Periodic Reporting for period 1 - scTRYPseq (Understanding the initiation virulence gene expression in African trypanosomes) Reporting period: 2019-04-01 to 2021-03-31 Summary of the context and overall objectives of the project African trypanosomes are parasitic protists which cause endemic disease in sub-Saharan Africa. Both humans and animals can be infected, leading to human or animal African trypanosomiasis (HAT or AAT). These parasites caused several epidemics over the 20th century, in addition to ongoing damage to agriculture on the African continent. Trypanosomes do not invade host cells, and are therefore directly exposed to the mammalian immune system. The evade both innate and adaptive immune responses the parasite has developed a system of antigenic variation, where the surface of the cell is covered in 11 million molecules of a tightly packed coat of variant surface glycoproteins (VSGs). Each cell expresses only one variant surface glycoprotein at a time, and this is periodically switched to evade new antibodies. The process of singular gene expression is termed monoallelic expression and this has two components, establishment and maintenance, i.e. how a single gene is selected for expression and how its singular expression is maintained throughout successive generations. The establishment of monoallelic VSG gene expression occurs in the salivary gland of the tsetse fly vector. This ensures that the parasite is prepared to infect the next host, as the VSG coat is then prepared to immediately protect the cell. The underlying dynamics of this process are not well understood. We want to deepen our understanding of this process. Previously, questions such as this were experimentally challenging, as parasites in the salivary gland are mixed populations, limited in number and growth arrested. Recent developments in single cell gene expression profiling have opened new avenues to access difficult questions such as this. Our project aims to use inDrop, a microfluidic droplet based technology to profile the transcriptomes of thousands of single cells as the parasites undergo development in the salivary gland of the fly. Furthering our understanding of the control of singular VSG gene expression is important in the context of HAT and AAT as this is a key virulence mechanism, without which the parasite cannot establish a new infection. In addition, singular antigen gene expression is a strategy used by other infectious organisms such as malaria and giardia parasites, and this work could be informative in the context of these parasites. More broadly, monoallelic gene expression is a broadly utilized strategy in biological systems, for instance in mammalian olfactory receptors which give us our sense of smell. Further understanding of these processes in a variety of organisms could therefore be informative. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Specifically, the main goals of this project were to:1) Establish an inDrop methodology for use with African trypanosomes.2) Apply inDrop to salivary gland derived parasites During this action, we have met both of the main goals (1 and 2) of the project. Goal three was not experimentally addressed as part of this action, however we were able to use publicly available genomic sequencing data to address the underlying aim of this goal.Goal 1: We initially established a methodology for inDrop using parasites from culture flasks. We took this initial step as these parasites are readily accessible and we are not limited in the numbers we could obtain. We generated single-cell transcriptomes for mixtures of two developmental stages of the trypanosome parasite cycle. This stage allowed us not only to establish experimental procedures for the encapsulation and barcoding of single trypanosomes, but also for the computational analysis of the resulting data. These experiments demonstrated that 1) we were able to perform inDrop with African trypanosomes and 2) we were able to informatically devonvolve different developmental states. This was an important precursor before transitioning to our second goal, where we needed to recover parasites from infected tsetse flies.Goal 2: We next used our methods for trypanosome inDrop to generate transcriptomes of parasites differentiating in the salivary glands of tsetse flies. This revealed several populations of cells corresponding to previously known trypanosome developmental biology in this organ. Clusters specifically corresponded to attached epimastigote, gamete and meiotic cells, pre-metacyclic and metacyclic populations. We interrogated the specific gene expression profiles of these clusters, finding that, for instance, metabolic remodelling of parasites from the production of ATP in the mitochondrion using the electron transport chain to glycolysis during this development had its inflection point following the asymmetric division of attached epimastigotes. Within these clusters we were able to delineate how trypanosomes switch on and establish monoallelic VSG gene expression. This occurs following differentiation to pre-metacyclic cells. Our data indicated that attached epimastigotes did not express VSG genes, as expected. Following differentiation to pre-metacyclics, the cells initiate expression of VSGs. We found that rather than initiate expression of a single VSG gene, pre-metacyclic cells initiate expression at multiple VSG loci, which then resolves into monoallelic gene expression in metacyclic cells. Finally we were able to use single molecule RNA-FISH in an in vitro model of trypanosome differentiation to confirm these findings using an orthogonal technique.These data have been compiled into a manuscript which is currently available on biorxiv (https://www.biorxiv.org/content/10.1101/2021.03.01.433049v1) Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Prior to this project, our understanding of the establishment of monoallelic VSG gene expression was limited. Our data provide the first insight into the dynamics of this process at a transcriptome wide level. Our analysis establishes the landscape which underpins the regulation of monoallelic gene expression in trypanosomes. This study therefore forms a foundation for further, functional studies into the factors which govern monoallelic VSG gene expression. These data not only inform us about the developmental progression of trypanosomes in the tsetse fly, but also on the regulation of an important process in the acquisition of virulence of this important parasite. Further, several other parasites such as malaria and giardia both utilise monoallelic expression of surface antigens to evade the host, which this study provide useful clues for further study. Establishment of monoallelic VSG expression at single cell resolution.
