Understanding Mechanisms of Motility in Trypanosoma brucei
Trypanosoma brucei are protozoan parasites that present a tremendous medical and economic burden on poverty-stricken areas of sub-Saharan Africa. These pathogens are estimated to affect nearly 60 million people and are considered one the world's most neglected diseases. A key aspect for parasite pathogenicity and transmission is the mechanism of motility. Recent advances have revealed that in addition to the ability to swim in incredibly viscous environments, T. brucei parasites are capable of forming multicellular communities upon surface exposure. Although this behavior is ubiquitous in microbiology, this is the first described pathogenic protozoa capable of coordinating motility for polarized movement as a multicellular group. Through both forward and reverse genetic screens, this study outlines efforts to define signaling pathways and genes required for this surface-induced behavior. In particular, cAMP has been implicated in this process and a biased screen was performed to identify cAMP effectors that are required for social motility.
At individual level, motility in T. brucei is achieved by a single flagellum that is attached along the length of the cell body. The T. brucei flagellum has a canonical 9 + 2 axoneme structure that is highly conserved in organisms with motile flagella. A previous phylogenetic study identified 50 genes as the Core components of Motile Flagella (CMF). This dissertation expanded the original study and compared 115 diverse eukaryotic genomes. CMF22 was identified as a broadly conserved gene that was present in almost all organisms with motile flagella but not in organisms that lack flagella or have immotile flagella. Biochemical fractionation, localization by epitope tagging and RNAi knockdown was used to characterize CMF22. These experiments have demonstrated that CMF22 is an axonemal protein required for propulsive motility. High-resolution cryo-electron tomography of CMF22 knockdown mutants revealed the requirement of CMF22 for assembly of the proximal lobe of the nexin-dynein regulatory complex (N-DRC). Finally, mutagenesis experiments of the putative IQ motif and AAA domain of CMF22 have elucidated a potential mechanism for CMF22 action in axonemal motility.
Altogether, this work reveals important findings of motility, both at the individual and multicellular level to enhance current understand of protozoan biology, social microbiology, and conserved mechanisms of flagellum motility.