Role of Flagellar Motility in Trypanosoma brucei Pathogenesis
The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. Parasite motility is suspected to contribute to infection and disease pathogenesis in the mammalian host. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. In the first part of my dissertation we identified a viable bloodstream-form motility mutant in 427-derived T. brucei and by adapting published approaches we set up mouse infection models of African trypanosomiasis. To assess the impact of trypanosome motility on infection in mice we used these mutants in a mouse infection model and showed that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. This presents the first ever investigation of the influence of parasite motility on infection of the mammalian host. Mutant cells used were derived from the laboratory-adapted strain 427-BSSM that causes an acute infection that progress rapidly in mice. This quick disease progression limits any reliable assessment of the CNS penetration, which commonly takes more than 14 days. To allow direct investigation of the requirement of parasite motility in the central nervous system (CNS) invasion, we have generated motility mutants in T. brucei strains that cause chronic infection in mice. Identification of motility mutants in pleomorphic BSF T. brucei that causes chronic infection will now make it possible for the first time to test if parasite motility is required for CNS penetration.
Traditionally, assessment of T. brucei infection is based upon examining parasitemia in blood and limited use of histochemistry to determine parasite presence in chemically-treated tissues. We have developed an advanced live-cell imaging approach using fluorescent T. brucei that will facilitate detailed dynamic studies of infection. This system enabled visualization of T. brucei ex vivo in mouse tissues as well as in vivo in whole live zebrafish embryos. Further validation of mCherry parasites at a microscale level revealed trypanosomes at single-cell resolution in ex vivo mouse tissues and in blood vessels of live fish. Hence, these systems have the potential for uncovering novel features of host-parasite interactions that could lead to drug, vaccine and diagnostics development, all of which is expected to ameliorate patient management in sleeping sickness.
A major roadblock to the study of the flagellum is a lack of facile methods for systematic mutational analysis of flagellar genes. We recently established systems for structure-function analysis of proteins in T. brucei, which has emerged as an excellent model to study the eukaryotic flagellum. Several flagellar proteins have been identified but there is scant information on molecular mechanisms underlying these proteins functions, individually or collectively. For instance, key amino acids and domains required for these proteins are for the most part unknown. To exploit the flagellum as a drug target it is crucial that we deepen understanding of molecular mechanisms of flagellum protein function. To start bridging this gap, we applied our structure-function system to define amino acids required for IFT88 and trypanin protein function in flagellum assembly and motility. Our studies tested amino acids that correspond to IFT88 mutations observed in human patients with defective cilia and showed which of these mutations are loss of function mutations versus polymorphisms. We have also uncovered key domains essential for the assembly and function of the dynein regulatory protein trypanin.
Altogether, these investigations broadly contribute to understanding T. brucei pathogenesis mechanisms and expand our knowledge of flagellum motility functions in trypanosomes, which are directly applicable to other flagellated protozoan parasites. In humans, the flagellum, also called a cilium, is required for normal development and physiology and genetic changes in flagellar genes cause many human heritable diseases. Thus, our studies are also relevant to eukaryotic cell biology in connection to human health and disease.