Abstract
The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.
Highlights
The eukaryotic flagellum is recognized as a major signaling center that acts as a cellular antenna to sense and transduce extracellular signals [1,2,3,4]
Purification of Intact, Membrane-enclosed Flagella from T. brucei in its Mammalian-infectious Form—The T. brucei flagellum is laterally connected to the cell body along most of its length by filamentous connectors and tightly apposed membrane-membrane contacts [40]
To determine if the same bias applied to flagellum surface and matrix proteins, we examined the phylogenetic distribution of “conserved” proteins in each data set, i.e. proteins that were encoded in the genome of at least one non-kinetoplastid organism (Fig. 9)
Summary
Lular adaptations that define the bloodstream-form life cycle stage, including changes in metabolism, morphology, and surface protein composition [23]. The flagellum emerges from the cytoplasm at the cell posterior and is laterally connected to the cell body by cytoskeletal filaments that connect the axoneme and PFR to subpellicular microtubules in the cell body and maintain tight apposition of the flagellar and cell surface membranes [39, 40]. These connections form a “flagellum attachment zone” (FAZ) that runs along most of the length of the flagellum, with a small distal portion of the flagellum extending free of the cell body. Our combined studies indicate that the trypanosome flagellum presents a diverse and dynamic signaling platform adapted for host-pathogen interaction
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