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Phenomics, Genomics and Genetics in Plasmodium Vinckei

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Phenomics, Genomics and Genetics in Plasmodium Vinckei Phenomics, Genomics and Genetics in Plasmodium vinckei Dissertation by Abhinay Ramaprasad In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia October, 2017 2 EXAMINATION COMMITTEE PAGE The dissertation of Abhinay Ramaprasad is approved by the examination committee Committee Chairperson: Prof. Arnab Pain Co-Supervisor: Prof. Richard Culleton Committee Members: Prof. Richard Carter, Prof. Takashi Gojobori, Prof. Xin Gao 3 ©October, 2017 Abhinay Ramaprasad All Rights Reserved 4 ABSTRACT Phenomics, Genomics and Genetics in Plasmodium vinckei Abhinay Ramaprasad Rodent malaria parasites (RMPs) serve as tractable models for experimental ge- netics, and as valuable tools to study malaria parasite biology and host-parasite- vector interactions. Plasmodium vinckei, one of four RMPs adapted to laboratory mice, is the most geographically widespread species and displays considerable phe- notypic and genotypic diversity amongst its subspecies and strains. The phenotypes and genotypes of P. vinckei isolates have been relatively less characterized compared to other RMPs, hampering its use as an experimental model for malaria. Here, we have studied the phenotypes and sequenced the genomes and transcriptomes of ten P. vinckei isolates including representatives of all five subspecies, all of which were collected from wild thicket rats (Thamnomys rutilans) in sub-Saharan Central Africa between the late 1940s and mid 1960s. We have generated a comprehensive resource for P. vinckei comprising of five high-quality reference genomes, growth profiles and genotypes of P. vinckei isolates, and expression profiles of genes across the intra-erythrocytic developmental stages of the parasite. We observe significant phenotypic and genotypic diversity among P. vinckei isolates, making them particu- larly suitable for classical genetics and genomics-driven studies on malaria parasite biology. As part of a proof of concept study, we have shown that experimental ge- netic crosses can be performed between P. vinckei parasites to potentially identify genotype-phenotype relationships. We have also shown that they are amenable to genetic manipulation in the laboratory. 5 ACKNOWLEDGEMENTS My deepest gratitude to my advisors, Professor Arnab Pain and Professor Richard Culleton. I thank Arnab for his unwavering support and guidance throughout my Masters and Doctoral studies and for always motivating me to achieve more. I thank him for the valuable research training he provided through several projects, his sage advice regarding my career decisions and for hosting awesome barbeques. I thank Richard for guiding me through the world of rodent malaria, personally training me in the wet lab work and for the many fruitful discussions we have had on diverse topics from malaria’s history to Bob Dylan. I would like to thank Hussein Abkallo for teaching me reverse genetics tech- niques and for all the laughs. Thanks to Severina Klaus for the outstanding work and for sharing the pain and glory of generating genetic crosses in P. vinckei. Also, for the limitless supply of German chocolate. I want to thank all members of the Pathogen Genomics group at KAUST and the Malaria Unit at NEKKEN for their support. I would also like to thank the Protozool- ogy lab at NEKKEN, KAUST Bioscience Core Lab and CBRC Dragon cluster for their support. Special thanks to Sarina, Fathia, Raeece, Jojo and Dr. Katsuhiko Mineta for their timely help in matters big and small. I would like to thank King Abdullah University of Science and Technology for their financial support through the KAUST fellowship award. Last but not least, I would like to thank those constants in the sea of variables - my parents, Chandrika and Prasad, for trading their dreams for mine, and my friends, Anu, Ice and Siva, for putting up with my hibernations, proofreading my write-ups and being the awesome friends that they are. 6 STATEMENT OF CONTRIBUTION The work presented in this thesis is a product of joint efforts made by several individuals apart from me. With profound gratitude towards them, I would like to mention here their contributions along with mine during the course of my doctoral study. The dissertation work was carried out at the Pathogen Genomics Lab, King Ab- dullah University of Science and Technology (KAUST) under the supervision of Prof. Arnab Pain (AP) and at the Malaria Unit, Institute of Tropical Medicine (NEKKEN), Nagasaki University under the supervision of Prof. Richard Culleton (RC). The project was jointly conceived by AP and RC. • Chapter 2 - Phenotyping (At the Malaria Unit) All animal work was carried out by Abhinay Ramaprasad (AR) under the supervision of RC. AR also carried out the parasitaemia counting and final presentation of the growth profiles. Transmission experiments were carried out by both AR and RC, where RC performed the mosquito dissections. • Chapter 3 - Genomes and Transcriptomes (At Malaria Unit and KAUST) All animal work, DNA and RNA isolations were carried out by AR. PacBio sequencing and Pacbio genome assembly were availed commercially from Macrogen Inc. Illumina PCR-free library preparation was performed by AR. All subsequent bioinformatic analyses were performed by AR under the super- vision of AP. The simplified protocol for RNAseq was formulated by AR. • Chapter 4 - Genetics (At Malaria Unit and KAUST) The four genetic cross experiments and LGS were performed jointly by AR, Ms. Severina Klaus (SK) 7 (University of Heidelberg) and RC. AR and SK took care of animal work and transmission to mosquitoes. RC performed mosquito dissection. qPCR was performed by SK. DNA isolation, library prep and sequencing for LGS were performed by AR. Bioinformatic analysis for LGS was carried out by Chris Illingworth (CI) (University of Cambridge), AR and Axel Martinelli (AM) (Hokkaido University). AR performed read mapping and SNP calling using scripts generated by AM. CI performed the mathematical modelling steps. Genetic manipulation experiments were done by AR, that includes plasmid design and construction, and parasite transfection. Cloning of transformed parasites were done by RC. Apart from this, I would like to acknowledge here Dr. Hussein Abkallo (Univer- sity of Edinburgh) and Dr. Kittisak Thawnashom (Naresuan University) who were generous in providing me with advice and training for the genetic manipulation experiments. In additional to the work presented in this dissertation, I have contributed to- wards other projects (some of which have resulted in publications listed below) during my doctoral study at the Pathogen Genomics Laboratory, KAUST and Malaria Unit, NEKKEN. 1. Abkallo HM, Martinelli A, Inoue M, Ramaprasad A, Xangsayarath P, Gitaka J, Tang J, Yahata K, Zoungrana A, Mitaka H, Acharjee A, Datta PP, Hunt P, Carter R, Kaneko O, Mustonen V, Illingworth CJR, Pain A, Culleton R. Rapid identification of genes controlling virulence and immunity in malaria parasites. PLoS Pathogens. 2017;13(7): e1006447. doi: 10.1371/journal.ppat.1006447 Contribution Transcriptome sequencing and analysis 2. Lu F, Culleton R, Zhang M, Ramaprasad A, Seidlein L, Zhou H, Zhu G, Tang J, Liu Y, Wang W, Cao Y, Xu S, Gu Y, Li J, Zhang C, Gao Q, Menard D, Pain A, Yang H, Zhang Q, Cao J. Emergence of Indigenous Artemisinin-Resistant Plasmodium falciparum in Africa. N Engl J Med. 2017;0028-4793. doi: 10.1056/NEJMc1612765. Contribution Determined geographical origin of ART resistant parasite by DNA sequencing and SNP analysis. 3. Ansari HR, Templeton TJ, Subudhi AK, Ramaprasad A, Tang J, Lu F, Naeem R, Hashish Y, Oguike MC, Benavente ED, Clark TG, Sutherland CJ, Barnwell JW, Culleton R, Cao J, Pain A. Genome-scale comparison of expanded gene 8 families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species. Int J Parasitol. 2016;S0020-7519(16)30135-7. doi: 10.1016/j.ijpara.2016.05.009. Contribution DNA sequencing, Genome assembly and annotation. 4. Moon RW, Sharaf H, Hastings CH, Ho YS, Nair MB, Rchiad Z, Knuepfer E, Ramaprasad A, Mohring F, Amir A, Yusuf NA, Hall J, Almond N, Lau YL, Pain A, Blackman MJ, Holder AA. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi. Proc Natl Acad Sci U S A. 2016;113(26): 7231-7236. doi: 10.1073/pnas.1522469113. Contribution Manual curation of gene models. 5. Roques M*, Wall RJ*, Douglass AP*, Ramaprasad A*, Ferguson DJ, Kaindama ML, Brusini L, Joshi N, Rchiad Z, Brady D, Guttery DS, Wheatley SP, Yamano H, Holder AA, Pain A, Wickstead B, Tewari R. Plasmodium P- Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes. PLoS pathogens. 2015;11(11):e1005273. doi: 10.1371/journal.ppat.1005273. (* equal contribution) Contribution Transcriptome sequencing and differential expression analysis. 6. Woo YH, Ansari H, Otto TD, Klinger CM, Kolisko M, Michalek J, Saxena A, Shanmugam D, Tayyrov A, Veluchamy A, Ali S, Bernal A, Del Campo J, Cihlar J, Flegontov P, Gornik SG, Hajduskova E, Horak A, Janouskovec J, Katris NJ, Mast FD, Miranda-Saavedra D, Mourier T, Naeem R, Nair M, Panigrahi AK, Rawlings ND, Padron-Regalado E, Ramaprasad A, Samad N, Tomcala A, Wilkes J, Neafsey DE, Doerig C, Bowler C, Keeling PJ, Roos DS, Dacks JB, Templeton TJ, Waller RF, Lukes J, Obornik M, Pain A. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. eLife. 2015;4. doi: 10.7554/eLife.06974. Contribution Manual curation of gene models. 7. Gornik SG, Febrimarsa, Cassin AM, MacRae JI, Ramaprasad A, Rchiad Z, McConville MJ, Bacic A, McFadden GI, Pain A, Waller RF. Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate. Proc Natl Acad Sci U S A. 2015;112(18):5767-72. doi: 10.1073/pnas.1423400112. PubMed PMID: 25902514. Contribution DNA and RNA sequencing 8. Guttery DS, Poulin B, Ramaprasad A, Wall RJ, Ferguson DJP, Brady D, Patzewitz E-M, Whipple S, Straschil U, Wright MH, Mohamed AMAH, Radhakrishnan A, Arold ST, Tate EW, Holder AA, Wickstead B, Pain A, Tewari R.
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