A Trypanosoma Brucei Orfeome-Based Gain-Of-Function Library

bioRxiv preprint doi: https://doi.org/10.1101/849042; this version posted November 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 A Trypanosoma brucei ORFeome-based Gain-of-Function Library 3 reveals novel genes associated with melarsoprol resistance 4 5 Carter M1†, Kim HS2†, Gomez S1, Gritz S1, Larson S1, Schulz D3*, Hovel-Miner GA1* 6 7 † - Co-first 8 * - Co-corresponding 9 10 1) The George Washington University 11 Department of Microbiology, Immunology, and Tropical Medicine 12 2300 Eye St., Washington, D.C, 20037 13 [email protected] 14 2) The Public Health Research Institute at the International Center for Public Health 15 New Jersey Medical School – Rutgers, The State University of New Jersey 16 225 Warren Street, Newark, NJ 07103-3535 17 [email protected] 18 3) Harvey Mudd College 19 Department of Biology 20 1250 N. Dartmouth Ave. 21 Claremont, CA 91711 22 [email protected] 23 bioRxiv preprint doi: https://doi.org/10.1101/849042; this version posted November 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 LIST OF ACRONYMS AND ABBREVIATIONS 25 ORF – Open reading frame 26 RIT – RNA-Interference Targeted 27 GOF – Gain-of-Function 28 RFU – Relative fluorescence units 29 DOX – doxycycline 30 SM – Single marker 31 LP – Landing pad 32 NGS – Next generation sequencing 33 GSH1 – g-glutamylcysteine synthetase 34 TR – Trypanothione reductase 35 T(SH)2 – Trypanothione 36 GSH2 – glutathione synthetase 37 ODC – Ornithine decarboxylase 38 TXN – Tryparedoxin 39 RR – Ribonucleotide reductase 40 41 42 43 44 45 46 bioRxiv preprint doi: https://doi.org/10.1101/849042; this version posted November 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 ABSTRACT 48 Trypanosoma brucei is an early branching protozoan that causes Human and Animal 49 African Trypanosomiasis. Forward genetics approaches are powerful tools for 50 uncovering novel aspects of Trypanosomatid biology, pathogenesis, and therapeutic 51 approaches against trypanosomiasis. Here we have generated a T. brucei ORFeome 52 consisting of over 90% of the targeted genome and used it to make an inducible Gain- 53 of-Function library for broadly applicable forward genetic screening. Using a critical drug 54 of last resort, melarsoprol, we conducted a proof of principle genetic screen. Hits arising 55 from this screen support the significance of trypanothione, a key player in redox 56 metabolism, as a target of melarsoprol and implicate novel proteins of the flagellum and 57 mitochondria in drug resistance. This study has produced two powerful new genetic 58 tools for kinetoplastida research, which are expected to promote major advances in 59 kinetoplastida biology and therapeutic development in the years to come. 60 61 62 63 64 65 66 67 68 69 bioRxiv preprint doi: https://doi.org/10.1101/849042; this version posted November 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 INTRODUCTION 71 Trypanosomatids are a major parasitic lineage that include the African 72 trypanosomes, American trypanosomes, and Leishmania spp. (family 73 Trypanosomatidae, order Kinetoplastida), which collectively cause death and disease in 74 millions of people living in tropical and sub-tropical regions (1). There are no vaccines 75 against this family of parasites and the limited number of anti-Trypanosomatid drugs 76 present ongoing challenges of host toxicity, complex treatment regimens, and 77 burgeoning drug resistance (2). These evolutionarily ancient eukaryotes have highly 78 divergent genomes from well-established model organisms with more than 50% of open 79 reading frames (ORFs) annotated as hypothetical (3). Trypanosomatid parasites appear 80 to have diverged from a shared ancestor around 100 million years ago, yet have 81 retained a high proportion of orthologous gene clusters and chromosomal synteny. Of 82 the 9,068 genes in the Trypanosoma brucei (African trypanosome) genome, 6158 are 83 orthologous with both Trypanosoma cruzi (American trypanosome) and Leishmania 84 major (3). Many of the nonsyntenic regions of the genome appear to be directly 85 associated with organism specific aspects of parasitism, such as surface antigen 86 variation, leaving many biological functions highly conserved among kinetoplastida 87 parasites (3). Trypanosomatids share essential subcellular structures, such as the 88 flagellum, glycosomes (specialized organelles of glycolysis), and the kinetoplast (a 89 network of mitochondrial DNA). While reverse genetics based on well-established 90 models can promote discrete advances, forward genetics approaches have the potential 91 to uncover important aspects of kinetoplastida biology shared among orthologous 92 genes. bioRxiv preprint doi: https://doi.org/10.1101/849042; this version posted November 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 T. brucei, the causative agent of Human African trypanosomiasis (HAT), has 94 traditionally been the most genetically tractable of the Trypanosomatid parasites. 95 Discoveries made in T. brucei have provided critical understanding to the biology of 96 other kinetoplastida as well as providing insights into eukaryotic evolution (4-7) and 97 uncovered broadly significant biological processes, such as GPI-anchors and 98 mitochondrial RNA-editing (8-10). For the past decade a whole-genome interfering-RNA 99 (RNAi) knock-down library has been the primary forward-genetics tool in T. brucei 100 resulting in identification of essential genes and genes associated with drug resistance 101 and pathogenesis, to name a few (11-13). A strength of the RNAi library and associated 102 RNA-Interference Targeted (RIT)-seq approaches is the identification of genes that 103 result in a loss-of-fitness phenotype (14). However, knockdown strategies may fail when 104 screening for a cellular event that requires a particular gene to be turned on rather than 105 turned off. Furthermore, they preclude identification of essential genes associated with 106 key processes (14). A Gain-of-Function library approach may be more effective in the 107 identification of genes whose expression is critical to the initiation of the event. For 108 example, Gain-of-Function screens have been employed to identify drug targets, as well 109 as resistance mechanisms (15-17), and in areas of basic biology have been critical for 110 discoveries in the areas of chromosome segregation and cell cycle, signal transduction, 111 transcriptional regulation, cell polarity, and stem cell biology (18). Thus, Gain-of- 112 Function screens are a powerful tool that can be used as a complement to loss-of- 113 function screening to uncover new biology in diverse areas. 114 Traditional methods of inducible gene library formation by cDNA synthesis and 115 cloning are not viable for T. brucei as most gene expression regulation in kinetoplastida bioRxiv preprint doi: https://doi.org/10.1101/849042; this version posted November 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 116 occurs post-transcriptionally, with 5’ and 3’UTRs playing a major role in determining 117 steady state levels of their associated transcripts (19). Existing overexpression libraries 118 for T. brucei, generated by whole-genome fragmentation methods have demonstrated 119 the usefulness of this approach, for example, in the identification of the benzoxaborole 120 drug target (polyadenylation specificity factor, CPSF3), despite their somewhat limited 121 ability to control individual ORF expression (20-23). In addition, generation of an 122 ORFeome provides a tool that can be applied to the downstream generation of multiple 123 whole-genome methodologies including yeast 2-hybrid libraries, tagging libraries, 124 inducible expression libraries for Gain-of-Function studies, and complementation for 125 loss-of-function mutant phenotypes (24-27). A T. brucei ORFeome would permit 126 production of an inducible Gain-of-Function library, which is expected to be an ideal 127 design for the identification of novel gene functions based on observable phenotypes in 128 large scale screens. 129 African trypanosomiasis is transmitted by the tsetse vector and characterized by 130 a stage one bloodstream infection (though other tissues are implicated (28)) and a 131 stage two central nervous systems (CNS) infection, which is invariably fatal if left 132 untreated. HAT is predominantly caused by T. b. gambiense, whose stage two 133 infections can be treated by NECT (nifurtimox/eflornithine combination therapy) (2) and 134 fexinidazole (29). To a lesser extent HAT is caused by T. b. rhodesiense, which rapidly 135 progresses to stage two infection and host death. Melarsoprol, an arsenical compound, 136 remains the only drug available to treat stage two T. b. rhodesiense infections despite 137 treatment challenges, high levels of host toxicity, and increasing reports of drug 138 resistance (30). Following melarsoprol

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