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Identification and characterization of novel flagellar cAMP signaling systems in the human-infectious parasite Trypanosoma brucei


African trypanosomes are devastating human and animal pathogens transmitted by tsetse flies between mammalian hosts. The trypanosome surface forms a critical host interface that is essential for sensing and adapting to diverse host environments. However, trypanosome surface protein composition and diversity remain largely unknown. In the following works, we identify the trypanosome cell and flagellar surface proteomes using surface labelling, affinity purification, and proteomic analyses of both insect-stage and mammalian bloodstream-stage Trypanosoma brucei. We identify a substantial number of novel proteins with unknown functionalities, indicating that the surface proteomes are larger and more diverse than previously anticipated. We demonstrate stage-specificity for a number of proteins, suggesting that the parasite surface undergoes fine-tuning in order to adapt to specific, but diverse, host environments.

Similar analyses were performed on the trypanosome flagellum, an essential and multifunctional organelle involved in motility, morphogenesis, and host-parasite interactions. Previous attempts to characterize flagellar composition were limited by the inability to purify intact flagellum. Using a combination of genetic and mechanical approaches in conjunction with surface labeling and affinity purification, we conducted independent analyses of the flagellum surface and matrix fractions. We identified a broad spectrum of proteins with predicted signaling functionalities, indicating that the flagellum is a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.

In procyclic, or insect-stage, parasites, we reported identification of several receptor-type adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei. Previously studied ACs were constitutively expressed or confined to bloodstream stage parasites. Using gene-specific tags, we find that ACs are glycosylated surface-exposed proteins that dimerize and possess catalytic activity. Notably, while some ACs were found to be distributed along the flagellum length, others specifically localized to the flagellum tip. Differential localization suggests that the membrane is organized into specific subdomains, suggesting a specific-role for cAMP signaling in procyclic-stage parasites.

Functional analyses of ACs were done in the context of socio-microbiological analyses. There are sophisticated systems for cell-cell communication that enable microbes to act as a group. In their native environment, T. brucei lives on host tissue surfaces, and in vitro cultivation on surfaces causes the parasites to actively assemble into densely packed communities, from which they coordinately migrate outwards in radial projections across the surface. This behavior is termed social motility (SoMo) due to analogies with bacterial systems. Functional analyses revealed that flagellar ACs cooperate with cAMP-specific phosphodiesterase to regulate trypanosomal social behaviors. This supports the hypothesis that ACs transduce extracellular signals and are involved in stage-specific signaling pathways. Experiments using cAMP analogues suggest that the phenotype is specific to cAMP, and not due to downstream metabolic byproducts. Notably, knockdown of only some ACs impacts social motility, indicating segregation of AC functions.

There are several possibilities for why only some ACs are involved in social motility. One of the most intriguing explanations has to do with the differential localization. Some ACs localize along the flagellum length, while others are specific to the tip. Such localization is novel, and this protein family is the first known example of transmembrane proteins in T. brucei, and one of the first in any system, to localize exclusively to the flagellar tip. Despite the importance of flagellar protein trafficking, flagellar targeting signals are virtually unknown. In these works, we investigate whether flagellar subdomain localization impacts AC functionality. Using protein truncations, chimeras, and point mutants, we identify an intracellular segment and specific residues required for flagellum and flagellum subdomain targeting. Strikingly, the social motility defect of a flagellum-tip AC mutant can be rescued by redirection of another AC from along the length to the flagellum tip. These results demonstrate the importance of protein targeting to specific subdomains within the flagellum, and implicates cAMP signaling at the flagellum tip as a key regulator of cell-cell communication required for social behavior.

These combined works identify and define the cell surface and flagellar proteomes, with in depth characterization and analysis of a group of novel cAMP signaling proteins. Through usage of the social motility assay, these works identify the first known regulators of trypanosomal social behavior, and the first direct evidence of cAMP functionalities in procyclic-stage parasites. Furthermore, signaling functionality is tied to differential localization of ACs to specific flagellar subdomains. Our works thus advance understanding of principles that govern protein targeting to flagellum subdomains and provides insight into T. brucei signaling mechanisms, both of which are poorly understood but fundamentally important features of flagellar and trypanosomal biology.

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