Work in the lab is focussed on the African trypanosome and its interaction with the host environment. 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, taking 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 chemotherapy (See WHO website and the blog post, ‘Why study African trypanosomes‘). The closely related but non-human infective T. b. brucei, T. congolense and T. vivax cause a disease in cattle known as N’agana. This 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 fly (Glossina spp), in which they undergo a complex developmental cycle during migration from the midgut to the salivary glands 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; in the case of the human infective forms, resistance to human serum trypanolytic factors that lyse the other trypanosomes, such as 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.
Work in the lab aims to develop our understanding of the parasite’s interaction with the host environment, in particular the uptake and intracellular transit of host- and parasite-derived molecules, and the consequences of such encounters for the parasite. We hope to use this knowledge to inform the development of novel interventions against HAT.
Principal areas of investigation:
1. Identifying the determinants of human and baboon serum sensitivity in T. b. brucei – what parasite-intrinsic factors define susceptibility to serum trypanolytic factors?
2. Nutrient uptake processes – how do they intersect with drug efficacy determinants and influence drug action?
3. Defining the efficacy determinants of the anti-leishmanials – identifying candidate efficacy determinants in T. brucei for follow-up in Leishmania, a related but less genetically tractable protozoan parasite.
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 Res, 2011 | PDF).
It is also possible to use the RNAi library to identify networks of proteins that determine the efficacy of a selective agent, such as a drug or other factor; this has previously been done for the five anti-HAT drugs currently in use (Alsford et al, Nature, 2012 | PDF). Suramin provides a great example of the level of detail that can be achieved: more than 50 proteins were shown to contribute to the efficacy of this drug, a subset of which are detailed in the figure below.
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 phenotype 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).