Sunday, December 8, 2024
12/8/2024
Project Summary/Abstract Trypanosoma brucei is a flagellated protozoan parasite responsible for sleeping sickness, a vector-borne disease that causes great human suffering and economic burden and is endemic to sub-Saharan Africa. In addition to its own medical importance, T. brucei represents a group of related human parasites. Because of easy genetic manipulation, T. brucei is also a valuable model organism for studying flagellum/cilium biology. To survive, be transmitted, and cause disease, T. brucei must sense and respond to environmental signals. This is especially important for a vector-transmitted parasite, because it must respond to multiple host environments. Sensing is achieved by signal transduction pathways that detect external changes and convert these into cellular responses. Little is known about these pathways and mechanisms in T. brucei, or other related pathogens – this is a critical knowledge gap and potential source of discovery of new treatments. Cumulative evidence shows the T. brucei flagellum is a critical platform for cAMP signaling that controls cell movement, cell-cell communication, chemotaxis, movement through tissues, transmission through the insect vector and pathogenesis in the mammalian host. It has also been demonstrated that the flagellum tip is an important domain for organizing cAMP signaling, and models hypothesize mechanisms for tight control over cAMP distribution, retaining cAMP near sites of generation and downstream effectors. Surprisingly, however, spatial arrangement of cAMP production and propagation has been directly examined. Moreover, almost nothing is known about effectors in the flagellum that transmit cAMP effects. Thus, fundamental questions regarding trypanosome cAMP signaling mechanisms remain unanswered. We are well-positioned to answer these questions by employing our state-of-the-art quantitative tools for phosphoproteomics of flagellar subdomains, functional signaling assays, and new tools for live imaging of cAMP fluctuations inside cells. Our specific aims are to (1) identify and functionally characterize cAMP-effectors and (2) test hypotheses for mechanisms of cAMP signal distribution and propagation through the flagellum. To identify proteins regulated by cAMP (1a), we established a platform to obtain the cAMP-dependent phosphoproteome and we will use this to find target proteins altered by cAMP. For functional analysis (1b), we will examine motility and signaling in mutants lacking the proteins or phosphorylation sites identified in part (1a). This aim will provide novel insights into the function of individual cAMP effectors as well as the broader role of phosphorylation as a mechanism of transducing cAMP signaling in trypanosomes. To determine distribution of cAMP, we will visualize cAMP fluctuations using a genetically encoded FRET sensor in live cells (2a). We will use this FRET-based system to study propagation of the cAMP signal in response to external stimuli and test tenets of the current flagellum microdomain cAMP signaling model (2b).
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