Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Tschudi, Christian
Abstract
Trypanosoma brucei is a protozoan parasite responsible for causing vector-borne diseases in vertebrate animals. The parasite is transmitted by the tsetse fly in sub-Saharan Africa. According to the Food and Agriculture Organization (FAO) of the United Nations, T. brucei significantly impacts Africa’s agriculture-based economy, causing approximately 3 million deaths in cattle per year estimated to cost up to $1.2 billion USD. The effects of trypanosomiasis on the economy are far-reaching as this reduces the supply of milk, meat, and draught power, and decreases soil fertility and employment income (Abro et al., 2021). Thus, there is a pressing need for research to better understand the biology of these pathogens and the mechanisms they use to survive within their hosts. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector (Diptera: Glossinidae), which among others involves changes in cell morphology, metabolism, signaling pathways, and gene expression. Consequently, these parasites have evolved adaptations to enable survival in both the gut and salivary glands of the tsetse fly, as well as in the bloodstream of their mammalian hosts. Upon feeding on an infected host, the tsetse fly ingests the slender, intermediate and stumpy bloodstream forms of the parasite. In the fly midgut, stumpy forms differentiate into non-infectious procyclic forms. Reacquisition of infectivity is achieved through metacyclogenesis, a complex developmental program culminating in the tsetse salivary glands with the generation of infectious metacyclics. Although the intricate nature of trypanosome development in the fly has been recognized for more than a century, research in this field has historically been limited due the inability to culture trypanosomes that undergo metacyclic development and metacyclic parasites could only be harvested through dissecting the tsetse fly. However, by overexpressing a single RNA-binding protein (RBP6) in non-infectious trypanosomes, the Tschudi laboratory recapitulated in vitro the events leading to acquisition of infectivity in the insect vector, allowing for previously inaccessible large-scale biochemical and genetic experiments. In this thesis, I screened for genes essential for metacyclogenesis and characterized a subset of the identified genes for their role in metacyclic development. To identify genes involved in metacyclogenesis, I performed an RNAi screen by individually targeting 86 transcripts by RNAi in the RBP6 overexpression cell line. I identified 22 genes that positively or negatively regulated the stepwise progression towards infectivity at different stages of development. Of the 22 identified genes, cold shock domain containing protein 1 (CSD1), CSD2, and zinc finger protein 45 (ZC3H45) were found not only to cause the most severe loss-of-function phenotypes on metacyclic production, but also induced rapid metacyclic production when overexpressed in procyclic parasites. Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP-seq) analysis of CSD1 and CSD2 indicated that these proteins target the same binding site “ANACAT”, bind primarily to the coding region of transcripts, and likely target identical transcripts. I also examined the effects that CSD1, CSD2, and ZC3H45 exerted on metacyclic production in relation to RBP10, whose overexpression resulted in rapid metacyclic production. I found that among these four regulators, CSD1 appeared to be located the furthest downstream in terms of its contribution to the process of parasite differentiation into the metacyclic form. Our studies on metacyclogenesis in this thesis expanded the network of known genes that contribute to metacyclic production and led to the discovery that CSD1, CSD2, and ZC3H45 are each sufficient in triggering rapid metacyclogenesis in vitro.
Recommended Citation
Toh, Justin, "Identification and characterization of regulators in the developmental process leading to infectious Trypanosoma brucei" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1033.
https://elischolar.library.yale.edu/gsas_dissertations/1033
