Understanding the evolution of the membrane trafficking system in diverse eukaryotes through comparative genomics and transcriptomics by Emily Katherine Herman A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cell Biology University of Alberta © Emily Katherine Herman, 2018 Abstract Single-celled organisms represent the majority of eukaryotic diversity. Recent advances in sequencing technologies have been critical for understanding the evolutionary biology and cell biology of microbial eukaryotes. Comparative genomic analyses have shown that many genes that underlie fundamental eukaryotic features (e.g. membrane trafficking, cytoskeleton) are conserved across the diversity of eukaryotes, suggesting that they have also maintained a similar function. However, many microbial eukaryotes have specialized lifestyles or behaviours, the evolutionary pressures of which may be observed in changes to gene content in a lineage; in either gene family expansion, divergence, or loss. Building on analyses of gene presence and absence, gene expression changes in relation to a specific cellular behaviour gives even more insight into the underlying cell biology of that process. The focus of this thesis is the membrane trafficking system, specifically the cellular machinery that underlies intracellular transport, endocytosis, and exocytosis. In the first Results chapter, comparative genomics is used to identify membrane trafficking components in three related organisms, one of which is free-living, while the other two are gut-associated endobionts and/or parasites. The purpose was to determine whether host-association contributes to sculpting of the trafficking system, as is the case in other eukaryotic parasites. In the second Results chapter, comparative genomics and transcriptomics are used to study how membrane trafficking underlies the process of encystation in the gut pathogens Entamoeba invadens and E. histolytica, which is critical for pathogenesis. The third results chapter looks at the biology of a unique behaviour in the haptophyte lineage: the secretion of large organic or calcium carbonate scales. Again, both comparative genomics and transcriptomics are used to understand how the membrane trafficking system contributes to this extensive secretory process. ii The last Results chapter takes a whole-genome approach to understanding pathogenesis in the free-living neuropathogenic amoeba Naegleria fowleri, as compared with its harmless relative, Naegleria gruberi. The purpose of comparative genomics and transcriptomics of N. fowleri and N. gruberi was primarily to identify pathogenicity factors. Although no single factor was identified that fully explains the difference in pathogenicity between the two Naegleria spp., two major outcomes were achieved. First, this analysis has generated a comprehensive look at the cell biology of N. fowleri during host infection. Secondly, it has produced a list of dozens of potential pathogenicity factors that can now be experimentally tested. An example of how in silico analyses can support functional work concludes this chapter, where evidence of a Golgi body in N. gruberi is shown for the first time. These –omics analyses have contributed significantly to understanding the biology of these lineages. They highlight patterns of retention, loss, and expansion of membrane trafficking machinery that may be related to unusual trafficking pathways or even novel organelles. They also allowed for comparisons of gene complement and expression between different lineages that have similar lifestyles, for example gut-associated parasites or endobionts (Entamoeba spp. and Blastocystis sp., Proteromonas lacertae), or organisms with a heavy secretory load (haptophytes and Entamoeba sp.). Common to all three transcriptomic analyses is the finding that transcriptional responses are complex, often involving differential regulation of paralogous genes. The data presented here have paved the way for future functional work in microbial eukaryotes, improving our depth of knowledge of membrane trafficking function in eukaryotes, and allowing us to fully appreciate unique cell biology in ecologically and medically relevant organisms. iii Preface (Mandatory due to collaborative work) The works presented in this thesis are the products of several research collaborations. Comparative genomics of the membrane trafficking system of three Blastocystis strains, found in Chapter 3 of this thesis, is the result of a collaboration with Andrew Roger and his lab at Dalhousie University. It has been published in Gentekaki E, Curtis BA, Stairs CW, Klimeš V, Eliáš M, et al. (2017) Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis. PLOS Biology 15(9): e2003769. E. Gentekaki, B. Curtis, C. G. Clark, and A. Roger were responsible for conceptualization and administration of the Blastocystis sp. genome project. The membrane trafficking system complement was analysed and described for the genome paper by Emily Herman and Alexander Schlacht, under the supervision of Joel Dacks. Other authors were responsible for other aspects of the genome project. The Proteromonas lacertae and Cafeteria roenbergensis data presented in Chapter 3 are the result of an ongoing collaboration with Andrew Jackson and his lab at the University of Liverpool. E. Herman performed all comparative genomic analyses of membrane trafficking and autophagy machinery in the genomes of P. lacertae and C. roenbergensis. The work performed in Chapter 4 is the result of a collaboration with Mark van der Giezen and his lab at the University of Exeter. It has been published as Herman E, Siegesmund MA, Bottery MJ, van Aerle R, Shather MM, Caler E, Dacks JB, and van der Giezen M. (2017). Membrane trafficking modulation during Entamoeba encystation. Scientific Reports 7:12854. M. van der Giezen and J. Dacks designed and supervised experiments; Maria Siegesmund and Mohammed Shather performed laboratory experiments; E. Herman, M. Siegesmund, Michael Bottery, Ronny van Aerle, and Elisabet Caler contributed to data analysis; E. Herman, M. Siegesmund, and M. Bottery organized, designed, and wrote the paper; and E. Herman, J. Dacks, and M. van der Giezen critically revised the manuscript. Specifically, E. Herman analysed the membrane trafficking system complement in E. invadens and E. histolytica, interpreted gene expression patterns for the trafficking complement, and helped write and revise the manuscript. iv Analysis of the membrane trafficking system of the haptophytes in Chapter 5 is part of a long-standing collaboration with Betsy Read and her lab at California State University San Marcos. The original analysis of the membrane trafficking machinery in Emiliania huxleyi was published as part of a genome project: Read BA, Kegel J, Klute MJ, Kuo A, Lefebvre SC, Maumus F, Mayer C, Miller J, Monier A, Salamov A, Young J, Aguilar M, Claverie JM, Frickenhaus S, Gonzalez K, Herman EK, Lin YC, Napier J, Ogata H, Sarno AF, Shmutz J, Schroeder D, de Vargas C, Verret F, von Dassow P, Valentin K, Van de Peer Y, Wheeler G; Emiliania huxleyi Annotation Consortium, Dacks JB, Delwiche CF, Dyhrman ST, Glöckner G, John U, Richards T, Worden AZ, Zhang X, Grigoriev IV. (2013) Pan genome of the phytoplankton Emiliania underpins its global distribution. Nature 499:209-213. B. Read coordinated the genome project and I. Grigoriev coordinated genome sequencing and analysis at the US DOE Joint Genome Institute. E. Herman and Mary Klute analysed the membrane trafficking system machinery and described the results of this analysis for the genome paper, under the supervision of J. Dacks. Other authors were responsible for other aspects of the genome project. Part of the comparative work in Chapter 5 with the haptophytes Gephyrocapsa oceanica and Isochrysis galbana specifically regarding adaptor protein evolution is published as Lee LJY, Klute MK, Herman EK, Read B, and Dacks JB. (2015) Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae. Protist 166:585-597. L. Lee and M. Klute performed comparative genomic analyses, B. Read generated sequence data and performed qPCR experiments. J. Dacks and B. Read supervised the project. E. Herman supervised Laura Lee and aided in data analysis and interpretation. As the corresponding author, E. Herman was heavily involved in manuscript writing and editing along with J. Dacks and B. Read, and corresponded with the journal during the manuscript review and publication process. Additional analysis of the membrane trafficking machinery of the haptophytes (including Chrysochromulina tobin), and gene expression under biomineralizing conditions, is part of an ongoing collaboration with B. Read and Xiaoyu Zhang. B. Read and X. Zhang produced genomic and transcriptomic data associated with E. huxleyi, G. oceanica and I. galbana. Analysis of membrane trafficking complement was performed by L. Lee and E. Herman, and analysis of biomineralization transcriptomic data specific to the membrane trafficking system v was performed by E. Herman. Elisabeth Richardson and E. Herman performed a comparative genomic analysis of membrane trafficking machinery in the publicly available genome of C. tobin. The data presented in Chapter 6 is the result of several collaborations. The N. fowleri V212 genome sequence and transcriptomic analysis was the result of a collaboration between our lab, Charles Chiu and his lab at the University of California San Francisco, Francine