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. I 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%) V 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.