 | rDc1a promotes opening of BgNav1 channels. (a) Comparison 29 ICK toxins, and a DALI search of the PDB returned no of the gating properties of BgNav1 before (black), and after (red), addition of structural homologues. First, rDc1a contains an additional 1 mM rDc1a. Shown are the normalized deduced conductance (G)–voltage disulphide bond (Cys42–Cys52) that appears to serve as a (filled circles) (G/Gmax) and steady-state inactivation (open circles; I/Imax) molecular staple which limits the flexibility of a disordered serine- relationships. Currents were elicited by 5-mV step depolarizations from a rich hairpin loop (residues 43–51) (Fig. 2b). Stapled hairpins of holding voltage of 90 mV or 100 mV, respectively. n ¼ 3–5, and error this kind have been observed in only a small number of spider bars represent s.e.m. Descriptive values resulting from Boltzmann fits can toxins30. Second, the extended N terminus of rDc1a along with an be found in the Results section. (b) Recovery from fast inactivation of WT unusually large loop between Cys26 and Cys40 enables the BgNav1 before (black), and after (red), addition of 1 mM rDc1a (red) formation of an amino-terminal (N-terminal) three-stranded determined by a double-pulse protocol to 30 mV with a varying time antiparallel b-sheet that is not found in any other knottin between pulses (0–50 ms). Values are reported in the Results section. (Fig. 2b,c). The uniqueness of the rDc1a structure can readily be Data represents n ¼ 4 experiments, and error bars represent the s.e.m. seen by comparison with the structure of As1a, a typical spider (c) On addition of 1 mM rDc1a to channels depolarized to 60 mV every 5 s venom–derived knottin that modulates the activity of insect NaV (red open circles) or 50 s (green open circles; holding voltage was 30 channels . The two toxins can be aligned over their core ICK 100 mV), BgNav1-mediated sodium currents become rapidly visible. regions with an rmsd of 1.9 Å (Fig. 2c) consistent with them both Channels completely recover after toxin washout. Inset shows current trace being members of the knottin family. However, when aligned before (black), and after (red), addition of 1 mM rDc1a at 60 mV (5 s over this ICK region, the N-terminal b-sheet of rDc1a is entirely pulses). Noticeable is the persistent current that appears at this voltage, separate to the region of structural overlap (Fig. 2c). Despite its even in control experiments. Abscissa scale is 10 ms, ordinate scale is structural uniqueness, the molecular surface of rDc1a contains a 0.3 mA. (d) Concentration dependence of rDc1a-induced current relatively uniform distribution of charged residues (Fig. 2d); potentiation determined from shifts in the midpoint of channel activation moreover, there are no distinct clusters of hydrophobic residues (V1/2). Line represents a fit of the data with the Hill equation resulting in a 7,31 that might mediate an interaction with lipid bilayers half-maximal concentration (EC50)of65±1 nM and a slope factor of (Supplementary Movie 1). 1.19±0.02; n ¼ 5 and error bars represent s.e.m.

rDc1a opens the German cockroach Nav channel BgNav1. result of the 22 mV shift in voltage-dependent opening of Diguetia spiders are generalist predators and their diet consists of BgNav1 (Fig. 3c). Moreover, rDc1a similarly affects the channel various insects ranging from small ants to sizeable prey such as when voltage-steps to 60 mV were applied every 50 s, suggesting 16,32 grasshoppers and cockroaches . As such, we decided to test that the toxin can access its binding site when BgNav1isinthe 12 whether rDc1a influences the gating properties of BgNav1, a resting state . Channels completely recovered following washout in well-studied Nav channel isoform cloned from the German toxin-free solution (Fig. 3c). The persistent nature of the emerging 8,21 cockroach . We functionally expressed BgNav1inXenopus current does not stem from the inhibition of fast inactivation by oocytes and applied various concentrations of rDc1a. At 1 mM, rDc1a since wild-type (WT) BgNav1 inherently possesses these 33 rDc1a produced robust opening of BgNav1 at voltages where the characteristics at mildly depolarizing voltages . Fitting the Hill channel is normally closed. This was achieved by a drastic shift in equation to the concentration dependence for toxin-induced channel activation to more negative potentials (V1/2 was shifted current potentiation, as determined by shifts in V1/2,yieldedan from 27.3±0.2 mV (mean±s.e.m.) (slope 4.0±0.2) to EC50 of 65±1 nM and a Hill coefficient of 1.19±0.02 (Fig. 3d), 49.4±0.4 mV (slope 3.5±0.3) in the presence of 1 mM suggesting that the toxin may interact with only one binding site. rDc1a; Fig. 3a) whereas steady-state inactivation (or channel Interestingly, 1 mM rDc1a did not affect any of the tested 34 availability) was only slightly affected (V1/2 was shifted from vertebrate Nav channel isoforms or the hERG channel , 52.2±0.1 mV to 55.1±0.1 mV in the presence of 1 mM a member of the cardiac voltage-gated potassium (Kv) channel rDc1a; Fig. 3a). The rate of recovery from fast inactivation was family and an Food and Drug Administration-mandated also not significantly altered (t is 2.1±0.1 ms versus 2.2±0.1 ms; screening target for potential off-target drug effects Fig. 3b). On addition of 1 mM rDc1a to channels depolarized to (Supplementary Fig. 2a). These results are consistent with the 60 mV every 5 s, sodium currents became rapidly visible as a report that Dc1a has no effect in mice when injected

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intraperitoneally or intracerebroventricularly at doses of 4.1 and a S3b S4 1.0 mg kg 1, respectively19. Kv2.1 BgNav1DI rDc1a interacts with the domain II paddle motif in BgNav1. K 2.1 Subsequently, we were interested in identifying the receptor sites v BgNav1 DII for rDc1a within insect Nav channels that result in such a profound shift in the voltage dependence of activation. Our Kv2.1 BgNav1 DIII experiments with BgNav1 suggest that rDc1a functions as an excitatory toxin to activate insect Nav channels at membrane Kv2.1 voltages where they are normally closed. Hence, the toxin may BgNav1 DIV interact with S3b–S4 paddle motifs within BgNav1 voltage sensors in a manner analogous to previously described b-scorpion toxins3,35,36. In order to test this hypothesis, we first needed to b DI DII DIII DIV establish whether BgNav1 contains paddle motifs with similar 37–39 functions as in Kv channels and in mammalian Nav channel isoforms where they have been identified (rNav1.2a, rNav1.4, 3,4 rNav1.8, rNav1.9, and hNav1.9) . To this end, we employed a previously reported approach in which specific S3b–S4 regions from each voltage-sensing domain of a Nav channel were transplanted into homotetrameric K channels (Fig. 4a)3,4,7. v cd1 After several attempts (Fig. 4a), we were able to generate Kv2.1 DI functional chimeras between BgNav1 and Kv2.1 using known 100 3,4 DII paddle motif boundaries (Fig. 4a,b). All of the constructs DIII max

I DIV contain the crucial basic residues that contribute to gating charge / (ms) I 40,41 τ 10 movement in Kv channels , suggesting that each of the four voltage-sensing domains in BgNav1 contain paddle motifs that are capable of sensing membrane voltage changes. 0 1 Examination of the conductance–voltage (G–V) relationships –100 –50 0 50 100 –150–100 –50 0 50 100 for the BgNav1/Kv2.1 chimeras reveals that each of the four V (mV) V (mV) voltage-sensor paddles has a distinct effect on the gating e K 2.1 DI DII DIII DIV properties of Kv2.1. This was evidenced by marked differences v in the midpoints of activation for the G–V relations for the domain I, II and IV constructs which are Z50 mV, 13±2 mV, and 9±2 mV, respectively (Fig. 4c). The G–V relationship for the domain III BgNav1/Kv2.1 construct is bimodal, suggesting that the functional coupling between the voltage-sensing domains Figure 4 | Transfer of the voltage sensor paddle motifs from BgNa 1to and the pore has been considerably altered. Although the v K 2.1. (a) Sequence alignment of the paddle region of K 2.1 with the underlying mechanism is unclear, it is possible that pore opening v v separate S3b–S4 regions of BgNa 1 arranged per domain. In BgNa 1, the in this particular chimera may occur when: (1) not all four voltage v v paddle motifs are not identical and are therefore coloured differently: sensors are fully activated42; or (2) each voltage sensor has purple, domain I paddle (DI); red, domain II paddle (DII); blue, domain III transferred only a portion of its charge across the hydrophobic paddle (DIII); green, domain IV paddle (DIV). In K 2.1, the paddle motifs are septum43–45. The latter scenario seems more likely since in the v identical and therefore have the same colour (black). Functional chimeras related Shaker K channel, all four voltage sensor need to be in the v are indicated with a green dot in front of the sequence whereas non- active position before the pore opens42,46. Moreover, intermediate functional chimeras are indicated with a red dot. (b,c) Transfer of the states have also been observed when measuring gating currents of BgNa 1 paddle motifs into K 2.1. Families of potassium currents (b) and tail the Shaker K channel in which a mutation immobilized voltage- v v v current voltage–activation relationships (c) for each chimeric construct sensor movement between the resting and activated states, (n 6; error bars represent s.e.m.). Holding voltage was 90 mV for DI, II, thereby shifting the voltage activation of the ionic currents47. ¼ and IV, and 120 mV for DIII, and the tail voltage was 60 mV Finally, it is worth mentioning that the domain III BgNa 1/K 2.1 v v ( 100 mV for DIII). Bars in b are 1 A and 100 ms. (d) Kinetics of opening construct may display multiple open states according to a model m and closing for K 2.1 channels containing paddle motifs from the four that has been described for N-type Ca channels48. v v domains of BgNa 1. Mean time constants ( ) from single-exponential ﬁts to Remarkably, domain IV paddle motifs from rNa 1.2a and v t v channel activation (ﬁlled circles) and deactivation (open circles) are plotted rNa 1.4 slow activation kinetics when transplanted into K v v as a function of the voltage at which the current was recorded. n 6, channels. These observations support the notion that the domain ¼ and error bars represent s.e.m. (e) Potassium currents elicited by IV paddle motif contributes substantially to the overall rate of depolarizations near the foot (less than 1/3 of maximal activation) of the voltage-sensor activation3,4. To explore whether the domain IV voltage–activation curve (c) for BgNa 1/K 2.1 chimeric constructs. Currents paddle motif serves a similar role in BgNa 1, we measured v v v are shown before (black) and following (coloured) addition of 1 M rDc1a. the kinetics of activation and deactivation of the four BgNa 1 m v rDc1a clearly affects only the DII chimera. paddle constructs in response to membrane depolarization and repolarization, respectively. Similar to previously studied chimeric channels, the activation and deactivation kinetics observed for the domain IV construct of BgNav1/Kv2.1 are slower over a wide additional evidence for the functional role of the domain IV voltage range when compared with the other domains (Fig. 4d), paddle motif in BgNav1 gating from experiments with BomIV, suggesting that this particular domain may indeed contribute a classic insect-selective a-scorpion toxin from the venom of 49–51 to fast inactivation in insect Nav channels . We obtained Buthus occitanus mardochei that inhibits fast inactivation in

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52 American cockroach neurons . To determine whether BomIV a 100 inﬂuences BgNav1 in a similar fashion, we applied 10 nM to channel-expressing Xenopus oocytes and observed that the toxin B. germanica indeed slows down fast inactivation (Supplementary Fig. 2b). 80 Subsequent testing on the BgNav1/Kv2.1 chimeras revealed that BomIV does not affect WT Kv2.1 or the domain I, II, and III 60 constructs. However, a dramatic toxin-induced inhibition of the domain IV chimera corroborates the importance of this particular 40 voltage-sensing domain in toxin binding and BgNav1 fast

inactivation (Supplementary Fig. 2b). Mortality at 24 h (%) We next investigated whether rDc1a modulates the activity of 20 any of the four BgNav1/Kv2.1 paddle chimeras and found that 1 mM rDc1a exclusively interacts with the domain II construct 0 whereas domains I, III, IV, and WT Kv2.1 are unaffected (Fig. 4e, Supplementary Fig. 2c). This result suggests that rDc1a 10–12 10–11 10–10 10–9 speciﬁcally targets the domain II voltage sensor within BgNav1 rDc1a (mol g–1) to inﬂuence channel opening. b rDc1a distinguishes between cockroach Nav1 channels. Since the domain II paddle motif in BgNav1 (ref. 21) is identical to that Knockdown >>5 nmol g–1 in PaNav1 (ref. 22), we expected a similar effect of rDc1a in 10,000 Lethality whole-cell patch-clamp recordings from American cockroach dorsal unpaired median (DUM) neurons. Surprisingly, 1 mM rDc1a had little effect on sodium current amplitude or kinetics in 1,000 )

DUM neurons as reﬂected in the threshold, or V1/2,ofNav –1 channel activation or the V1/2 of steady-state inactivation measurements (Supplementary Fig. 3). Furthermore, there were 100 (pmol g

no significant use-dependent effects on sodium current amplitude 50 or kinetics. Dose Due to the unexpectedly weak effects of 1 mM rDc1a on 10 voltage-dependent activation of P. americana DUM neurons compared with BgNav1 channels from B. germanica, acute toxicity assays were expanded to include both cockroach species 1 (Fig. 5a,b). Remarkably, these bioassays revealed lethal toxicity of rDc1a in B. germanica compared with only mild, reversible effects Lucilia Musca Blattella Periplaneta in P. americana. At doses up to 5 nmol g 1, rDc1a generated cuprina domestica germanica americana only minor spastic contractions of the abdomen, some shaking Figure 5 | Acute toxicity of rDc1a in two cockroach families. and evidence of reduced motor activity in P. americana. Even (a) Dose–response curve for lethal effects of rDc1a determined 24 h after though these effects were completely reversible, a very small injection into German cockroaches (B. germanica; family Blattellidae, closed percentage of cockroaches did develop paralysis after 24 h (7±7% circles). Data were fitted according to Equation 5 in Methods and represent knockdown, n ¼ 3). One explanation for these observations may mean±s.e.m. of three independent experiments. (b) Comparison of the 53 be toxin binding to a subset of Nav channel splice variants or KD50 (open columns) and LD50 (grey columns) doses at 24 h following the presence of endogenous pharmacologically active auxiliary injection of rDc1a in L. cuprina, M. domestica and B. germanica. Note: doses 54 subunits . Conversely, B. germanica underwent dose-dependent up to 5 nmol g 1 failed to produce any signs of knockdown or lethality up to flaccid or contractile paralysis, and lethality was observed after 72 h post injection in American cockroaches (Periplaneta americana; family 8 h at all doses tested (Fig. 5a). The KD50 and LD50 values at 24 h Blattidae). Data represent mean±s.e.m. of three independent experiments. post injection were 3.0±1.2 nmol g 1 and 4.2±3.4 nmol g 1 (n ¼ 3), respectively. These observations suggest that residues in regions outside of the domain II paddle motif may underlie the remarkably different sensitivity of BgNav1 and PaNav1 YE to rDc1a. rDc1a was no longer sufficient to open BgNav1 whereas this concentration generated large inward sodium currents The S1–S2 loop modulates Nav channel sensitivity to rDc1a. when applied to WT channels (Fig. 6c, inset). When surveying Sequence alignment of BgNav1 with PaNav1 reveals a highly higher concentrations of rDc1a, it becomes clear that conserved S1–S4 voltage sensor in domain II with only three concentrations of more than 1 mM are required to achieve a YE amino acid substitutions (Fig. 6a; Supplementary Fig. 4). Of potentiation with BgNav1 that is comparable to the WT B those, two residues in the S1–S2 loop are potentially accessible for channel (EC50 65 nM; Fig. 6c). Although we cannot exclude the a peptide toxin applied from the extracellular environment possibility that rDc1a interacts with other PaNav1 domains at (His805 and Asp812 in BgNav1 which correspond to a Tyr and such concentrations, administering high doses of the toxin is Glu in PaNav1, respectively). On mutating these residues in indeed mildly toxic to P. americana (Fig. 5a,b). Thus, our YE BgNav1 to their PaNav1 counterparts, the resulting BgNav1 mutagenesis experiments have uncovered a novel region within construct functionally expressed in Xenopus oocytes with a insect Nav channels that helps determine their sensitivity to voltage-dependent activation V1/2 of 41.5±0.3 mV and a spider toxins. This locus contributes to the drastically reduced steady-state inactivation V1/2 of 56.4±0.2 mV (Fig. 6b). sensitivity of the American cockroach to rDc1a from the venom Unfortunately, a functional PaNav1 clone was unavailable to of the desert bush spider whereas its German counterpart is undertake gain-of-function experiments. Strikingly, 100 nM highly susceptible.

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a BgNav1

PaNav1

bc1 1 –25

–30

–35 max

max wt (mV) G I / / I 1/2 G –40 V

–45

0 0 –50 Mutant –100 –60 –20 1 10 100 1,000 V (mV) rDc1a (nM)

Figure 6 | Residues outside of the DII paddle motif contribute to rDc1a binding. (a) Sequence alignment of the DII S1–S2 regions of BgNav1 and PaNav1 (See Supplementary Fig. 4 for more details). In the extracellular loop connecting S1 to S2, all residues are identical except the H805Y and D812E YE substitutions (grey background). The T797M substitution within S1 is unlikely to be toxin-accessible. (b) Gating properties of the mutant BgNav1 channel. Shown are the normalized deduced conductance (G)–voltage (ﬁlled circles) (G/Gmax) and steady-state inactivation (open circles; I/Imax) relationships. Descriptive values can be found in the Results section. Currents were elicited by 5 mV step depolarizations from a holding voltage of 90 mV or YE 100 mV, respectively. n ¼ 3–5, and error bars represent s.e.m. (c) Afﬁnity measure for rDc1a interaction with BgNav1 (red) compared with WT BgNav1 (grey). Concentration dependence for toxin-induced current potentiation (as determined by shifts in V1/2) is shown. It is clear that alterations in YE channel opening on rDc1a exposure are less pronounced with the BgNav1 mutant when compared with WT BgNav1. n ¼ 5 and error bars represent YE s.e.m. Inset shows premature channel opening after addition of 100 nM rDc1a to WT BgNav1 (grey) whereas BgNav1 is not yet affected (red). Current traces were evoked by a 50-ms depolarization to 55 mV from a holding potential of 90 mV.

Discussion toxin residues in strategic positions to stabilize the domain II The initial goal of this study was to explore the mechanism voltage sensor in an activated position. This hypothesis is underlying the efficacy of Dc1a, a potent insecticidal peptide strengthened by the observation that the substitution of two toxin produced by desert bush spiders. To this end, we first residues in the domain II S1–S2 loop within mammalian Nav1.2 produced recombinant rDc1a and determined its solution channels decreases sensitivity to the functionally related structure using heteronuclear NMR (Figs 1 and 2). Next, we b-scorpion toxin CssIV by a factor of four59. Another possible tested rDc1a on the heterologously expressed German cockroach mechanism is that rDc1a binding to the domain II S1–S2 loop in channel BgNav1 and discovered that the toxin dramatically BgNav1 allosterically influences channel opening. Such a promotes channel opening (Fig. 3). By taking advantage of the hypothesis was postulated when a domain III S1 splice variant portable nature of S3b–S4 paddle motifs within voltage-sensing of BgNav1 revealed an increased susceptibility to the insect- 3,4,7,38 domains , we demonstrated that such motifs also exist in selective b-scorpion toxin Lqh-dprIT3 (ref. 60). However, one each of the four voltage-sensing domains of BgNav1, and that caveat is that a depolarizing prepulse is needed to potentiate the rDc1a selectively interacts with the paddle motif in domain II, a maximal effect of CssIV and Lqh-dprIT3 (refs 49,50). Similar to feature that it shares with an extensive list of animal toxins that the structurally unrelated spider toxin Magi5 which activates rat 3,4,12,55,56 61 target this particular paddle motif , albeit with a high Nav1.2 channels by binding to the domain II voltage sensor , degree of insect selectivity (Fig. 4). Remarkably, our insect assays rDc1a does not require such a prepulse. revealed that German cockroaches are highly susceptible to rDc1a In summary, the current study provides a molecular explana- whereas the closely related American cockroach is virtually tion for the remarkable insect selectivity of Dc1a, the most potent insensitive despite the fact that the domain II paddle motif in insecticidal toxin identified in the venom of the desert bush BgNav1 is identical to that in PaNav1 (Fig. 5; Supplementary spider. Moreover, we have uncovered a novel toxin receptor site Fig. 4). To elucidate the machinery responsible for this within insect Nav channels that provides a new framework for the discrepancy, we mutated two residues in the S1–S2 loop that design of molecules capable of targeting specific insect families. differ between BgNav1 and PaNav1 (His805 and Asp812) and This knowledge may be used in the future to develop insecticides YE found that the susceptibility of the resulting BgNav1 construct that target specific insect pests without affecting beneficial insects to rDc1a is dramatically reduced (Fig. 6). Interestingly, or endangering human health. mammalian Nav channel isoforms possess a Tyr and Ser at the corresponding positions, which presumably contributes to their Methods insensitivity to rDc1a; however, their domain II paddle motif Chemicals. All chemicals were purchased from Sigma-Aldrich Australia (Castle differs as well (Supplementary Fig. 4). Hill, NSW, Australia), Sigma-Aldrich USA (St Louis, MO, USA), or Merck It is interesting to consider potential mechanisms that may Chemicals (Kilsyth, Victoria, Australia) with the exception of IPTG and streptomycin (Life Technologies, Victoria, Australia), tetrodotoxin (Alomone Labs, Israel), underlie the insect-family specificity of rDc1a. For example, the and high-performance liquid chromatography (HPLC)-grade acetonitrile (RCI 13 15 b3–b4 hairpin (Fig. 2b) often houses the pharmacophore in Labscan, Bangkok, Thailand). C6-glucose and NH4Cl were from Sigma-Aldrich 57 spider ICK toxins that target voltage-gated ion channels , but the Australia. Recombinant His6-TEV protease (EC 3.4.22.44) was produced in-house 62 unusual architecture of rDc1a (Fig. 2c) and its unique ability to using a published protocol . dramatically promote opening of insect Nav channels (Fig. 3) suggests that it might interact with the voltage sensors of these Production of recombinant rDc1a. A synthetic gene encoding Dc1a, with codons optimized for expression in Escherichia coli, was produced and cloned into a channels in a unique manner. As such, the domain II S1–S2 loop variant of the pLIC-MBP expression vector by GeneArt (Invitrogen, Regensburg, may play a role in positioning the toxin into a water-filled cavity Germany). This vector (pLIC-NSB3) encodes a MalE signal sequence for 58 adjacent to the S3–S4 paddle motif , thereby placing particular periplasmic export, a His6 tag for affinity purification, a maltose-binding protein

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(MBP) fusion tag to aid solubility, and a tobacco etch virus (TEV) protease provided with dry food and water. The paralytic activity was then determined over recognition site directly preceding the codon-optimized Dc1a gene23. The plasmid a 24-h period. For each acute toxicity assay up to fourteen doses of rDc1a (n ¼ 10 encoding Dc1a was transformed into E. coli strain BL21(lDE3) for recombinant insects per dose) and the appropriate control (insect saline; n ¼ 10 insects) were toxin production. Protein expression and purification were performed as described used. The assay was repeated three times. Median knockdown (KD50) and median 30 66 previously with minor modifications. In summary, cultures were grown in Luria- lethal (LD50) doses were calculated as described previously and averaged to Bertani broth at 37 °C with shaking. Toxin gene expression was induced with produce KD50 and LD50 values. 500 mM IPTG at an OD600 of 1.0–1.1, then cells were grown at 20 °C for a further 12 h before collecting by centrifugation for 15 min at 10,500 g. For production of uniformly 13C/15N-labelled rDc1a, cultures were grown in minimal medium Electrophysiological measurements on DUM neurons. DUM neurons were supplemented with 13C -glucose and 15NH Cl as the sole carbon and nitrogen isolated from unsexed adult American cockroaches (P. americana) as described 6 4 previously30. Briefly, terminal abdominal ganglia were removed and placed sources, respectively. The His6-MBP-toxin fusion protein was extracted from the bacterial periplasm by cell disruption at 27 kPa (TS Series Cell Disrupter, Constant in normal insect saline (NIS) containing (in mM): NaCl 180, KCl 3.1, N-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) 10 and D-glucose 20. Systems Ltd, Northants, UK), and then captured by passing the extract (buffered in 1 40 mM Tris, 450 mM NaCl, pH 8.0) over Ni-NTA Superflow resin (Qiagen, Ganglia were then incubated in 1 mg ml collagenase (type IA) for 40 min at Chadstone, Australia). Proteins bound non-specifically were removed by washing 29 °C. Following enzymatic treatment, ganglia were washed three times in NIS and triturated through a fire-polished Pasteur pipette. The resultant cell suspension was with 10 mM imidazole then the fusion protein was eluted with 600 mM imidazole. 1 The eluted fusion protein was concentrated to 10 ml and the buffer was exchanged then distributed onto 12-mm diameter glass coverslips pre-coated with 2 mg ml to remove imidazole. Reduced and oxidized glutathione were then added to a final concanavalin A (type IV). DUM neurons were maintained in NIS supplemented concentration of 0.6 mM and 0.4 mM, respectively, to maintain TEV protease with 5 mM CaCl2, 4 mM MgCl2, 5% foetal bovine serum and 1% penicillin and streptomycin (Life Technologies, Victoria, Australia), and maintained at 29 °C, activity and promote folding of the protein. Approximately, 100 mgofHis6-tagged TEV protease was added per mg of rDc1a, then the cleavage reaction was allowed 100% humidity. Ionic currents were recorded in voltage-clamp mode using the whole-cell patch-clamp technique employing version 10.2 of the pCLAMP data to proceed at room temperature for 12 h. The cleaved His6-MBP and His6-TEV were precipitated by addition of 1% trifluoroacetic acid (TFA), then the sample was acquisition system (Molecular Devices, Sunnyvale, CA). Data were filtered at centrifuged at 16,000 g. The supernatant was filtered using a 0.45-mm syringe filter 5–10 kHz with a low-pass Bessel filter with leakage and capacitive currents (Millipore, MA, USA) and subjected to further purification using RP-HPLC. subtracted using P-P/4 procedures. Digital sampling rates were set between 15 and RP-HPLC was performed on a Vydac C18 column (250 4.6 mm, particle size 25 kHz depending on the length of the protocol. Single-use 0.8–2.5 MO electrodes 5 mm) using a flow rate of 1 ml min 1 and a gradient of 20–45% Solvent B (0.043% were pulled from borosilicate glass and fire-polished prior to current recordings. TFA in 90% acetonitrile) in Solvent A (0.05% TFA in water) over 30 min. rDc1a Liquid junction potentials were calculated using JPCALC, and all data were compensated for these values. Cells were bathed in external solution through a contains a non-native N-terminal serine residue (a vestige of the TEV protease 1 cleavage site), making it one residue longer than native Dc1a. continuous pressurized perfusion system at 1 ml min , while toxin solutions were introduced via direct pressurized application via a perfusion needle at B50 mlmin 1 (Automate Scientific, San Francisco, CA). Control data were not Mass spectrometry. Toxin masses were confirmed by MALDI-TOF mass acquired until at least 20 min after whole-cell configuration was achieved to spectrometry using a Model 4700 Proteomics Bioanalyser (Applied Biosystems, eliminate the influence of initially fast time-dependent shifts in steady-state CA, USA). RP-HPLC fractions were mixed (1:1 v-v) with a-cyano-4 inactivation resulting in NaV channel current (INa) rundown. All experiments were 1 hydroxycinnamic acid matrix (5 mg ml in 50/50 acetonitrile/H2O) and performed at ambient temperature (20–23 °C). To record INa, the external bath MALDI-TOF spectra were acquired in positive reflector mode. All reported solution contained (in mM): NaCl 80, CsCl 5, CaCl2 1.8, tetraethylammonium þ masses are for monoisotopic [M þ H] ions. chloride 50, 4-aminopyridine 5, HEPES 10, NiCl2 0.1, and CdCl2 1, adjusted to pH 7.4 with 1 M NaOH. The pipette solution contained (in mM): NaCl 34, CsF 135, 0 0 MgCl2 1, HEPES 10, ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetraacetic Structure determination. Several buffer conditions were screened to optimize acid 5, and ATP-Na2 3, adjusted to pH 7.4 with 1 M CsOH. To eliminate any quality of NMR data recorded for rDc1a, including 20 mM phosphate buffer, pH influence of differences in osmotic pressure, all internal and external solutions were 6.0; 20 mM MES buffer, pH 6.0 and 20 mM acetate buffer pH 5.0. Initial buffer adjusted with sucrose to 400±5 mOsmol l 1. Experiments were rejected if there screening revealed the acetate buffer, pH 5.0 was optimal for acquisition of NMR were large leak currents or currents showed signs of poor space clamping. data. Recombinant 15N/13C-labelled rDc1a was dissolved in 20 mM acetate buffer, Off-line data analysis was performed using Axograph X version 1.3 or Clampfit 2 pH 5.0 to a final concentration of 350 mM. 5% H2O was added, then the sample 10 (Molecular Devices, USA). Voltage–activation relationships were obtained by was filtered using a low-protein-binding Ultrafree-MC centrifugal filter (0.22 mm measuring steady-state currents elicited by stepwise depolarizations of 5–10 mV pore size; Millipore, MA, USA) and 300 ml was added to a susceptibility matched from a holding potential of –90 mV and calculating peak conductance (GNa) using 5 mm outer-diameter microtube (Shigemi Inc., Japan). NMR data were acquired at the following equation: G ¼ INa/(Vm–Erev) where G is peak conductance, INa is 25 °C using a 900 MHz NMR spectrometer (Bruker BioSpin, Germany) equipped peak inward sodium current, Vm is the test potential and Erev is the reversal with a cryogenically cooled probe. 3D and 4D data used for resonance assignments potential. Reversal potentials were individually estimated for each data set67 by were acquired using non-uniform sampling. Sampling schedules that approximated fitting the INa-V data with the following equation: INa ¼ [1 þ exp(–0.03937 z 26 1 the signal decay in each indirect dimension were generated using sched3D . (Vm–V1/2))] g (Vm–Vrev) where z is the apparent gating charge, g is a Non-uniform sampling data were processed using the Rowland NMR toolkit factor related to the number of channels contributing to the macroscopic whole- (www.rowland.org/rnmrtk/toolkit.html) and maximum entropy parameters cell INa, Vm is equal to the voltage potential of the pulse, and V1/2 is the voltage at were automatically selected as previously described63. 13C- and 15N-edited half-maximal activation. The normalized conductance was fitted to a two-state HSQC-NOESY (mixing time of 200 ms) experiments were acquired using uniform Boltzmann function of the form: G/Gmax ¼ 1/(1 þ exp((–0.3937 z (Vm–V1/2))) 2 sampling. All experiments were acquired in H2O containing 5% H2O. Dihedral where G,z,Vm and V1/2 have the same meaning. The voltage dependence of 64 angles (f, c) were derived from TALOS þ chemical shift analysis and the steady-state Nav channel inactivation (hN/V) data were normalized to the restraint range for structure calculations was set to twice the estimated s.d. The maximum peak current in the control or maximum peak current and fitted using Gly9–Pro10 and Arg36–Pro37 peptide bond was determined to be in the trans the following Boltzmann equation: hN ¼ A/(1 þ exp((Vm–V1/2)/k)) where A is the conformation on the basis of characteristic NOEs and the Ca and Cb chemical fraction of control maximal peak INa (value of 1.0 under control conditions), V1/2 is shifts of Pro residues. NOESY spectra were manually peak picked and integrated, the midpoint of steady-state inactivation, k is the slope factor and Vm is the then peak lists were automatically assigned, distance restraints extracted and an prepulse voltage. The rate of recovery from fast inactivation data were fitted to the ensemble of structures calculated using the torsion angle dynamics package following single-exponential function: I/Imax ¼ 1 exp(–t/t) where t is the time CYANA 3.0 (ref. 25). The tolerances used for CYANA 3.0 were 0.025 p.p.m. in the constant of current recovery. Dose–response curves to determine LD50 and KD50 direct 1H dimension, 0.03 p.p.m. in the indirect 1H dimension and 0.3 p.p.m. for values were fitted using the following form of the logistic equation: y ¼ 1/(1 þ [x]/ 13 15 nH the heteronucleus ( C/ N). During the automated NOESY assignment/structure Dose50) where x is the toxin dose, nH the Hill coefficient (slope parameter) and calculation process, CYANA assigned 92% of all NOESY crosspeaks (2,271 out Dose50 is the median inhibitory dose causing lethality (LD50) or knockdown of 2,469). (KD50). Non-linear curve-fitting of data were performed using GraphPad Prism version 6.00c for Macintosh (GraphPad Software, San Diego) or Origin 8 (OriginLab, MA, USA). Comparisons of two sample means were made using a Insecticidal assays . rDc1a was dissolved in insect saline and injected into the paired Student’s t-test and differences were considered to be significant if Po0.05. ventro-lateral thoracic region of adult sheep blowflies (Lucilia cuprina;), adult All data are presented as mean±s.e.m. of n independent experiments. houseflies (M. domestica), adult American cockroaches (Periplaneta americana) and adult German cockroaches (Blattella germanica)65. Injections were made using a 1.0 ml Terumo Insulin syringe (B-D Ultra-Fine, Terumo Medical Corporation, Electrophysiological measurements on oocytes. Chimeras and channel mutants Maryland, USA) with a fixed 29 G needle fitted to an Arnold hand micro-applicator were generated using sequential PCRs with Kv2.1D7 (ref. 68) and BgNav1 (ref. 21) (Burkard Manufacturing Co. Ltd., England). A maximum volume of 2 mlwas as templates. The Kv2.1D7 construct contains seven point mutations in the outer injected per L. cuprina, M. domestica and B. germanica and 5 ml for the larger vestibule, rendering the channel sensitive to agitoxin-2. The DNA sequence of all P. americana. Thereafter, flies were individually housed in 2-ml tubes and provided constructs and mutants was confirmed by automated DNA sequencing and cRNA with 10% sucrose while cockroaches were housed in closed Petri dishes and was synthesized using T7 polymerase (mMessage mMachine kit, Life Technologies,

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USA) after linearizing the DNA with appropriate restriction enzymes. Channel 21. Dong, K. A single amino acid change in the para sodium channel protein is constructs including hNav1.1–hNav1.7 and hERG were expressed in Xenopus associated with knockdown-resistance (kdr) to pyrethroid insecticides in 69 oocytes by themselves or, in the case of BgNav1, together with the TipE subunit German cockroach. Insect Biochem. Mol. Biol. 27, 93–100 (1997). (1:5 molar ratio), and studied following 1–2 days incubation after cRNA injection 22. Moignot, B., Lemaire, C., Quinchard, S., Lapied, B. & Legros, C. The discovery (incubated at 17 °C in 96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl2 and 1 of a novel sodium channel in the cockroach Periplaneta americana: evidence 1.8 mM CaCl2,50mgml gentamycin, pH 7.6 with NaOH) using two-electrode for an early duplication of the para-like gene. Insect Biochem. Mol. 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(Johns Hopkins University/Lieber Institute for Brain Development), and Marie-France 56. Xiao, Y., Jackson, 2nd J. O., Liang, S. & Cummins, T. R. Common molecular Martin-Eauclaire and Pierre E Bougis (University of Marseille) for sharing BgNav1/TipE, þ determinants of tarantula huwentoxin-IV inhibition of Na channel voltage Kv2.1, hNav1.4, hNav1.5, hERG and BomIV, respectively. We thank Sandipan sensors in domains II and IV. J. Biol. Chem. 286, 27301–27310 (2011). Chowdhury and Carlos Villalba-Galea for helpful discussions and Geoff Brown 57. Saez, N. J. et al. Spider-venom peptides as therapeutics. Toxins 2, 2851–2871 (Department of Agriculture, Fisheries and Forestry, Brisbane) for supply of blowflies. (2010). Parts of the research in this publication were supported by the Australian Research 58. Krepkiy, D. et al. Structure and hydration of membranes embedded with Council (Discovery Grant DP130103813 to G.F.K.) and the National Institute of voltage-sensing domains. Nature 462, 473–479 (2009). Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) 59. Zhang, J. Z. et al. Structure-function map of the receptor site for b-scorpion under award number R00NS073797 (F.B.). toxins in domain II of voltage-gated sodium channels. J. Biol. Chem. 286, 33641–33651 (2011). 60. Song, W. et al. Substitutions in the domain III voltage-sensing module enhance Author contributions the sensitivity of an insect sodium channel to a scorpion b-toxin. J. Biol. Chem. N.S.B., S.D., M.M., V.H., J.G., G.M.N., G.F.K. and F.B. designed research; N.S.B., S.D., 286, 15781–15788 (2011). M.M., V.H., J.G., J.W., G.M.N., G.F.K. and F.B. performed research; N.S.B., S.D., M.M., 61. Corzo, G. et al. Distinct primary structures of the major peptide toxins from the V.H., G.M.N., G.F.K. and F.B. analysed data; N.S.B., S.D., M.M., V.H., G.M.N., G.F.K. venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium and F.B. wrote the paper. channel. FEBS Lett. 547, 43–50 (2003). 62. Fang, L. et al. An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization. Protein Expr. Purif. Additional information 51, 102–109 (2007). Accession codes: Complete chemical shift assignments for rDc1a have been deposited in 63. Mobli, M., Maciejewski, M. W., Gryk, M. R. & Hoch, J. C. An automated tool BioMagResBank under accession code 19666. for maximum entropy reconstruction of biomolecular NMR spectra. Nat. Methods 4, 467–468 (2007). Supplementary Information accompanies this paper at http://www.nature.com/ 64. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS þ : a hybrid method naturecommunications for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009). Competing financial interests: The authors declare no competing financial interests. 65. Eitan, M. et al. A scorpion venom neurotoxin paralytic to insects that affects Reprints and permission information is available online at http://npg.nature.com/ sodium current inactivation: purification, primary structure, and mode of reprintsandpermissions/ action. Biochemistry 29, 5941–5947 (1990). 66. Herzig, V. & Hodgson, W. C. Neurotoxic and insecticidal properties of venom How to cite this article: Bende, N. S. et al. A distinct sodium channel voltage-sensor from the Australian theraphosid spider Selenotholus foelschei. Neurotoxicology locus determines insect selectivity of the spider toxin Dc1a. Nat. Commun. 5:4350 29, 471–475 (2008). doi: 10.1038/ncomms5350 (2014).

6 Metarhizium Genetic Engineering

Viruses, microorganisms and fungi that cause diseases to insects have been studied and evaluated for many years, but very few of them are widely used as a bioinsecticdes. The reasons for this failure are poor efficacy, inconsistency in performance and inability to compete with chemical insecticides140. Better understanding of fungal pathogenesis in insects and the introduction of genetic manipulation techniques is allowing construction of transgenic strains with improved efficacy. Most studies have utilized entomopathogenic fungi themselves as a source of genes to improve efficacy141. For example, insertion of a transgene encoding cuticle degrading protease Pr1 into the genome of Metarhizium anisopliae resulted in a 25% improvement in LT50 (time to kill 50% of test insects) towards Manduca sexta as compared to the wild type strain142. In another example, overexpression of chitinase CHIT1, which is capable of degrading chitin in the insect cuticle, improved the virulence of Beauveria bassiana by 23%143.

Surprisingly, in spite of their potential for improving the efficacy of entompathogens, insecticidal peptides have not been widely exploited141. However, it was recently demonstrated that engineering M. anisopliae to express AaIT, a NaV channel blocker from scorpion venom, improved fungal performance against the lepidopteran Manduca sexta and the malaria vector Anopheles gambiae by 22-fold and 9-fold, respectively117. Toxins from Australian funnel-web spiders found to improve performance even more than AaIT in this system (GF King and R St Leger, unpublished data). Hence, we decided to examine whether the efficacy of Metarhizium strains against mosquitoes and triatomid bugs could be significantly improved by engineering then to express highly insecticidal spider-venom peptides (ISVPs).

33 6.1 Metarhizium Strain selection

There are >2000 Metarhizium strains available through the ARS collection of Entomopathogenic Fungal Cultures (ARSEF). Metarhizium strains generally have a broad host range, with the primary exception being M. acridum which is specific to acridids, To date, it has not proved possible to isolate Metarhizium strains with activity wholly restricted to mosquitoes (dipterans) or tritomid bugs (hemipterans)106. After an extensive search of the ARSEF catalogue and experimental validation, we selected M. anisopliae-1548 for triatomid bugs. M. anisopliae-1630 which also known as M. anisopliae var. anisopliae (Mestch.) Sorokin. This strain was originally isolated by Dr N. Maniania in 1989 from tsetse fly near Kendu Bay, western Kenya144. M. anisopliae-1548 was isolated from the adult black rice bug, Scontinophara coarctata (Hemiptera) in 1984 at Arbor Lan, Palawan, Philippines. These wild-type strains were acquired from ARSEF.

6.2 ISVP selection for Metrhizium genetic engineering

An efficient E. coli periplasmic expression system was developed to produce sufficient amount of ISVPs for insecticidal and functional bioassays. Based on the blowfly toxicity assays described in

Chapter 2, we shortlisted five ISVPs for engineering into Metarhizium, namely the NaV channel blocker U2-CUTX-As1a (As1a), the KCa/CaV blocker ω/κ-hexatoxin-Hv1a (Hybrid), the NaV channel activator µ-DGTX-Dc1a (Dc1a), the CaV channel blocker ω-hexatoxin-Hv1a (Hv1a), and U1-AGTX- Ta1a (Ta1a), whose molecular target remains to be determined. In addition, the scorpion neurotoxin AaIT was chosen as a positive control for comparison with the five ISVPs.

34 6.3 Material and Methods

6.3.1 Vector construction

Vector pPK2-bar-GFP-pMCL1 was kindly provided by Prof. Raymond St Leger, University of Maryland, USA. A two-point mutation was performed on this vector to enable subcloning of synthetic IVSP genes using AvrII and EcoRV restriction sites. Synthetic genes encoding the selected ISVPs (i.e., Hybrid, Hv1a, As1a, Ta1a, and Dc1a), with codons optimised for expression in Metarhizium, were produced and cloned into a variant of pPK2-bar-GFP-pMCL1 vector in order to produce the ISVP-encoding vectors pPK2-bar-GFP-pMCL1-Hybrid, pPK2-bar-GFP-pMCL1- Hv1a, pPK2-bar-GFP-pMCL1-As1a, pPK2-bar-GFP-pMCL1-Ta1a, pPK2-bar-GFP-pMCL1-Dc1a and pPK2-bar-GFP-pMCL1-AaIT. The IVSP genes are linked to a pMCL1 promoter in order to target their expression to the hemocoel of infected insects. Co-expression of GFP enables verification of the specificity of this promoter, while the herbicide gene and kanamycin resistance genes provide selective growth of transformants (See Figure 6.1).

Figure 6.1: Map of the vector pPK2-bar-GFP-pMCL1-Hybrid. The construct contains a herbicide resistance gene (bar), a kanamycin resistance gene, and it encodes GFP (green fluorescent protein) in addition to the Hybrid IVSP. The AvrII and EcoRV restriction sites were used for subcloning IVSP genes.

35 6.3.2 Metarhizium transformation

Metarhizium strains were transformed using Agrobacterium tumefaciens strain AGL-1 harbouring the binary vector pPK2-bar-GFP-pMCL1 (AGL1::pPK2-bar-GFP-pMCL1) using a protocol described previously145 with minor modifications. In summary, an AGL1::pPK2-bar-GFP-pMCL1- ISVP colony from a 2–3 day old plate was inoculated into 10 ml of liquid broth (LB) medium containing 50 µg/ml kanamycin, followed by incubation at 270C for 16–20 h under constant agitation (250 rpm). Picked colonies were tested for the presence of the gfp gene by performing

PCR using gfp primers. Once an OD600 of 0.4–0.8 was attained, the liquid culture was diluted to an

OD600 of 0.15 with induction medium containing 200 µM acetosyringone (AS, a virulence gene 0 inducer) (IMAS solution; Appendix 6.1) and then re-incubated at 27 C for 4–5 h (OD600 ~0.4–0.8) under constant agitation (250 rpm). Meanwhile Metarhizium conidia from a 12-day-old plate were harvested into 5 ml of 0.05% sterile Triton-X (Triton-X helps to separate spores). The mixture was vortexed for 2 min then filtered through sterile glass wool to remove mycelial mass. The conidia concentration was calculated using a haemocytometer (Reichert Bright-Line, USA) and readjusted to a final concentration of 1×106 conidia/ml with IMAS solution. 100 µl of this conidial suspension was mixed with an equal volume of AGL1::pPK2-bar-GFP-pMCL1-ISVP cells (OD600 ~ 0.4–0.8) by gentle inversion and the mixture was spread evenly on black filter paper (autoclaved Whatman 0.18mm thickness) deposited onto freshly poured IMAS plates (Appendix A). All steps were performed under sterile conditions.

After 2 days of co-cultivation at 27 °C, the black filter paper was transformed onto freshly poured M-100 plates (Appendix A) containing 400 µg/ml cefotaxime and 200 µg/ml glufosinate-ammonium PESTANAL (phosphinothricin, PPT). Cefotaxime was used to kill the remaining Agrobacterium cells and the PPT was used as a herbicide-selective agent. After 1 day incubation at 27 °C, the black filter paper was overlaid with freshly prepared M-100 agar containing 200 µg/ml PPT and the plates were incubated again at 27 °C. Putative recombinants started to become visible after 5–6 days and were collected every day or so until the plates were fully covered and no more individual recombinants could be picked up. Recombinant colonies were transferred to M-100 plates and grown for 1 week. The 1-week-old colonies, which had bright fluorescence, were selected for further culturing on Potato Dextrose Agar (PDA) plates. As a positive control, the same Metarhizium strain was transformed with AGL1::pPK2-bar-GFP-pMCL1 without using the selective agent PPT. This control indicated that the fungus is viable on the medium used for transformation (IMAS and M-100). As a negative control, the same Metarhizium strain was transformed with 100 µl IMAS solution instead of AGL1::pPK2-bar-GFP-pMCL1. This control indicated that only transformed cells carrying the PPT resistance gene (bar) could grow on the selection plates (M- 100 + 200 µg/ml PPT). Methods for all chemicals and medium plates used for transformation are 36 detailed in Appendix A. Basic spore viability and germination test was conducted for all transgenic Metarhizium strains by incubating spore mix on Teflon coated slides for 48 h at 300C. Germination of transgenic Metarhizium was compared with wild type strains. The colonies of transgenic Metarhizium, which produced equal amount of spores as of wild type strains, were preserved and used for further culturing.

6.3.3 Transgenic Metarhizium genomic DNA extraction

In order to validate the presence of ISVP genes in the Metarhizium genome, genomic DNA extraction was performed. The extraction procedure was based on a protocol previously described146 with few changes. In summary, a scoop of Metarhizium transformant was transferred into a sterile eppendorf tube containing 500 µl of fungal DNA extraction buffer [0.2 M TrisCl (pH 7.5) + 0.5 M NaCl + 10 mM EDTA (pH 8.0) + 1% SDS]. A micro-homogeniser (Thomas Scientific, USA) was used to disrupt the cells. To this cell lysate, 500 µl of phenol-chloroform-isoamylalcohol (25:24:1) was added, then the mixture was vortexed for 5 min at room temperature (RT). The mixture was then centrifuged at RT/10,000 rpm for 10 min, and the aqueous top layer (essentially containing DNA) transferred to new sterile eppendorf tube. Chloroform (500 µl) was then added to remove any residual phenol, then the solution was centrifuged at RT/10,000 rpm for 10 min. 10 µl of RNase solution was added then the mixture was incubated at 370C for 1 h to degrade RNA. 500 µl of phenol-chloroform-isoamylalcohol was added to separate degraded RNA from DNA, then the solution was centrifuged at RT/10,000 rpm for 10 min and the aqueous top layer transferred to new sterile eppendorf tube. 500 µl of 100% ethanol was added and mixed by gentle inversion to precipitate DNA. The sample was then centrifuged at RT/12000 rpm for 10 min, then the tube was decanted to remove the ethanol and the remaining pellet was dissolved in ultra pure water. This genomic DNA was used as a template for PCR. PCR products from each of the transformants (M1630-As1a, M1630-Dc1a, M1630-Ta1a, M1630-Hv1a, M1630-Hybrid, M1548-As1a, M1548- Dc1a, M1548-Ta1a, M1548-Hv1a, M1548-Hybrid) were electrophoresed on agarose gel; DNA bands were excised and sequenced to confirm the presence of ISVP transgenes in transformed Metarhizium strains. The primers used in this study and their respective usage are summarised in Table 6.2.

Table 6.1: Primers used in this study and their respective usage.

Primers Sequences Usage of Primers

ISVP-5 TAA TTC GTT CCT GGC TCA AAT TCT TTT C To amplify ISVPs present in genomic DNA of transformed ISVP-3 TCA AGC TGT TTG ATG ATT TCA GTA ACG Metarhizium

GFP-5 GGATCCTTACTTGTACAGCTCGTCCA To amplifyi GFP in transformed Agrobacterium colony GFP-3 CTCAGAATGGTGAGCAAGGGCGAG

ISVP-qPCR-5 GTTCTCGCCCTTT CGGGCTTG To detect ISVP gene expression ISVP-qPCR-3 ATCTCGGTGACGGGCAGGAC GPD-qPCR-5 CGATGTCATCTCCAACGCTTCCTGC To detect GPD gene expression TTCTGGGTGGCGGTGTAGGAG GPD-qPCR-3

6.3.4 Characterization of expression of ISVP’s in Metarhizium

The expressions of ISVP’s in Metarhizium were determined with qPCR as described previously117 with minor modifications. Transformed Metarhizium strains (M1630-As1a, M1630-Dc1a, M1630- Ta1a, M1630-Hv1a, M1630-AaIT and M1630-Hybrid) were cultured in Sabouraud dextrose broth (SDB) for 2 days. The grown mycelial inoculums for each strain from SDB cultures were incubated with M. sexta hemolymph at 27 0C for 2 hours. Same amount of mycelia were also collected before incubation and used as a control. To extract RNA, mycelium was separated from hemolymph and total RNA was isolated using the RNeasy Mini Kit (Qiagen) and genomic DNA was eliminated with the RNAse free DNAse set (Qiagen). 1 ug of total RNA was used for cDNA synthesis (ThermoFisher Scientific), and 10 ng of cDNA was used for qPCR. GPD gene (glyceraldehyde-3- phosphate dehydrogenase) was used as a reference. qPCR was performed with the Maxima SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific) using a Lightcycler 480 (Roche, Indianapolis, IN). Each sample was tested in triplicate on a 96 well PCR plate. Relative transcript levels of toxin gene versus the GPD gene (ΔCp) were calculated for each sample. The changes of the relative transcript levels in 2h versus 0h of hemolymph incubation were then calculated (ΔΔCp) for each toxin strains.

6.3.5 Mosquitocidal Bioassay

Spores from a 12-day-old plate of each transformed strain (M1630-As1a, M1630-Dc1a, M1630- Ta1a, M1630-Hv1a, M1630-AaIT and M1630-Hybrid) and the wild-type strain (M1630-WT) were harvested into 5 ml of 0.01% sterile Tween-80, vortexed and filtered using glass wool to remove

38 the mycelial mass. The spore concentration was calculated using a haemocytometer (Reichert Bright-Line, USA) and readjusted to a final concentration of 1×106 spores/ml. 5 ml of this spore suspension was added to a sprayer (details) for application to test mosquitoes (See Figure 6.2). Fungi were applied by spraying spore suspensions onto cold (4 0C) anesthetized adult female mosquitoes (Anopheles gambiae). Briefly, mosquitoes were placed on filter paper in a Petri dish, which was kept on ice to maintain anaesthesia, then mosquitoes were sprayed with spore solution. A separate sprayer was used for each strain only once.

Figure 6.2: Growth and sporulation of wild type and transgenic M. anisopliae- 1630 strains expressing selected ISVPs. The panel shows 12-day-old PDA plates of (a) transgenic M. anisopliae 1630-As1a; (b) transgenic M. anisopliae 1630-Ta1a; (c) transgenic M. anisopliae 1630-Hv1a; (d) transgenic M. anisopliae 1630-Dc1a; (e) transgenic M. anisopliae 1630-Hv1a; (f) transgenic M. anisopliae 1630-Hybrid and; (g) wild-type M. anisopliae 1630 strain. Panel (h) shows the sprayers used for mosquitocidal bioassays filled with spore solution (106 spores/ml) of wild type, transgenic strains and control (0.01% Tween-80) treatment.

Twenty mosquitos were used for each treatment, and three replicates were performed. Each group of 20 mosquitoes was sprayed eight times with a suspension of 106 spores/ml from a distance of at least 1 foot. This technique delivers approximately 45 spores per mosquito147. In total, six transformed strains, namely M1630-As1a, M1630-Dc1a, M1630-Ta1a, M1630-Hv1a, M1630- Hybrid, and M1630-AaIT, were assayed, with the wild-type isogenic strain also included for comparison. 0.01% Tween-80 treatment was used as a control. In order to test for synergistic effects, a combination experiment was performed in which 2 ml of 106 spores/ml of M1630-Dc1a was mixed with an equal amount of 106 spores/ml of M1630-Hybrid. The mixture was vortexed and sprayed on mosquitoes.

39 The treatment and control mosquitoes were provided 15% glucose diet (soaked into cotton balls) in a plastic cage (Figure 6.3A). The mosquito cages were designed to allow removal of dead mosquitoes during the course of treatment (Figure 6.3B). The mosquito cages were maintained at 260C with 60% relative humidity. Mortality was recorded every 12 h. To confirm mortalities were due to Metarhizium infection, dead mosquitoes were transferred to a Petri dish containing a wet paper towel to provide humidity for fungal growth (Figure 6.3C). The mosquitoes that were killed by other factors were not considered for determination of LT50 values. The LT50 for all transgenic strains were calculated using the SPSS statistical package (IBM, USA).

Figure 6.3: Mosquito bioassay. Panel A shows the treatment cages used for the bioassay. Cotton balls soaked in 15% glucose were provided. Panel B shows the design of the mosquito cage, which allowed removal of dead mosquitoes through a cage window covered with a rubber sheet to restrict flying of live mosquitoes. Panel C shows a Petri dish containing dead mosquitoes from one of the treatment protocols, with a wet paper towel providing humidity for fungal growth

Mosquito blood feeding tendency was tested during the course of these experiments. The blood – feeding experiments were conducted by inserting sweaty socks in treatment cages at every 24 h interval. Mosquitos those are not attracted towards socks are considered as they stopped blood feeding.

40 6.4 Results and Discussion

6.4.1 Genetic transformation of Metarhizium

Designing suitable genetic engineering methods always represents a challenge. Over the past few years, transformation methods such as electroporation, Agrobacterium mediated transformation, and biostatic transformation have been assessed for fungal transformation. All techniques need case-by-case optimization prior to application to fungal strains of interest148. Another important factor to be considered is selection of promoter to direct expression of the transgene of interest, in our case ISVP transgenes. We selected the MCL1 (Metarhizium collagen-like-1) promoter, pMCL1, to direct expression of ISVPs to the insect hemolymph149. We optimized and employed Agrobacterium-mediated transformation using Agrobacterium tumefaciens strain AGL-1. ISVP transgenes were chemically synthesized with the MCL1 signal peptide to allow secretion and cloning in the common transfer plasmid pPK2-bar-GFP-pMCL1. The selected ISVP transgenes were individually cloned into M. anisopliae strain 1630 (mosquito strain) and M. anisopliae strain 1548 (triatomid bug strain). Three colonies of each recombinant Metarhizium were preserved. PCR primers were designed to track the presence of ISVPs in the recombinant Metarhizium; DNA fragments from the PCR reactions were extracted from agarose gels and sequenced (See Figure 6.4). The sequencing results confirmed the presence of ISVP transgenes in each of the transformed strains.

Figure 6.4: Agarose gel showing PCR-based detection of ISVP transgenes in transformed M. anisopliae-1630. Lane 1, PCR product from M1630-As1a; Lane 2, PCR product from M1630-Dc1a; Lane 3, PCR product from M1630-Hv1a; Lane 4, PCR product from M1630-Hybrid; Lane 5, PCR product from M1630-Ta1a. IN each case, the size of the PCR product matches that expected for each IVSP transgene. Lane M contains DNA size markers, with the size in bp indicated.

41 6.4.2 ISVP gene expression is induced by insect hemolymph

To confirm the ISVP genes in transgenic Metarhizium strains can be induced by insect hemolymph, qPCR was performed from samples collected before (0 hour) and 2 hours after incubation with M. saxta hemolymph. All 5 transgenic Metarhizium strains (M1630-As1a, M1630- Ta1a, M1630-Dc1a, M1630-Hv1a, M1630-Hybrid, M1630-AaIT) had higher ISVP expression after 2h incubation in hemolymph than before incubation (See Figure 6.5). Primers used in this study are listed in Table 6.1.

Figure 6.5: Quantification of ISVPs in transgenic Metarhizium. qPCR analysis of transgenic Metarhizium revealed the ISVP expression is induced by Manduca hemolymph. After 2h incubation of transgenic strains in Manduca hemolymph, transcript levels of As1a is increased by 4.44 fold, Dc1a by 2.78 fold, Hv1a by0.44 fold, Hybrid by 1.35 fold and Ta1a by 6.11 fold.

6.4.3 Transgenic M. anisopliae-1630 is more virulent to mosquitos

The virulence of each transformant was compared by calculating the median lethal time LT50 (i.e., the time taken to kill 50% of the test mosquito population). Each mosquito received ~45 spores147. The majority of the mosquitoes started dying from day 2 of the treatment. Metarhizium sporulated on >98% of the dead mosquitos under humid conditions, confirming that the reason for the death of mosquitos was fungal (Metarhizium) infection. When dead mosquitoes were observed under a fluorescence microscope, bright green fluorescence from co-expressed GFP was visible in the abdomen of the mosquitoes, confirming that the pMCL1 promoter was directing transgene expression to the insect hemolymph (see Figure 6.6, C&D). After 5–7 days of treatment, fungal growth was visible on treated mosquitoes whereas there was no fungal growth on control mosquitoes (See Figure 6.6, A&B).

All of the strains expressing ISVPs killed mosquitoes much faster than the isogenic wild-type strain. Expression of As1a, Ta1a, Dc1a, Hv1a, Hybrid or AaIT transgenes reduced the LT50 by 32.8%, 33.40%, 56.30%, 48.5%, 52.2% and 41% respectively (See Figure 6.7). Expression of the Dc1a and Hybrid IVSPs caused mosquitoes to die significantly faster than expression of As1a, Ta1a, and AaIT. Metarhizium expressing As1a or Ta1a killed mosquitoes with similar speed. The

LT50 values for each of the recombinant Metarhizium strains are listed in Table 6.2. All of the

42 recombinant Metarhizium strains showed a greater reduction in blood feeding than the wild-type strain. If mosquitoes do not blood feed they cannot transmit disease, and hence the reduction in blood feeding is just as important as the reduction in time it takes to kill mosquitoes.

Table 6.2: Bioassay of transgenic M. anisopliae strains against A. gambiae.

Treatment Strain Dose LT50 (Days ± STD) M1630-As1a 106 spores/ml 6.39 ± 0.27 M1630-Ta1a 106 spores/ml 6.34 ± 0.13 M1630-Dc1a 106 spores/ml 4.16 ± 0.21 M1630-AaIT 106 spores/ml 5.61 ± 60 M1630-ω-Hv1a 106 spores/ml 4.90 ±0.52 M1630-ω/κ -Hv1a (Hybrid) 106 spores/ml 4.55 ± 0.80 M1630-Wild type 106 spores/ml 9.52 ± 0.41 M1630-Dc1a+M1630-Hybrid 106 spores/ml 3.82 ± 0.26

Figure 6.6: Effect of transgenic Metarhizium on mosquitoes. Panel A shows a control mosquito without infection; Panel B shows an infected mosquito 7 days after treatment with transgenic Metarhizium expressing Dc1a; Panel C is a photo taken under white light of a mosquito 3 days after infection with transgenic Metarhizium expressing Dc1a; Panel D is the same mosquito shown in panel C, but the photo was taken using a fluorescence microscope. The green fluorescence from GFP is present only in the abdomen of mosquito, confirming that the pMCL1 promoter directs transgene expression to the insect hemolymph.

Figure 6.7: Bioassay of transgenic Metarhizium against mosquitoes (A. gambiae). The LT50 of each of the transgenic Metarhazium strains is significantly improved compared to the wild-type strain LT50 of 9.5 days. A) The LT50 of the M1630-As1a (green) is 6.4 days; B) LT50 of the M1630-AaIT (orange) is 5.6 days; C) LT50 of the M1630-Ta1a (purple) is 6.3 dyas; D) LT50 of the M1630-Hv1a (pink) is 4.9 days; E) LT50 of the M1630-Hybrid (cyan) is 4.6 days and F) LT50 of the M1630-Dc1a (red) is 4.2 days.

Combined effect on blood feeding efficiency and killing potency of mosquito is significantly improved in transgenic Metarhizium strains. For example, at day 4, M1630-Dc1a knocked down > 95% of test mosquitos and M1630-As1a, M1630-Ta1a, M1630-AaIT, M1630-Hybrid, M1630-Hv1a knocked down ~ 80% of test mosquitos compare to ~40% of mosquitos with WT strain. (See figure 6.8)

Figure 6.8 Combined effects on blood feeding efficiency and killing potency of transgenic and wild type Metarhizium strains at day 4. Histogram illustrates percentage of total affected (mosquito died + mosquito stop blood feeding) test mosquitos. M1630-Dc1a knocked down > 95% of test mosquitos and M1630-As1a, M1630-Ta1a, M1630-AaIT, M1630- Hybrid, M1630-Hv1a knocked down ~ 80% of test mosquitos compare to only ~40% of mosquitos with WT strain.

As Dc1a targets NaV channels whereas the Hybrid toxin targets CaV and KCa channel, we decided to examine whether these two toxins might have synergistic effects. Thus, mosquitoes were sprayed with an equal quantity of the Metarhizium strains expressing Dc1a and Hybrid toxin; this combination reduced the LT50 to 3.8 days, which was lower than any of the single IVSP-expressing Metarhizium strains (See Figure 6.9 & Table 6.2). But, it is statistically not significant. In ideal case, it is important to expresses these two toxins in a single Metarhizium strain to confirm if they act synergistically to kill mosquitoes.

Figure 6.9 Combined effect of M1630-Dc1a and 100 M1630-Hybrid. Equal amount of M1630-Dc1a and

M1630-Hybrid sprayed on mosquito resulted in LT50 of 80 3.8 days which is lower than any single treatment with Control 60 WT M1630-Hybrid (4.6 days) and M1630-Dc1a(4.2days). Dc1a 40 Hybrid Mortality % 20 Dc1a+Hybrid

0 1 2 3 4 5 6 7 8 9 10 11 Days post-infection

45 6.4.4 Transgenic M. anisopliae -1548

The bug specialist strain M. anisopliae-1548 was successfully transformed to express each of the selected ISVPs (i.e., As1a, Dc1a, Ta1a, Hv1a and Hybrid). Each transformed strain (M1548-As1a, M1548-Dc1a, M1548-Hv1a, M1548-Hybrid and M-1548-Ta1a) were grown on PDA plates and spores were preserved for bioassays (See Figure 6.10). Genomic DNA extraction and sequencing results confirmed the presence of ISVPs transgenes in each transgenic strain. In future experiments we aim to examine the efficacy of these transgenic strains against triatomid bugs in comparison to the wild-type isogenic M. anisopliae-1548 strain.

Figure 6.10: Growth and sporulation of

transgenic M. anisopliae-1548 strains

expressing selected ISVPs. 12-day-old PDA

plates showing growth of: (a) transgenic M. anisopliae 1548-As1a; (b) transgenic M. anisopliae 1548-Ta1a; (c) transgenic M. anisopliae 1548-Dc1a; (d) transgenic M. anisopliae 1548-Hv1a; and (e) transgenic M. anisopliae 1548-Hybrid

46 6.4.5 Recombinant Metarhizium as a bioinsecticide

More than 1000 known species of entomopathogens are major mortality factors for insects. Some of these entomopathogens offer environmental advantages in addition to the ability to tackle insect populations that have become resistant to chemical insecticides. However, fungal pathogens have been underused because of their longer kill times compared to chemical insecticides and inconsistency in results140. In this study we significantly enhanced the virulence of Metarhizium strains by engineering them to express ISVPs. Although these transgenic strains need to pass stringent ecological laws and field trials, current data suggest that they will be safe under field conditions. The ISVPs are insect-selective and safe to humans and other vertebrates. Injection of high doses of these IVSPs into mice is non-toxic150, 151, 152. Furthermore, expression of the ISVPs will be limited to the hemocoel of infected insects due to use of the insect-hemolymph specific promoter, pMCL1. This will restrict the release of ISVPs into the environment117.

Despite the environmental safety of fungal entomopathogens, the cost of production and the requirement for high spore doses have limited their use on large-scale insect vector control programs. The recombinant ISVP-expressing Metarhizium strains produced in this study are more virulent than the wild-type strain, which reduces both the kill time and the effective dose. This means the infection rate would be increased and equivalent insect kill could be achieved with fewer spores, which translates into reduced costs.

Even though the ISVPs we used have different molecular targets (NaV, CaV and KCa channels), they all increased fungal virulence. Moreover, a synergistic effect can be obtained with two Metarhizium strains expressing different toxins (Dc1a and Hybrid) with different molecular targets. This raises the possibility of engineering Metarhizium strains that express two or more ISVPs which, in addition to decreasing kill time and spore dose, is likely to reduce the development of target-site resistance since in order to become resistant insects would need to evolve resistance mutations on multiple molecular targets simultaneously153, 14.

At present, commercially available fungal insecticidal products are mainly used as a preventive measure148. But, reduction in insect survival time and feeding damage increases possibility of applying transgenic Metarhizium as a primary treatment. Considering the scope of this study we tested transgenic Metarhizium only in mosquitoes. But, in theory, the same technique could be employed for other disease vectors and insect crop pests.

7 Summarizing Discussion

7.1 Research findings

The long-term goal of the research described in this thesis is to develop bioinsecticides to control vectors of human diseases, with a particular emphasis on malaria and Chagas disease. To this end, transgenes encoding insecticidal spider venom peptides (ISVPs) were used to improve the insecticidal potency of the entomopathogenic fungus M. anisopliae with a view to improving the potential of M. anisopliae as a commerically viable bioinsecticide.

7.1.1 Recombinant ISVP production

The first objective of this thesis was the recombinant production of ISVPs. This provided a means to produce sufficient amount of each ISVP for structural and functional studies. The multiple cysteine residues present in each of the ISVPs provided a particular challenge as they can form non-native disulfide bond connectivities154. As described in Chapter 2, an efficient E. coli recombinant expression system was developed to overcome these difficulties. The overall success rate using this system for disulfide rich peptide production is ~75%124. Here, however, we noted a slightly lower (~65%) success rate of ISVP expression (see Table 7.1). Throughout this project, expression conditions were systematically modified in an attempt to optimise the expression of the each ISVP. The failure of these extensive efforts for some ISVPs suggests that alternative peptide expression systems or chemical synthesis approaches need to be employed to produce the remaining ISVPs for further studies. Although solid-phase peptide synthesis offers a good alternative, a range of folding conditions typically need to be screened to enable formation of the correct native disulfide-bond connectivity and in some cases such screens prove fruitless in providing high yields of natively folded peptide155. Moreover, isotopic labelling of the peptide with 15N and 13C for heteronuclear NMR is currently prohibitively expensive using chemical synthesis. Yeast expression systems have proven to be very successful for production of disulfide-rich venom peptides from snakes and scorpions156,157. While this may ultimately provide a good alternative approach for production of unlabelled ISVPs, the costs of 13C labelling for NMR studies remains high for yeast expression systems.

7.1.2 Insect bioassay of ISVPs

The second objective of this thesis was to investigate the insecticidal activity of recombinantly produced ISVPs in mosquitoes and triatomine bugs. To achieve an unbiased comparison of the insecticidal potency of peptide toxins, it is very critical to test all compounds using the same experimental conditions (e.g., injection volume, dose of toxin, species of test insect, life stage of the test insect, injector used for injection, food provided to test insect etc.136). Even factors such as temperature and relative humidity at which the insect kept after toxin injection can have a significant impact on the outcomes158. The ISVPs studied here were previously tested using a wide range of test insects and experimental conditions. Thus it was imperative to assay the selected ISVPs under the same experimental conditions, in the same test species, in order to quantify their relative toxicities.

As described in Chapter 2, seven of the 11 target ISVPs were produced in sufficient amounts to perform insecticidal assays. ISVPs were tested against sheep blowflies and then against female anopheline mosquitoes and triatomine bugs. Our unbiased comparisons of seven ISVPs indicated that the following toxins were the best candidates for engineering of toxin transgenes into Metarhizium: As1a, ω/κ-hexatoxin-Hv1a (Hybrid), Dc1a, Hv1a, and Ta1a. Outcome of insect bioassays are summarised in table 7.1.

Table 7.1 Summary of the insect bioassays of selected ISVPs

ISVPs LD50 Blowflies (L Cuprina) LD50 Mosquitoes (A. LD50 Triatomine (R. prolixus) aegypti)

ω/κ -hexatoxin-Hv1a 259 ± 17 pmol/g 37.4 ± 9.7 pmol/g 15.8 ± 4.5 pmol/g

ω-hexatoxin-Hv1a 278 ± 7 pmol/g 64.4 ± 3.5 pmol/g 47.4 ± 14.5 pmol/g

κ-hexatoxin-Hv1c 463 ± 22.7 pmol/g – –

U1-AGTX-Ta1a 198 ± 33 pmol/g – –

µ-DGTX-Dc1a 231 ± 32 pmol/g – –

49 7.1.3 Mechanism of action of ISVPs

Determining the mechanism of action of the five ISVPs selected for Metarhizium transformation was one of the priorities of this thesis. The molecular targets of ω/κ-hexatoxin-Hv1a (Hybrid)137 and ω-hexatoxin-Hv1a159 were previously determined. Thus, as described in Chapters 3, 4 and 5, we used electrophysiology to find the molecular targets of the remaining ISVPs. We used patch-clamp electrophysiological recordings of ion channel currents in dorsal unpaired median (DUM) neurons isolated from the terminal abdominal ganglion of the American cockroach Periplaneta americana and also using two-electrode voltage-clamp electrophysiology to examine the effect of some ISVPs on the German cockroach (Blattella germanica) NaV channel heterologously expressed in Xenopus oocytes. These experiments revealed that As1a and Sf1a blocks insect NaV channels whereas

Dc1a is a potent activator of insect NaV channels. NaV channel block by As1a and Sf1a was voltage-independent. As1a appears to be a pore blocker that plugs the outer vestibule of the Nav channel and to a lesser extent acts on Cav channel. Sf1a appears to be selective Nav channel pore blocker. In striking contrast, Dc1a promotes opening of insect NaV channels by interacting with the domain II voltage sensor and shifting the voltage-dependence of activation to more negative potentials. The molecular target of Ta1a remains elusive (see Table 7.2), despite extensive testing against common ion channels targets.

Knowing the molecular targets of the ISVPs allows exploration of the effect of combining ISVPs with different modes of action. For example, the preliminary data shown in Chapter 6 suggest that a synergistic effect could be obtained by engineering a Metarhizium strain that expressed two different ISVPs (Dc1a and Hybrid) with different molecular targets. In addition to decreasing the kill time and spore dose, it is likely that such synergistic combinations would substantially reduce the likelihood of the targeted insect pest developing target-site resistance, since the insects would need to evolve resistance mutations on multiple molecular targets simultaneously160,161.

7.1.4 Structure determination of ISVPs

As described in Chapters 3, 4 and 5, we employed NMR spectroscopy to determine high-resolution 3D structures of selected ISVPs. For proteins smaller than 10 kDa, which includes the vast majority of venom polypeptides, NMR spectroscopy is the dominant approach for structure determination162. The E. coli-based recombinant protein expression system we used to produce the toxins allows facile incorporation of isotopic labels, introduced by expressing the peptide toxin 15 13 in a defined minimal medium enriched with NH4Cl and C-glucose as the sole nitrogen and carbon sources, respectively. Thus, replacing these nuclei with stable and NMR-sensitive 13C and 15N isotopes enabled the use of a wide variety of 3D/4D heteronuclear NMR experiments162. The 3D structure of Hybrid, ω-Hv1a, κ-Hv1a, and Ta1a were previously determined. During the course

50 of this project we determined the 3D structure of As1a, Dc1a and Sf1a (See Table 7.2). The quality of the NMR structures can be compared in terms of precision and stereochemical quality. Based on the measurement of precision (RMSD values) and stereochemical quality (MolProbity score), the 3D structures of As1a, Sf1a and Dc1a rank as high-resolution structures162.

Table 7.2 Summary of the outcomes of ISVP characterisation in this thesis.

Toxin E. coli Yield of pure High resolution Mode of action expression toxin 3D structure

ω/κ -hexatoxin- Yes +++ Yes Cav & Kv blocker Hv1a

ω-hexatoxin-Hv1a Yes +++ Yes Cav blocker

κ-hexatoxin-Hv1c Yes +++ Yes Kv blocker

U1-AGTX-Ta1a Yes +++ Yes In progress

µ-DGTX-Dc1a Yes ++ Yes Nav activator

U2-CUTX-As1a Yes +++ Yes Nav blocker

U2-SGTX-Sf1a Yes +++ Yes In progress

µ-AGTX-Aa1d Yes (Multiple - No Nav blocker isomers) U1-PLTX-Pt1a Yes (Multiple - No Unknown isomers U -CUTX-As1c Yes (Insoluble) - No Unknown 1

δ-CNTX-Pn1a Yes (Multiple - No Nav blocker isomers - no expression; ++ good expression of soluble peptide; +++ excellent expression of soluble peptide

51 7.1.5 Metarhizium genetic engineering

The key objective of this thesis was to express potent ISVPs in selected Metarhizium strains using genetic engineering techniques. We employed Agrobacterium-mediated transformation of Metarhizium using Agrobacterium tumefaciens strain AGL-1. As described in Chapter 6, we successfully transformed a panel of the most potent ISVPs (Hybrid, ω-Hv1a, As1a, Dc1a, Ta1a & AaIT) into Metarhizium strains suitable for infection of mosquitoes (M. anisopliae strain 1630) or triatomine bugs specialist (M. anisopliae strain 1548) using a specially designed construct pPK2- bar-GFP-pMCL1. The highly expressed pMCL1 promoter was chosen as it specifically directs expression of ISVP to the insect hemolymph163. This genetic modification of M. anisopliae-1630 reduced the time required for achieving mortality in mosquitoes. For example, genetically modified

(GM) M. anisopliae-1630-Dc1a reduced the LT50 to ~4.1 days compare to ~9.5 days for wild-type

M. anisopliae-1630 Future experiments will be aimed at determining the LC50 (concentration to kill 50% of test population) for all GM M. anisopliae strains. Based on previous experience with GM Metarhizium expressing AaIT164 and Hybrid toxin (St. Leger & King, unpublished data), it is most likely that the LC50 values against mosquitoes for GM Metarhzium expressing ISVPs will be 20–30 fold lower than wild-type strains.

The major obstacles for widespread use of entomopathogenic fungi are, first, the time required to achieve insect mortality and second, the high cost in comparison to chemical insecticides. Here we found that increasing the virulence of GM Metarhizium resulted in a reduction in LT50 and, most likely, LC50 values. These factors will decrease the dose of Metarhizium required for effective mosquito control and thereby minimise the cost.

Restricting ISVP expression to hemolymph has safety consideration. It will restrict release of ISVP in environment during saprophytic growth of the fungi163,164. GM fungi need to pass stringent environmental and ecological criteria to become a practical means of insect control. Entomopathogenic fungi strains have a wide host range (exception, M. acridum). So their effect on non-target insect host needs rigours risk benefit analysis. For example, though DDT is harmful to non-target insect species and humans, it is still used for endemic malaria control165. GM fungi have the potential to pass the existing stringent safety laws, but the major hurdle will be political and social resistance. Involving political and commercial establishment in educating people about GM fungus could help to improve social acceptance166. As there is increased demand for more effective and sustainable alternatives to conventional chemical insecticides, development of GM fungi that significantly reduce target insect populations and have no hazardous effect on the environment (ISVP expression specific to insect hemolymph) will gain social and regulatory acceptance.

52 Thus, the GM entomopathogenic fungi address the limitation of wild type entomopathogenic fungus (longer time and cost) and opens up the possibility of development of bioinsecticide.

7.2 Future perspectives

Although biological control of insects using entomopathogenic fungi is a well-established technology, it is only employed commercially on a large scale against agricultural insect pests; it is currently not used to control disease vectors such as mosquitoes and triatomine bugs. For example, in China and Brazil, more than 1 million hectares of cropland is treated annually with fungal biopesticide167. In Australia, Metarhizium based products are commonly used for locust control168. Thus, knowledge about implementation and production of fungal biopesticides for control of crop pests is available and this information will be useful for successful deployment of GM entomopathogenic fungi to control vectors of malaria and Chagas disease.

7.2.1 Combining GM Metarhizium & insecticides

A combination of pyrethroid insecticides, permethrin and entomopathogenic fungi (Metarhizium anisopliae or Beauveria bassiana) proved to be synergistic against highly pyrethroid-resistant strains of A. gambiae169. When used alone, Metarhizium or Beauveria took 9 days to cause 100% mortality, but when combined with permethrin ~70% mortality was achieved in 4 days169. It would be informative to conduct similar studies with GM Metarhizium and panel of conventional insecticides against resistant strains of mosquitoes and triatomine bugs.

7.2.2 GM Metarhizium expressing multiple ISVPs

With established technology for genetic engineering of Metarhizium, it would be facile to produce GM Metarhizium expressing multiple ISVPs. Priority would be given to ISVPs with different molecular target in order to achieve synergistic effects. Expressing ISVPs with different molecular targets also has an advantage of reducing development of target-site resistance160. Further, peptides with anti-plasmodial properties could be considered for engineering in to Metarhizium along with ISVPs, which would provide dual action to control malaria at vector as well as parasite level.

53 7.2.3 GM Metarhizium to control agriculture pest

In this thesis, GM Metarhizium was developed to control mosquitoes and triatomine bugs. But similar techniques could be employed to develop Metarhizium strains for targeting major agriculture pests. Although fungal bioinsecticdes have been used for decades, they claim only a small share of global pesticide market106. The main reason is the long time required to achieve mortality and it is expected that an increase in the market share will be proportional to reducing this time170. Engineering ISVP transgenes into Metarhizium strains that target insect pests could overcome this barrier by reducing the kill time.

7.2.4 Optimizing fungal production and efficacy

Optimization of fungal culture conditions such as temperature, culture medium and incubation time could enhance spore yield and spore quality171,172. One of the most important factors for successful field implementation of GM Metarhizium is the long-term effectiveness under field conditions. Several factors such as high temperature and UV-light can negatively affect spore shelf life and insecticidal activity173,174. Spore persistence can vary significantly among fungal strains and even between isolates of the same strains173. The fungal strains we used in this thesis (M. anisopliae- 1630 and M. anisopliae-1548) were selected on the basis of high virulence to mosquitoes and triatomine bugs, respectively. Extensive screening for more persistent fungal strains could be a valuable approach to improve long-term efficacy. Another option to enhance fungal persistence is genetic engineering of fungal strains to express highly efficient Halobacterium salinarum Cyclobutane Pyrimidine Dimers (CPD) photolyase. For example, expression of CPD photolyase increased photo repair > 30-fold in M. robertsii and B. bassiana. The transgenic strains were more resistant to sunlight and retained their virulence175. Long-lasting formulations have also been used to improve fungal spore tolerance to UV radiation and dryness due to high temperature176. These protective formulations include foam formulations177, oil-based suspension178, solvents with sunscreen adjuvants179 and photo-protective anionic dyes180. Microencapsulation techniques have also been explored to prolong fungal spore sustainability181. Thus, successful field implementation of fungal entomopathogens depends not only on the virulence of the fungus but also on its persistence and mass production efficiency; these factors remain to be explored in the context of the more virulent GM strains developed here.

54 7.2.5 Field trials of GM Metarhizium

Evaluation of the efficacy of GM Metarhizium strains in the field under realistic environmental conditions will be the decisive step towards successful deployment of these bioinsecticides for insect vector control. Ambient temperature and relative humidity could both have a significant impact on fungal growth and virulence. Moreover, disease vectors (mosquitoes and triatomine bugs) can behave differently in different climates, which may affect their susceptibility to the fungus.

Field trials take a long time and are expensive. Instead, a good starting point would be semi-field studies that provide near-natural realistic environmental conditions. For example, for mosquito trials, a malaria sphere could be created consisting of experimental huts (like traditional houses), sugar sources (plants, sugar baits), breeding sites (water in plastic containers) and natural climate conditions creating mosquito habitat141,182. For using GM Metarhizium as part of an integrated vector control (IVM) strategy, it will be important to test their combined impact with other insecticides.

7.3 Conclusion

Overall, this thesis has contributed towards the development of entomopathogenic fungi for controlling vectors of human disease. Laboratory trials demonstrated that transgenic M. anisopliae- 1630 expressing ISVPs are significantly more virulent than the wild-type fungus, with drastically reduced LT50 values against malaria vectors. Moreover, in addition to reducing LT50, all GM M. anisopliae strains reduced blood-feeding frequency relative to wild-type fungus. The transgenic M. anisopliae-1630 strains expressing Dc1a and hybrid toxin stand out as the best strains in these trials.

Based on studies with ISVPs alone (Chapter 6), we expect to achieve similar success with transgenic M. anisopliae-1548 strains we constructed that are targeted against triatomine bugs. Thus, it is anticipated that the results described in this thesis will serve as a useful guide for future experiments aimed at development of bioinsecticides for controlling other vectors of human diseases, such as tsetse flies and ticks.

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65 Appendices

Appendix A

Stock Solutions and Medium Plates

1) 2.5X MM salts for Induction Medium

Salts 2.5X (1litre)

KH2PO4 (Potassium phosphate monobasic) 3.625 g

K2HPO4 (Potassium phosphate dibasic) 5.125 g NaCl 0.375 g

MgSO4 (Magnesium sulfate anhydrous) 1.250 g

CaCl2.2H2O (Calcium chloride dihydrate) 0.165 g

FeSO4.7H2O (ferrous sulfate) 0.0062 g

(NH4)2SO4 (ammonium sulfate) 1.250 g

2) 1 M MES Stock Solution (25X Buffer) Dissolve 19.52 g MES (2-(N-morpholino)ethanesulfonic acid) in 80ml dH2O. Adjust pH of the solution to 5.3 with 5M KOH. Bring volume to 100 ml. Sterilize solution by filtering through 0.22um filter. Store in 9 ml aliquots at -200C. 3) 5M KOH Dissolve 7.013g KOH in dH2O. Bring final volume to 25 ml.

4) 10mM Acetosyringone (AS) [50X Virulence Gene Inducer] Add 19.62 mg of AS to 10 ml of sterile water. Stir for 1 hour. Adjust the pH to 8.0 with 5 M KOH. Filter sterile and store in 5 ml aliquots at -200C

5) M-100 Trace Element Solution

Trace Element For 500 ml Solution

H3BO3 30 mg

MnCl2.4H2O 70 mg

ZnCl2 200 mg

Na2MoO4.2H2O 20 mg

FeCl3.6H2O 50 mg

CuSO4.5H2O 200 mg

dH2O To 500 ml final volume

6) M-100 Salt Solution

Chemicals For 1 Litre Solution

KH2PO4 16g

Na2SO4 4g

KCl 8g

MgSO4.7H2O 2g

CaCl 1g 2 M-100 trace element 8ml solution

dH2O To 1 litre final volume

7) Induction Medium

Ingredients For 200ml medium

1X MM salts 80 ml of 2.5X stock

10 mM Glucose/Dextrose 0.36 g

0.5% Glycerol 1 ml

dH2O To 192 ml to final volume

Autoclave. Cool to 500C and then add: 40mM MES 8 ml of 1 M stock solution

Add AS (3 5-dimethoxy-4-hydroxyacetophenone) to liquid induction medium as needed just before use.

8)Induction Medium plates

Ingredients For 400ml medium

1X MM salts 160 ml of 2.5X stock

5 mM Glucose/Dextrose 0.36 g

0.5% Glycerol 2ml

dH2O To 384 ml to final volume

1.5% Agar 6 g

Autoclave. Cool to 500C and then add 16 ml of 1 M stock solution 40mM MES

9) Induction Medium Plates with 200uM Acetosyringone

Make Induction Medium plate as mentioned above, but reduce dH2O to 376 ml and add 8 ml of 10mM AS after autoclaving and cooling the medium.

10) M-100 Plates with 400ug/ml Cefotaxime and 200 ug/ml PPT

Ingredients For 1 litre medium

67 Glucose 10 g

KNO3 3 g

M-100 Salt Solution 62.5 ml

dH20 To 1 litre final volume

1.5% Agar 15 g

After autoclaving and cooling the medium add cefotaxime to final concentration of 400ug/ml and PPT to final concentration of 200ug/ml

68 Appendix B

Methods for deployment of spider venom peptides as bioinsecticides

Published book chapter - Advances in Insect Physiology

69 CHAPTER EIGHT

Methods for Deployment of Spider Venom Peptides as Bioinsecticides

Volker Herzig*, Niraj S. Bende*, Md. Shohidul Alam*, H. William Tedford†, Robert M. Kennedy†, Glenn F. King* *Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia †Vestaron Corporation, Kalamazoo, Michigan, USA

Contents 1. Introduction 390 2. Spider Venom Peptides as Bioinsecticides 391 3. Transcytosis of Spider Venom Peptides Across the Insect Gut Epithelium via Fusion to Molecular Transport Vehicles 394 4. Use of Entomopathogens for ISVP Delivery 397 4.1 Entomopathogens as bioinsecticides 397 4.2 Entomopathogens as a delivery vehicle for ISVPs 398 5. In Planta Expression of Spider Venom Peptides 399 6. ISVP Mimetics 401 6.1 Synthetic mimics of venom peptides 401 6.2 Hv1a pharmacophore analysis and modelling 402 6.3 Optimisation and mechanism of action of the Type II mimic VNX-440 405 6.4 Conclusions from development of Type-II mimetics of Hv1a 407 7. Outlook 407 Acknowledgements 408 References 408

Abstract Despite intensive control measures, insect pests cause enormous damage to crops and stored grain. In addition, insects vector some of the world's most devastating human diseases, including malaria, dengue and Chagas disease. Chemical insecticides remain the dominant method of controlling insect pests but the arsenal of extant insecticides is rapidly diminishing due to the evolution of resistance in pest species and a more difficult regulatory environment due to heightened concern about the potential adverse impacts of chemical insecticides on human health and the environment. Along with predatory beetles, spiders are the most successful insect predators on the planet and

their venoms contain a diverse array of small, disulfide-rich insecticidal peptides. In addition to being potent and highly selective for insects, they collectively offer very diverse pharmacology and should degrade to innocuous breakdown products in the field. However, their major disadvantage is a low level of intrinsic oral activity. Consequently, much research has been directed towards improving the oral activity of these peptide toxins or, alternatively, obviating the oral activity problem altogether by incorporating transgenes encoding the toxins into suitable biological delivery vehicles. Here, we discuss recent advances in both of these areas. We also discuss an approach that merges the advantages of small-molecule and peptide-based insecticides, namely using the pharmacophore of an insecticidal spider-venom peptide to rationally develop a small-molecule mimetic that has improved oral activity, but retains activity for a novel insecticide target.

1. INTRODUCTION

The human population is projected to reach to 9.3 billion by 2050 (Bloom, 2011). This expansion of the population will create enormous pressure on food production since there is limited scope for increasing the amount of cultivated land (Oerke, 2006). Despite intensive control measures, herbivorous insects reduce world crop yields by 10–14% (Oerke, 2006) and damage as much as 30% of stored grain (Boyer et al., 2012), and hence their control is a key factor in determining food productivity. Increases in human population density combined with global warming and a dramatic escalation in international travel has also placed considerable pressure on the public health sector to control the spread of insects that vector human diseases such as malaria, dengue and Chagas disease. Unfortunately, the arsenal of chemical insecticides available to combat insect pests is in rapid decline. This is largely due to the fact that most chemical insecticides target a handful of enzymes, receptors and ion channels in the insect nervous system, and consequently their long-term use has led to widespread resistance in pest species (King and Hardy, 2013; Smith et al., 2013). Concerns about the adverse impact of certain classes of chemical insecticides on the environment and human health have also led to widespread de-registrations and use cancellations; for example, over the period 2005–2009, the U.S. Environmental Protection Agency (EPA) de-registered or limited the use of 169 insecticides, while only nine new insecticides were registered during the same time period (Windley et al., 2012). In simple terms, we have to combat an increasing insect pest problem with a rapidly diminishing chemical arsenal. Spider Venom Peptides as Bioinsecticides 391

2. SPIDER VENOM PEPTIDES AS BIOINSECTICIDES

Spiders are among the most successful and abundant insect predators on the planet. They play a key role in keeping insect populations at bay in a wide range of ecosystems (Nyffeler and Sunderland, 2003). During more than 300 million years of co-evolution with insects, spiders not only evolved sophisticated ways of using silk for prey capture (Blackledge et al., 2009; Vollrath and Selden, 2007), but also acquired powerful venoms that rapidly paralyse insect prey. Spider venoms are complex chemical cocktails of salts, small organic molecules, peptides and proteins, but the dominant components in most spider venoms are small, disulfide-rich peptides (King and Hardy, 2013; Kuhn-Nentwig et al., 2011). Individual spider venoms can contain more than 1000 small peptides (Escoubas et al., 2006). These peptides simultaneously attack virtually every component of the synaptic machinery in the central or peripheral nervous system of envenomated prey, which is tantamount to “shock-and-awe” at the molecular level (Ikonomopoulou and King, 2013; King and Hardy, 2013). Given that more than 44,500 extant species of spiders have been described to date (Platnick, 2013), then a conservative estimate of 250 peptides per venom indicates that as a group spider venoms are likely to contain >10 million bioactive peptides (King, 2011), the vast majority of which are expected to be insecticidal. Hence, spider venoms provide an almost inex- haustible source of insecticidal molecules. To date, more than 200 disulfide- rich insecticidal spider-venom peptides (ISVPs) have been sequenced, ranging in size from 3.3 to 9.0 kDa and containing three to six disulfide bonds (King and Hardy, 2013). Several of these peptides are devoid of vertebrate activity and sufficiently potent against pest insects (i.e. LD50 <1500 pmol/g) and to warrant serious consideration as bioinsecticides (Table 8.1). Almost all ISVPs isolated to date contain a structural motif known as an inhibitor cystine knot (King et al., 2002; Pallaghy et al., 1994) that provides these peptides with remarkable chemical and thermal stability as well as resistance to proteases (King and Hardy, 2013; Saez et al., 2010). Thus, in contrast with most other peptides and proteins, biological stability is not a concern with respect to use of ISVPs as bioinsecticides. ISVPs should be stable during long periods of storage as well as in the field, and they should degrade to innocuous breakdown products. Spider-venom peptides can also be produced using a range of heterologous expression systems that enable cost-effective large-scale production (Klint et al., 2013). Table 8.1 Properties of ISVPs with potential for development as bioinsecticidesa Mass Disulfide Molecular Phyletic LD50 Oral Vertebrate Rational name Synonyms Species ArachnoServer IDb (kDa) bonds target specificity (pmol/g)c activity activity d e μ-AGTX- μ-Agatoxin Agelenopsis 381 4.20 4 NaV D, L, O 30 (D) N.D. No Aa1d IV aperta channel d μ-CUTX- Aptotoxin Apomastus 403 3.76 4 NaV D, L 133 (L) N.D. N.D. As1a 3; Aps III schlingeri channel

U1- Aptotoxin Apomastus 407 8.31 4 N.D. L 2 (L) N.D. N.D. CUTX-As1c 6; Aps VI schlingeri

U1- Peptide A Calisoga sp. 28 4.30 4 N.D. L 551 (L) N.D. No NETX-Csp1a d d μ-DGTX- DTX9.2 Diguetia 352 6.39 4 NaV B, D, L 380 (L) N.D. No Dc1a canities channel d d κ-HXTX- J-ACTX- Hadronyche 174 3.76 4 KCa B, C, D, 91 (D) Low No Hv1c Hv1c versuta channel L, O d ω-HXTX- V-1 Hadronyche 193 4.05 3 CaV B, C, D, 77 (D) Low No Hv1a versuta channel H, L, O d d ω-HXTX- – Hadronyche 204 4.48 3 CaV B, C, 160 (C) N.D. No Hv2a versuta channel D, O Γ-CNTX- Tx4(5-5); Phoneutria 279 5.17 5 Glu B, D, O <100 N.D. No Pn1a Pn4A nigriventer receptor (D) (NMDA subtype) δ-CNTX- Tx4(6-1); Phoneutria 280 5.24 5 NaV B, D 36 (D) N.D. No Pn1a PnTx4(6-1) nigriventer channel

δ-AMATX- δ-palutoxin Pireneitega 301 4.03 4 NaV D, H, L 2347 (L) No N.D. Pl1a IT1 luctuosa channel d U1- Plectoxin- Plectreurys 405 5.07 5 N.D. D, L 14 (L) N.D. N.D. PLTX-Pt1a V/VI tristis

U2- Toxin SF12; Segestria 117 5.12 4 N.D. L 1365 (L) N.D. No SGTX-Sf1b Toxin F5.7 florentina

U1- OAIP-1 Selenotypus 1495 3.81 3 N.D. B, C, L 2 (C) Moderate N.D. TRTX-Sp1a plumipes d U1- TaITX-1 Tegenaria 349 5.68 3 N.D. C, D, L 780 (L) N.D. No AGTX-Ta1a agrestis aTable is modified and updated from King and Hardy (2013) and includes only ISVPs reported to have no toxicity on vertebrates. Phyletic specificity refers to the insect orders in which activity has been reported: Blattaria (B), Coleoptera (C), Diptera (D), Hemiptera (H), Lepidoptera (L) and Orthoptera (O). NaV, voltage-gated sodium channel; CaV, voltage-activated calcium channel; KCa , calcium-activated potassium channel. bRefers to the toxin ID number in the ArachnoServer database. To navigate to the toxin card, use http://www.arachnoserver.org/toxincard.html?id¼ID-number. c LD50 value is given for the insect order (in parentheses) in which the peptide was found to be most potent. dV. Herzig, N. Bende, and G.F. King (unpublished data). eN.D., not determined. 394 Volker Herzig et al.

The biggest potential disadvantage of ISVPs is that they are not orally or topically active. Spiders have solved the delivery problem by evolving fangs that act like hypodermic syringes to deliver their venoms into the haemolymph of prey, thus enabling ISVPs to access their targets in the insect nervous system. We have found that the LD50 values for ISVPs fed to insects are about 100-fold higher than when they are injected (Hardy et al., 2013; King and Hardy, 2013), presumably due to a slow rate of absorption in the insect gut, as observed previously for insect neuropeptides (Audsley et al., 2008) and disulfide-rich peptides from scorpion and snake venoms (Casartelli et al., 2005). Nevertheless, the low level of intrinsic oral activity of ISVPs is not restricted to insects, as other arthropod pests such as ticks were found to be sensitive to oral administration of ω-hexatoxin-Hv1a (hereafter Hv1a) (Mukherjee et al., 2006). There is growing evidence that some peptides and proteins can access paracellular and transcellular transport routes in the insect midgut. Septate junctions in insects are leaky for molecules smaller than 5 kDa (Casartelli et al., 2005; Skaer et al., 1987), and this paracellular route might explain the observed uptake of insect neuropeptides and ISVPs (Audsley et al., 2007; Hardy et al., 2013). However, size alone is not the sole determinant of uptake rate, as the 0.9-kDa insect neuropeptide cydiastatin-4 is translocated across the midgut of the lepidopteran Manduca sexta at a rate 42 times lower than for Hv1a, a 4-kDa ISVP (V. Herzig, N. Audsley, and G.F. King, unpublished data). In addition to the paracellular route, the translocation of large proteins such as albumin across the insect midgut epithelium involves active energy-dependent processes such as transcytosis (Casartelli et al., 2005). Even though our understanding of transport routes across the insect midgut is still in its infancy, it is clear that some peptides can pass undegraded through the midgut, consistent with the observed insecticidal effects of ISVPs after oral administration. Further research in this area might facilitate the rational introduction of chemical modifications and/or the use of formulations that will enhance the oral activity of ISVPs and facilitate their direct deployment as bioinsecticides. However, the remainder of this chapter will focus on alternative methods of delivering ISVPs to target insect species as well as the option of developing orally active small-molecule ISVP mimetics.

3. TRANSCYTOSIS OF SPIDER VENOM PEPTIDES ACROSS THE INSECT GUT EPITHELIUM VIA FUSION TO MOLECULAR TRANSPORT VEHICLES

ISVPs appear to be able to access the insect haemolymph via paracellular transport. Although this route of uptake is extremely inefficient, Spider Venom Peptides as Bioinsecticides 395 it could be enhanced by formulation with appropriate solvents and adjuvants. However, a more generic approach for improving the oral activity of these peptides might be to fuse them to a protein that is capable of being transcytosed across the insect gut epithelium. These fusion proteins would thus serve as molecular transporters that could ferry ISVPs across the insect gut. This approach was first demonstrated via fusion of ISVPs to a plant lectin. Lectins are protease-resistant, carbohydrate-binding proteins that are widely distributed in animals, plants and micro-organisms. Insecticidal plant lectins play an important role in defence against herbivorous insects (Michiels et al., 2010). Galanthus nivalis agglutinin (GNA), a small 14-kDa mannose-specific lectin from the snowdrop plant, is taken up by receptor-mediated endocytosis in the insect midgut after binding to aminopeptidase N, a membrane- bound glycoprotein. Subsequent transcytosis leads to its accumulation in haemolymph, Malpighian tubules, fat bodies, ovarioles and the central nerve cord (Fitches et al., 2001, 2012)(Fig. 8.1). Thus, the transcellular uptake of GNA can be exploited as a mechanism for delivering ISVPs to the insect haemolymph and nervous system (reviewed in Bonning and Chougule, 2014). For example, the oral activity of both Hv1a and the ISVP U2- SGTX-Sf1a (SFI1) was improved dramatically by fusion to the N-terminus of GNA. Second instar cabbage moths, Mamestra brassicae, fed on cabbage leaves coated with 0.2% Hv1a–GNA fusion protein suffered 85% mortality after 10 days, whereas control mortality was <20% (Fitches et al., 2012). Similarly, although neither GNA nor SFI1 was toxic when fed to fourth instar tomato moths, Lacanobia oleracea, the inclusion of a GNA–SFI1 fusion protein as 2.5% of dietary protein was insecticidal to first instar larvae, causing 100% mortality after 6 days, and to fifth instar larvae, causing 20% mortality and threefold reduced weight gain compared to controls (Fitches et al., 2004). An alternative fusion-protein approach recently introduced for specific delivery of ISVPs to aphids (Bonning et al., 2014) relies on the fact that aphids and other sap-sucking hemipterans vector a large number of plant viruses. Luteoviruses are specifically vectored by aphids; the virus is ingested during feeding on an infected plant and then it persists in the aphid without replicating. Remarkably, the virus is able to move from the aphid gut into the haemocoel via transcytosis, which relies on specific recognition of the viral coat protein (CP) via gut receptors followed by receptor-mediated endocytosis (Bonning and Chougule, 2014). Thus, as for GNA, the virus CP can be used as a delivery vehicle for ISVPs. Hv1a has very little intrinsic oral activity against aphids (Pal et al., 2013), but it was recently demonstrated to be highly orally active against a wide range of aphids when fused to the 396 Volker Herzig et al.

Figure 8.1 Summary of methods for delivering ISVPs to the insect haemocoel: (1) Some ISVPs appear to be able to access the haemocoel via paracellular uptake through leaky septate junctions. (2) ISVPs can be fused to carrier proteins such as snowdrop lectin (GNA) and the coat protein of pea enation mosaic virus, which are recognised by specific receptors on gut epithelial cells. These fusion proteins can be taken up via receptor- mediated endocytosis and delivered to the haemocoel by transcytosis. (3) Baculovirus can be engineered to encode an ISVP transgene. Upon recognition by specific gut receptors, budded virions infect gut epithelial cells and other body tissues and release ISVPs. (4) Entomopathogenic fungi such as Metarhizium and Beauveria can also be engineered to encode an ISVP transgene. Fungal spores (conidia) adhere to the insect cuticle, germinate and then invade the integument. The fungus then proliferates in the haemocoel in the form of wall-less blastopores, which produce and release ISVPs. Adapted from Bonning and Chougule (2014).

C-terminus of the CP of pea enation mosaic virus (PEMV); the CP–Hv1a fusion protein was even orally active against the soybean aphid, Aphis gly- cines, which is not a vector for PEMV (Bonning et al., 2014). The CPs from two other luteoviruses, barley yellow dwarf virus and soybean dwarf virus, are also effective vehicles for ISVP delivery (Miller and Bonning, 2007). A significant advantage of the GNA and CP delivery methods is that the fusion proteins can be produced cheaply by recombinant techniques, and transgenes encoding the fusion proteins can be engineered into entomopathogens and plants (see Sections 4 and 5). GNA is a highly generic Spider Venom Peptides as Bioinsecticides 397 delivery vehicle as it has been used to deliver insecticidal peptides to lepidopterans, hemipterans, coleopterans and dipterans. However, GNA does have deleterious effects on some parasitoids, although only at very high doses (Gatehouse et al., 2011). Thus, it will be important, especially in the context of integrated pest management programs, to establish whether attachment to GNA alters ISVP selectivity. In contrast with GNA, CP is a highly specific delivery vehicle that is currently limited to aphids and possibly other sap- sucking hemipterans. However, development of a deeper molecular understanding of the mechanism of CP recognition and uptake in the insect gut might allow this technology to be adapted to the control of other insect pests (Bonning and Chougule, 2014).

4. USE OF ENTOMOPATHOGENS FOR ISVP DELIVERY 4.1. Entomopathogens as bioinsecticides Entomopathogens, which can be bacterial, viral or fungal, are pathogens that kill or seriously disable insects. They play a vital role in natural regulation of insect pests (Sandhu et al., 2012). A wide range of entomopathogens have been studied and used as bioinsecticides, including the soil bacterium Bacillus thuringiensis (Bt), baculoviruses and entomopathogenic fungi such as Metarhizium and Beauveria species. Bt is a naturally occurring spore-forming bacterium from the family Bacillaceae. It is widespread in soil and lethal to a range of insects due to the production of pore-forming Cry toxins (also known as δ-endotoxins) (Gatehouse et al., 2011; Pardo-Lo´pez et al., 2013; also refer to Chapters 2, 3 and 6). Cry toxins are toxic to specific species of insects, especially larval stages, and they have proved to be safe to humans and other vertebrates. Heterologously expressed Cry toxins supply the insect-resistance trait in Bt transgenic crops (see Section 5; Chapter 6). Most Bt species are only active against Lepidoptera, Diptera and Coleoptera, although the subspecies Bt israelensis is effective against the larval stage of mosquitoes (Nartey et al., 2013; Chapter 3). Baculoviruses are pathogenic to the larval stage of a limited range of insects, mostly within the orders Lepidoptera, Hymenoptera and Coleoptera (Bonning and Hammock, 1996; Inceoglu et al., 2006). As for Bt, baculoviruses have to be ingested by the insect to cause mortality. Although wild-type baculoviruses have been used successfully for control of soybean and forest pests, they have not competed successfully with chemical insecticides. This is because baculoviruses take 5–14 days to kill infected insects, 398 Volker Herzig et al. during which time the insect continues to feed and cause crop damage (Bonning and Hammock, 1996; Inceoglu et al., 2006). There are 400–500 species of entomopathogenic fungi and some have been used, without modification, for biocontrol of pest insects for 150 years (Bailey and Boyetchko, 2010; Shah and Pell, 2003). Compared to other micro-organisms, fungi infect a broader range of insects including lepidopterans, homopterans, hymenopterans, coleopterans and dipterans (Vilcinskas et al., 1997). The EPA have registered more than 100 fungus-based insect control products since 1995 (De Faria and Wraight, 2007; Shah and Pell, ® 2003). Mycotrol , for example, is a Beauveria-based mycoinsecticide used for control of whiteflies, thrips, mealybugs and aphids (De Faria and ® Wraight, 2007), while Green Muscle contains spores of Metarhizium ® acridum, which are selectively pathogenic to locusts. Green Muscle showed no detrimental effects on non-target organisms and it is now registered and recommended for locust control by the United Nations (Lomer et al., 2001). However, as for baculoviruses, a major disadvantage of entomopathogenic fungi compared with chemical insecticides is their slow kill time (typically >7 days).

4.2. Entomopathogens as a delivery vehicle for ISVPs Since ISVPs are gene-encoded mini-proteins, transgenes encoding these peptides could be engineered into a variety of entomopathogens, which would mitigate two of their potential disadvantages. First, in this scenario, ISVPs would be produced systemically in the insect host after entomopathogen infection, and consequently their low level of oral activity would not be an impediment to toxin deployment. Second, the phyletic selectivity of the ISVP would become less important as the range of affected insects would be determined primarily by the host range of the selected entomopathogen. Off-target effects on predators and parasitoids could be limited by choosing entomopathogens with restricted host selectivity; for example, M. acridum exclusively infects locusts in the suborder Caelifera, making it valuable as a locust-specific bioinsecticide. Transgenic baculovirus have been engineered that express insecticidal venom peptides from sea anemones, scorpions or spiders; in all cases, the toxin transgene reduced the time between virus application and cessation of feeding or death. The most dramatic improvement in insecticidal activity resulted from incorporation of a transgene encoding an ISVP (Maggio et al., 2010). The potency and speed of kill of entomopathogenic fungi can also be Spider Venom Peptides as Bioinsecticides 399 enhanced by engineering them to express insecticidal venom peptides. For example, the time as well as the dose required for Metarhizium anisopliae to kill the tobacco hornworm, M. sexta, and the dengue vector, Aedes aegypti, was reduced when the fungus was engineered to express the scorpion- venom peptide AaIT (Wang and St Leger, 2007). Entomopathogenic fungi are desirable insect control agents as they directly parasitise arthropods via penetration of the host integument, which sets them apart from bacteria and viruses, which must be ingested to gain host entry (Bailey and Boyetchko, 2010; Roberts, 1973). Engineering entomopathogenic fungi to express ISVPs removes two of the major impediments to their commercial development—the kill time is dramatically decreased and the spore dose required for effective insect control is lowered, thereby reducing application costs.

5. IN PLANTA EXPRESSION OF SPIDER VENOM PEPTIDES

The introduction of genetically modified (GM) crops in the mid- 1990s revolutionised global crop production. As of 2012, 170 million hectares of GM crops were grown by 17.3 million farmers in 30 countries ( James, 2012). More than 90% of growers are small resource-poor farmers in developing countries ( James, 2012). Four GM crops currently dominate global agriculture: cotton (81% of all cotton planted globally), soybeans (81%), maize (35%) and canola (30%) ( James, 2012). The introduction of insect-resistant corn and cotton expressing insecticidal Cry toxins from Bt dramatically reduced insecticide use, improved crop yields and helped conserve beneficial natural enemies (Que et al., 2010; Tabashnik et al., 2013; also refer to Chapter 6). However, constitutive expression of the toxin in transgenic plants has expedited resistance development in a number of pest species; field-evolved resistance to Bt corn has been reported for the maize stalk borer, Busseola fusca; the Western corn rootworm, Diabrotica virgifera virgifera; and the fall armyworm, Spodoptera frugiperda; while resistance to Bt cotton has been reported for the corn earworm, Helicoverpa zea, and the pink bollworm, Pectinophora gossypiella (Gassmann et al., 2014; Tabashnik et al., 2013; this chapter). In China, 2.6% of the population of the cotton bollworm, Helicoverpa armigera, are now resistant to Cry1Ac cotton, which might be a prognostic indicator of future widespread resistance (Tabashnik et al., 2013). Moreover, since Cry toxins primarily target lepidopterans and coleopterans, other insects (especially sap-sucking hemipterans) that were only secondary pests prior 400 Volker Herzig et al. to the introduction of Bt crops have now become a major pest on some crops (Bonning and Chougule, 2014). Resistance to Bt crops can be delayed through the use of non-GM ref- uges and/or by engineering Bt plants to express additional insecticidal genes that act via different mechanisms, an approach known as pyramiding or trait stacking (Moar and Anilkumar, 2007; Que et al., 2010; Chapter 6). ISVP transgenes are good candidates for trait stacking with Bt since (i) they have completely different mechanisms of action; (ii) they are likely to be synergised by Cry toxins which lyse midgut epithelial cells (Sobero´n et al., 2007), a property that should facilitate translocation of ISVPs into the insect haemocoel; and (iii) whereas Bt toxins are largely specific for the insect orders Lepidoptera and Coleoptera, ISVPs with complementary selectivity, particularly against sap-sucking hemipterans, can be selected for trait stacking. Attempts to engineer plants expressing ISVPs began almost two decades ago with the demonstration that transgenic tobacco expressing ω-HXTX- Ar1a, a 37-residue insect-specific calcium channel blocker from the Australian funnel-web spider, Atrax robustus, had enhanced resistance to H. armigera ( Jiang et al., 1996). Transgenes encoding this ISVP (or its orthologue Hv1a) have subsequently been engineered into cotton (Omar and Chatha, 2012), tobacco (Khan et al., 2006) and poplar (Cao et al., 2010). All of these ISVP-expressing transgenic plants were shown to have significantly increased resistance to insect pests. For example, the mortality of second instar H. armigera fed on transgenic tobacco expressing Hv1a was 75–100% after 72 h compared to 0% for larvae fed on untransformed plants, regardless of whether the ISVP was expressed under the strong 35S promoter (Khan et al., 2006) or weaker phloem-specific promoters (Shah et al., 2011). It has even been claimed that transgenic cotton expressing ® Hv1a is as effective as Bollgard II cotton in controlling major cotton pests (Omar and Chatha, 2012). Tobacco plants engineered to express U7-HXTX-Mg1a (Magi-6), a 36-residue ISVP from the venom of the hexathelid spider Macrothele gigas were found to be significantly more resistant than wild-type plants against S. frugiperda (Hernandez-Campuzano et al., 2009). More recently, transgenic Arabidopsis expressing Hv1a fused to the CP of PEMV were shown to be resistant to a diverse range of aphid species (Bonning et al., 2014). Thus, ISVP transgenes appear to have significant potential either as a standalone insect-resistant plant trait or for trait stacking with Cry toxins. Spider Venom Peptides as Bioinsecticides 401

6. ISVP MIMETICS 6.1. Synthetic mimics of venom peptides Venom peptides present structural characteristics that are far from the norm for synthetic agrochemicals and drugs (Tice, 2001), and their size and polar- ity impede passive diffusion through cell membranes. Instead, their oral bioavailability to targets beyond the gut lumen relies on paracellular or active transport (see Section 2). Given the diversity and potential complexity of mechanisms that can mediate such paracellular/active transit through the physical barriers of the gut wall, it seems unlikely that a simple set of general rules could be derived that describe the oral bioavailability of venom peptides. While the oral activity of ISVPs might be improved by chemical modifications (e.g. PEGylation and acylation) or by packaging them in liposomes, microparticles and/or nanoparticles, these are complex and expensive modifications that would significantly increase production costs. An alternative approach to improving oral activity is to create peptidomimetics wherein the functionally critical chemical features of the peptide are displayed on a smaller structural scaffold with better pharmaco- kinetic properties. This alternative has been most extensively pursued for the conotoxin family of venom peptides. The physicochemical properties of Ziconotide/ ® Prialt (ω-conotoxin MVIIA), a cone snail venom peptide used for management of intractable pain, result in insignificant oral bioavailability and consequently this drug requires intrathecal administration (Bingham et al., 2010). In order to avoid the complexity of this drug delivery method, efforts have been undertaken to design small-molecule mimetics that might show oral bioavailability and blood–brain barrier permeability (Brady et al., 2013). Peptidomimetics that could surmount these challenges are classifiable into three types (Ripka and Rich, 1998): (Type I) molecules in which peptide bonds are replaced with more tractable bioisosteres; (Type II) molecules which use moieties that are not structurally equivalent to the native peptide to interact with the receptor; and (Type III) molecules which utilise a scaffold that can spatially display the critical amino acid residues in the same way as the native peptide. Several papers have been published on the design of Type-III mimetics of ω-conotoxins due to the medical and commercial need (Baell et al., 2001, 2004; Menzler et al., 2000). Despite early progress, the resultant conotoxin mimetics were much less potent than the native peptide (Brady et al., 2013). 402 Volker Herzig et al.

Recently, we took a more aggressive approach in the design of Type-II mimetics of Hv1a (Tedford et al., 2013). Hv1a is a 37-residue ISVP that blocks insect but not mammalian voltage-gated calcium (CaV)channels(Fletcher et al., 1997; Tedford et al., 2004b). It is harmless when injected into newborn mice (Atkinson et al., 1998) and a closely related homologue (Ar1a) is inactive against nerve–muscle preparations from chicken and rat (Chong et al., 2007). We developed 3D models of the Hv1a pharmacophore and deployed them as search terms for an in silico screen of a database of millions of commercially available compounds. Hits from this screen were sorted and filtered as part of a multiple-round selection process, ultimately yielding an “Hv1a mimetic” collection that was tested for activity against A. aegypti and Spodoptera exigua. This testing identified several families of viable prototype molecules, including the VNX-000440 (VNX-440) family of triazine molecules that were further optimised by iterative synthesis and analogue testing.

6.2. Hv1a pharmacophore analysis and modelling The 3D solution structure of Hv1a comprises a core region rich in β turns and disulfide bonds (residues 4–21) and a β hairpin (residues 22–37) that projects from the disulfide-rich core (Fletcher et al., 1997). Site-directed mutagenesis and synthetic truncates were used to elucidate the Hv1a pharmacophore (i.e. the set of key structural features contributing to insecticidal activity, presumably by directing interactions with one or more classes of insect CaV channels) (Tedford et al., 2001, 2004a). The primary pharmacophore residues (Pro10, Asn27 and Arg35) form a small, contiguous patch of 200 A˚ 2 on one face of the toxin (Tedford et al., 2001, 2004a). To build a viable peptide pharmacophore model, key functionalities of the critical amino acids were converted into pharmacophoric features and mapped out in three dimensions (Fig. 8.2). Analyses of the structure of Hv1a and several related peptides suggested that certain chemical features not identified previously by scanning mutagenesis, such as the Gly8 carbonyl oxygen and the hydrophobic Val33 isopropyl functionality, are involved in Hv1a activity. This conclusion was taken into account while building different versions of the pharmacophore model: the Val33 side chain was represented in two versions of the pharmacophore model as a hydrophobic feature, while the backbone carbonyl oxygen of Gly8 was included as a hydrogen-bond acceptor (HBA) in one of the models (Table 8.2 lists features built into each version of the pharmacophore models that were used for database searches). Spider Venom Peptides as Bioinsecticides 403

Figure 8.2 Development of small-molecule mimetics of Hv1a. (A) Richardson schematic of Hv1a (first molecule in the PDB file 1AXH) showing the key chemical features that were used for pharmacophore modelling (i.e. the side chains of Pro10, Asn27, Val33, and Arg35 and the backbone carbonyl of Gly8). Hydrogen, carbon, oxygen and nitrogen atoms are shown in white, orange, red and blue, respectively. The N- and C-termini are labelled. (B) Bioassay screening hit VNX-440. (C) Three-dimensional stick representation of VNX-440; atoms are coloured as in (A) except that carbons are grey and chlorine atoms are green. Superimposed on VNX-440 are representations of the eight chemical features of the residues that were included in pharmacophore models of the peptide. Cyan spheres indicate hydrophobic features matching the hydrophobic side chains of Val33 and Pro10; magenta spheres and sticks indicate hydrogen-bond donor (HBD) features projecting from Asn27 and Arg35; green spheres and sticks indicate hydrogen-bond acceptor (HBA) features projecting from Asn27 and Gly8 (leftmost and rightmost residues, respectively). (D) Optimised lead compound VNX-443, an analogue of VNX-440.

Available SAR data suggested that the most functionally important residue of Hv1a is Arg35 (Tedford et al., 2004a). This residue contributed three features to the pharmacophore models: two hydrogen-bond donors (HBDs) and one positively ionisable group. Asn27 contributed two features: one HBD and one HBA. Pro10 and Val33 presumably mediate hydrophobic interactions with CaV channels targeted by the peptide; hence, hydrophobic features represented their side chains in the pharmacophore. Gly8, which adopts an unusual backbone configuration (backbone dihedral angles: ϕ¼90 and ψ ¼20), is conformationally restricted by the cystine knot in a way that allows the Gly8 carbonyl oxygen to contribute to the toxin pharmacophore. 404 Volker Herzig et al.

The five toxin residues—Gly8, Pro10, Asn27, Val33 and Arg35—whose substituents were used to derive features of the pharmacophore models— are illustrated in the context of the overall structure of Hv1a in Fig. 8.2A. The minimal set of pharmacophore features is formed by Arg35, Pro10 and Asn27, yielding a 6-feature pharmacophore (see “6 point 1”, Table 8.2). The pharmacophore model was deployed to search the Accelrys Chemicals Available for Purchasing (CAP) database. Low-energy conformers of the compounds were measured and scored for their fit to chemical features of the pharmacophore. The “8-point” model proved too stringent as a search term, so two 6-feature models were constructed and used to query the CAP database. After searches using all three versions of the pharmacophore (Table 8.2), 8773 compounds were identified as “hits” that showed a substantial structural match to the Hv1a pharmacophore model. A set of physical properties considered relevant for this exercise were then used to filter, sort and annotate the hit compound set. This resulted in the purchase of 1370 unique compounds that were each tested for insecticidal activity against A. aegypti larvae. This led to the identification of several families of compounds, including one containing the screening hit VNX-440 (Fig. 8.2B). VNX-440 is a commercially available compound that was previously shown to have modest antimicrobial activity (McKay et al., 2006). The remarkable fit of VNX-440 to the pharmacophore model used for in silico screening, presented in Fig. 8.2C, can be described as follows: a nitrogen of the triazine core ring and the oxygen of the morpholine functionality of the compound overlap with the HBA features of the pharmacophore; two amine substituents on the triazine core of the compound overlap with the model’s HBD features; the basic nitrogen of the morpholine overlaps with

Table 8.2 Chemical features of key Hv1a residues used to build 3D pharmacophore models Arg35 Asn27 Val33 Pro10 Gly8 Model HBD HBD PI HBA HBD HPH (Cβ, HPH (Cβ, HBA version (Nη1) (Nη2) (Nε) (Oδ1) (Nδ2) Cγ1, Cγ2) Cγ,Cδ) (O) “8-point” Y Y Y Y Y Y Y Y “6-point 1” Y Y Y Y Y N Y N “6-point 2” N Y Y Y Y Y Y N

HBD, hydrogen-bond donor; PI, positively ionisable group; HBA, hydrogen-bond acceptor; HPH, hydrophobe. Spider Venom Peptides as Bioinsecticides 405 the cationic feature; and, finally, the remaining anilide has modest overlap with the two hydrophobic features. 6.3. Optimisation and mechanism of action of the Type II mimic VNX-440 Iterative analoguing, guided by the results of bioassays performed against A. aegypti and S. exigua, allowed us to further characterise the VNX-440 family of hits and to develop a lead molecule (VNX-443; Fig. 8.2D) with significantly improved activity compared with the initial screening hit. The structural changes between VNX-440 and the latter analogue are summarised as follows (cf. Fig. 8.2B and D): first, the amine of the anilide that roughly matched the hydrophobic features in the model was excised to provide a direct connection of a phenyl group to the triazine core. Second, the anilide that only matched a single HBD feature was replaced with het- erocycles to improve water solubility. Third, the hydrophilic tail was altered to change the positioning and basicity of the amine. These alterations retained a match with HBD and cationic features of the model for this pen- dant group and increased insecticidal activity. In aggregate, these structural changes increased insecticidal activity as much as 2200-fold (against S. exigua) and resulted in a class of novel compounds (Kennedy and Steinbaugh, 2013). To confirm that VNX-440 acted against insects via a mechanism similar to Hv1a, its effect on insect ion channels was examined using whole-cell voltage-clamp recordings from dorsal unpaired median (DUM) neurons harvested from the ventral nerve cord of the American cockroach, Peri- planeta americana (Tedford et al., 2013). Figures 8.3A and B show examples of the effects of 100 μM VNX-440 on IBa (a measure of CaV channel activity) recorded from clamped DUM neurons. Control superfusion with bath solution had no effect on IBa, but superfusion with 100 μM VNX-440 reduced IBa dramatically, with maximum inhibition reached within 10 min of exposure to VNX-440. The IC50 for inhibition of peak IBa by VNX-440 was estimated from the dose–response curve to be 23.1 μM(Fig. 8.3C), which is only 20 times higher than the IC50 (1.08 μM) reported for inhibition of high voltage- activated CaV channels by Hv1a (Chong et al., 2007). Unfortunately, this activity was not replicated when VNX-443 was tested in this system. Regardless, VNX-443 and various other analogues in this series caused no adverse effects in an acute oral toxicity study in which rats were dosed at 100 mg/kg (R.M. Kennedy, unpublished results). 2+ Figure 8.3 Inhibition of Ba currents (IBa) in American cockroach DUM neurons by VNX-440. (A) IBa recorded at 10 mV: black trace shows control IBa, green trace shows IBa during superfusion with bath solution and the red trace shows IBa following with superfusion with 100 μM VNX-440. (B) Time course of peak IBa values recorded at a test potential of 10 mV after addition of 100 μM VNX-440. (C) Dose–response curve for VNX-440 inhibition of peak IBa at a test potential of 10 mV. (D) I–V curves for IBa obtained before, and 10 min after, application of 100 μM VNX-440. Spider Venom Peptides as Bioinsecticides 407

6.4. Conclusions from development of Type-II mimetics of Hv1a Our in silico modelling and screening using the pharmacophore of Hv1a led to a small-molecule hit (VNX-440) that showed properties consistent with mimicry of the native peptide’s biological activity. To our knowledge, this remains the only published example of the discovery of an insecticidal lead compound through testing of a small set of candidate compounds chosen to match the pharmacophore of a peptide. However, since the screening lead VNX-440 was optimised solely via an in vivo assay (with insect mortality as the read-out), it is not entirely clear if fidelity was maintained with respect to the peptide’s mechanism of action, or if another property was optimised. An additional consideration is that while VNX-443 has good per os activity, it has only limited contact activity in adult mosquitocidal tests. The physicochemical properties enabling topical bioavailability are considerably stricter than for oral bioavailability and may not allow full coverage of the 200 A˚ 2 pharmacophoric hot spot on peptides such as Hv1a.

7. OUTLOOK

An unmodified ISVP developed by Vestaron Corporation was recently approved by the U.S. EPA for use against cabbage looper, Tricho- plusia ni, marking the start of new era of peptide-based insecticides. ISVPs have many properties that make them attractive as insect control agents: they are potently insecticidal, highly selective for insects, stable to extremes of temperature and should degrade in the field into innocuous breakdown products. Although they typically have low levels of intrinsic oral activity, simple formulation can improve their oral activity sufficiently to make them competitive with chemical insecticides. The oral activity of ISVPs can also be markedly improved by fusing them to proteins that ferry them via transcytosis across the insect gut, including plant lectins and the CP of insect-vectored viruses. Transgenes encoding these fusion proteins could be incorporated into crop plants either as a standalone insect-resistance trait or they could be pyramided with Bt transgenes in order to reduce the likelihood of resistance development and expand the range of susceptible insects. Entomopathogens can also be engineered to express transgenes encoding ISVPs or ISVP fusion proteins with enhanced oral activity. This approach has the advantage that the selectivity of the ISVP can be tailored by taking advantage of the limited host range of many entomopathogens. 408 Volker Herzig et al.

Thus, in summary, there are a wide variety of methods by which ISVPs can be deployed as natural insecticides. We also recently demonstrated for the first time that the pharmacophore of ISVPs can be used to rationally develop small-molecule mimetics with improved oral activity, thereby providing another approach by which these peptides can be exploited for the control of insect pests. ACKNOWLEDGEMENTS The authors would like to thank the Australian Research Council for financial support. R.M. Kennedy was funded in part by a grant from the Foundation for the National Institutes of Health through the Vector-based Transmission of Control: Discovery Research (VCTR) program of the Grand Challenges in Global Health initiative. REFERENCES Atkinson, R.K., Howden, M.E.H., Tyler, M.I., Vonarx, E.J., 1998. Insecticidal toxins derived from funnel web (Atrax or Hadronyche) spiders. U.S. Patent No. 5,763,568. Audsley, N., Matthews, J., Nachman, R., Weaver, R.J., 2007. Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae. Gen. Comp. Endocrinol. 153, 80–87. Audsley, N., Matthews, J., Nachman, R.J., Weaver, R.J., 2008. Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro. Peptides 29, 286–294. Baell, J.B., Forsyth, S.A., Gable, R.W., Norton, R.S., Mulder, R.J., 2001. Design and synthesis of type-III mimetics of ω-conotoxin GVIA. J. Comput. Aided Mol. Des. 15, 1119–1136. Baell, J.B., Duggan, P.J., Forsyth, S.A., Lewis, R.J., Lok, Y.P., Schroeder, C.I., 2004. Syn- thesis and biological evaluation of nonpeptide mimetics of ω-conotoxin GVIA. Bioorg. Med. Chem. 12, 4025–4037. Bailey, K.L., Boyetchko, S.M., 2010. Social and economic drivers shaping the future of biological control: a Canadian perspective on the factors affecting the development and use of microbial biopesticides. Biol. Control 52, 221–229. Bingham, J.P., Mitsunaga, E., Bergeron, Z.L., 2010. Drugs from slugs—past, present and future perspectives of ω conotoxin research. Chem. Biol. Interact. 183, 1–18. Blackledge, T.A., Scharff, N., Coddington, J.A., Szuts, T., Wenzel, J.W., Hayashi, C.Y., Agnarsson, I., 2009. Reconstructing web evolution and spider diversification in the molecular era. Proc. Natl. Acad. Sci. U.S.A. 106, 5229–5234. Bloom, D.E., 2011. 7 billion and counting. Science 333, 562–569. Bonning, B.C., Chougule, N.P., 2014. Delivery of intrahemocoelic peptides for insect pest management. Trends Biotechnol. 32, 91–98. Bonning, B.C., Hammock, B.D., 1996. Development of recombinant baculoviruses for insect control. Annu. Rev. Entomol. 41, 191–210. Bonning, B.C., Pal, N., Liu, S., Wang, Z., Sivakumar, S., Dixon, P.M., King, G.F., Miller, W.A., 2014. Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids. Nat. Biotechnol. 32, 102–105. Boyer, S., Zhang, H., Lemperiere, G., 2012. A review of control methods and resistance mechanisms in stored-product insects. Bull. Entomol. Res. 102, 213–229. Brady, R.M., Baell, J.B., Norton, R.S., 2013. Strategies for the development of conotoxins as new therapeutic leads. Mar. Drugs 11, 2293–2313. Spider Venom Peptides as Bioinsecticides 409

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Supplementary data- Nature Communication, 2014

Supplementary Figures

Supplementary Fig. 1: Effect of rDc1a on L. cuprina and M. domestica Dose–response curves for lethal effects of rDc1a determined 24 h after injection into sheep blowflies (L. cuprina; open circles) and houseflies (M. domestica; filled circles). Data were fitted according to equations indicated in Methods section.

Supplementary Fig. 2: rDc1a does not influence human Nav channels a, Effect of 1 µM rDc1a on hNav1.1–hNav1.7 and hERG (Kv11.1). Representative Nav channel currents were elicited at voltage ranging from –40 mV to –20 mV depending on the studied isoform (holding voltage was −90 mV). No significant toxin-induced effects (red) were seen at any of the tested voltages (n = 4). Normalized conductance-voltage (G/Gmax) relationships of hERG channels were determined by voltage steps from –100 mV to +50 mV in 10-mV increments for 2 s followed by a pulse to –60 mV for 2 s from a holding potential of –90 mV (n = 4). Shown are a representative control trace (black) and a trace in the presence of 1 µM rDc1a (red) at a voltage of -20 mV. b, First panel: effect of 10 nM BomIV, an insect- targeting scorpion α-toxin capable of inhibiting Nav channel fast inactivation, on inward BgNav1 currents evoked by a 50 ms depolarization to -30 mV from an oocyte membrane holding potential of -90 mV. BomIV clearly slows down fast inactivation to the extent that a persistent sodium ion current becomes visible at the end of the depolarizing pulse. Other panels: potassium currents elicited by depolarizations near the foot (less than 1/3 of maximal activation) of the voltage-activation curve for WT Kv2.1 and BgNav1/Kv2.1 chimeric constructs. Currents are shown before (black) and in the presence of 1 µM BomIV. Our results reveal that Bom IV interacts with the DIV paddle motif in BgNav1. Consequently, this result supports a role for the BgNav1 domain IV voltage sensor in fast inactivation. c, Tail current voltage– activation relationships for each chimeric construct (n = 3; error bars represent SEM) before (black) and following (colored) addition of 1 µM rDc1a. rDc1a clearly affects only the DII chimera. Holding voltage was −90 mV for DI, II, and IV, and –120 mV for DIII, and the tail voltage was −60 mV (−100 mV for DIII).

Supplementary Figure 3: Lack of effect of rDc1a on cockroach DUM neuron PaNav1 a, Typical effects of 1 µM rDc1a on PaNav1. Traces show superimposed control (black) and toxin (grey and shaded) currents elicited by a 50-ms depolarizing test pulse (Vtest) as shown in the voltage protocol above the current traces. The lack of effect on INa amplitude and time course is highlighted in the inset. The dotted line represents zero current. b, Effect of rDc1a on the voltage-dependence of PaNav1 channel activation. Nav channel currents were elicited by the test pulse protocol shown above the GNa-V relationships. Currents recorded in the presence of 1 M rDc1a were normalized to the maximum inward INa in controls, converted into normalized GNa-V relationships and fitted with a Boltzmann function. Data shows GNa before (closed circles) and after (open circles) application of 1 µM rDc1a. Data were taken from panel (c) and fitted using linear regression. c, Lack of a use-dependent effect of rDc1a on PaNav1. Effects of 1 µM rDc1a on use-dependent decline in INa during 20 depolarizing test pulses from –90 mV to –10 mV at 30 Hz using the test pulse protocol at the top of the panel. Currents were normalized to the peak INa amplitude of the first pulse in the train in the absence (closed triangles), and presence (open circles and shaded), of 1 µM rDc1a. d, Effect of rDc1a on steady-state Nav channel inactivation (h∞). Steady-state inactivation was determined using a two-pulse protocol (see inset panel d). Peak INa, recorded during Vtest, were expressed as a fraction of maximum control INa and plotted against prepulse potential. Panel (d) shows the proportion of INa that is available for activation under control conditions (closed circles), and during perfusion with 1 µM rDc1a (open circles and shaded). All data are expressed as the mean ± SEM (n = 3).

Supplementary Fig. 4: Sequence alignment of domain II voltage-sensors Putative transmembrane segments (S1-S6) within the DII voltage-sensing domain of BgNav1 (Blatella germanica), PaNav1 (Periplaneta americana), MdNav1 (Musca domestica), and rNav1.2a are indicated with a green bar above the relevant amino acids. The BgNav1 residue count is indicated in italics. The H to Y and D to E substitutions discussed in the manuscript are highlighted in red against a gray background whereas the domain II S3b-S4 paddle motifs are indicated with a box. The third residue that differs between PaNav1 and BgNav1 is located in the S1 transmembrane segment and is indicated in red without a gray background. Underlined residues in the S1-S2 loop region were identified previously as 1 playing a role in β-scorpion toxin binding to rNav1.2a .

Supplementary Table 1: Structural statistics for the ensemble of rDc1a structuresa.

Experimental restraintsb Interproton distance restraints Intraresidue 267 Sequential (|i–j| = 1) 253 Medium range (|i–j| 5) 106 Long range (|i–j| 5) 276 Disulfide-bond restraints 12 dihedral-angle restraints 44 dihedral-angle restraints 46 Total number of restraints per residue 17.6 Violations of experimental restraintsc 0 R.m.s. deviation from mean coordinate structure (Å)d All backbone atoms 0.48 ± 0.09 All heavy atoms 0.90 ± 0.15 Backbone atoms, residues 3–42, 52–56 0.27 ± 0.07 Heavy atoms, residues 3–42, 52–56 0.70 ± 0.11 Stereochemical qualitye Residues in most favored Ramachandran region (%) 99.4 ± 1.2 Ramachandran outliers (%) 0.0 ± 0.0 Unfavorable sidechain rotamers (%) 9.2 ± 3.0 Clashscore, all atomsf 0.0 ± 0.0 Overall MolProbity score 1.24 ± 0.12 a All statistics are given as mean ± S.D. b Only structurally relevant restraints, as defined by CYANA, are included. c There were no violations of any distance or dihedral-angle restraints in the final ensemble. d Mean r.m.s. deviation calculated over the entire ensemble of 20 structures. e According to MolProbity (http://molprobity.biochem.duke.edu) f Defined as the number of steric overlaps >0.4 Å per thousand atoms

Supplementary Reference

1. Zhang, J.Z. et al. Structure-function map of the receptor site for β-scorpion toxins in domain II of voltage-gated sodium channels. J. Biol. Chem. 286, 33641–33651 (2011).