Research

Cultured bloodstream form Trypanosoma bruceiWork in the lab is focussed on the African trypanosomes. This group of extracellular protozoan parasites includes Trypanosoma brucei gambiense and T. b. rhodesiense, which cause Human African Trypanosomaiasis (HAT or sleeping sickness) in West/Central and East Africa, respectively. Though the duration and progression of the two forms of the disease varies (West African HAT is regarded as chronic and can take years to progress to late stage CNS involvement, while East African HAT can reach this stage in a matter of weeks), both forms of HAT are typically fatal without treatment (See WHO website and the blog post, ‘Why study African trypanosomes‘). The related but non-human infective T. b. brucei, T. congolense and T. vivax cause a disease in livestock known as N’agana, which is widespread throughout sub-Saharan Africa, where it has a significant impact on food production. All the African trypanosomes are transmitted between mammalian hosts by the bite of the tsetse  (Glossina spp), in which they undergo a complex developmental cycle during migration from the midgut to the salivary glands or mouth parts via the proventriculus.

There are several virulence factors that promote infection in mammals, including: antigenic variation, which enables the parasite to persist in the bloodstream indefinitely, or until the host succumbs;  resistance to human serum trypanolytic factors by T. b. gambiense and T. b. rhodesiense (these lyse the animal trypanosomes, T. b. brucei, T. vivax and T. congolense); differentiation, which allows the trypanosome to limit its population growth, as well as to adapt its physiology in preparation for transmission to the tsetse fly vector; and, emergent drug resistance, which is affecting the efficacy of the limited set of difficult to administer toxic drugs currently available.

In addition to being pathogenic parasites, trypanosomes are extremely divergent eukaryotes. Classified as members of the Kinetoplastida, a group within the eukaryotic supergroup, the Excavata, trypanosomes possess many features that are strikingly different to the more widely studied Opisthokonta, such as yeast, C. elegans, Drosophila, Xenopus and mammals. Therefore, understanding their biology in relation to these model organisms can inform our understanding of the last eukaryotic ancestor, as well as the constraints and flexibility evidenced the different evolutionary paths taken in the development of particular features.

Work in the lab is focused on two areas…

Understanding the trypanosome’s interaction with its host environment, in particular the uptake and intracellular transit of host-derived molecules, such as drugs and innate immune factors, and the consequences of such encounters for the parasite. This has focused our attention on the parasite’s endo-lysosomal system, its contribution to toxin uptake and transit, and its functional regulation.

Understanding the regulatory cross-talk between the two competing demands for RNA polymerase-I in bloodstream-form trypanosomes: rRNA and VSG mRNA. Significant progress has been made in understanding the regulation and maintenance of monoallelic VSG expression, but comparatively little is known about rRNA transcriptional regulation. Following the discovery that only a subset of rDNA arrays are transcriptionally active in BSF T. brucei, we are now developing the molecular tools to enable us to decode the protein networks responsible for this transcriptional repression.

Principal areas of investigation:

1. Having identified 63 proteins that contribute to the anti-trypanosomal action of apoL1, including multiple ubiquitin modifiers and several membrane trafficking proteins, we are now exploring the contributions of these proteins to apoL1 sensitivity and to endo-lysosomal function.
2. Identifying anti-kinetoplastid drug efficacy determinants, i.e. what parasite proteins drive drug action, including transporters, activators and their associated regulatory networks. This chemico-genomics approach can lead to insights into protein function beyond roles in drug action.
3. Understanding the regulation of rRNA transcription and how this intersects with RNA polymerase-I transcription of the major surface antigen mRNA in bloodstream-form (VSG) and insect-stage (procyclins) T. brucei.

Applying forward genetics in T. brucei

We use a genome-scale RNAi library to identify previously unkown factors that contribute to these areas of trypanosome biology. This methodology has previously been used to identify the genes/transcripts required for growth in culture as bloodstream, procyclic or differentiating cells (Alsford et al, Genome Research, 2011).

The RNAi library can also be used to identify parasite protein networks that contribute to a trypanocidal agent’s efficacy. We initially applied this approach to the five anti-HAT drugs currently in use (Alsford et al, Nature, 2012) and more recently used human serum and apoL1 selection to identify 63 apoL1 sensitivity determinants (Alsford et al, PLoS Pathogens, 2014; Currier et al, PLoS Pathogens, 2018), leading to insights into apoL1 and TLF mode of action (see figure below).

Models of TLF1 (A) and apoL1-TLF2 (B) uptake and intracellular transit (from Currier et al, PLoS Pathogens e1006855)

Selective screens, such as the one described above, are only the first stage of the process, and not an end in themselves. The roles and contributions of the key proteins identified in each screen are validated using a variety of techniques available in the lab, depending on the character of the protein(s) under investigation. These include phenotypic analysis following specific stem-loop RNAi or gene deletion, tagged-protein localisation, and over-expression of wild type and mutant proteins, all of which provide insights into protein function. All these approaches are enabled by the suite of molecular tools that we have in the lab (these are freely available to the wider T. brucei research community on request)