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

Identification of Positive Chemotaxis in the Protozoan Pathogen Trypanosoma brucei.

To complete its infectious cycle, the protozoan parasite Trypanosoma brucei must navigate through diverse tissue environments in both its tsetse fly and mammalian hosts. This is hypothesized to be driven by yet unidentified chemotactic cues. Prior work has shown that parasites engaging in social motility in vitro alter their trajectory to avoid other groups of parasites, an example of negative chemotaxis. However, movement of T. brucei toward a stimulus, positive chemotaxis, has so far not been reported. Here, we show that upon encountering Escherichia coli, socially behaving T. brucei parasites exhibit positive chemotaxis, redirecting group movement toward the neighboring bacterial colony. This response occurs at a distance from the bacteria and involves active changes in parasite motility. By developing a quantitative chemotaxis assay, we show that the attractant is a soluble, diffusible signal dependent on actively growing E. coli Time-lapse and live video microscopy revealed that T. brucei chemotaxis involves changes in both group and single cell motility. Groups of parasites change direction of group movement and accelerate as they approach the source of attractant, and this correlates with increasingly constrained movement of individual cells within the group. Identification of positive chemotaxis in T. brucei opens new opportunities to study mechanisms of chemotaxis in these medically and economically important pathogens. This will lead to deeper insights into how these parasites interact with and navigate through their host environments.IMPORTANCE Almost all living things need to be able to move, whether it is toward desirable environments or away from danger. For vector-borne parasites, successful transmission and infection require that these organisms be able to sense where they are and use signals from their environment to direct where they go next, a process known as chemotaxis. Here, we show that Trypanosoma brucei, the deadly protozoan parasite that causes African sleeping sickness, can sense and move toward an attractive cue. To our knowledge, this is the first report of positive chemotaxis in these organisms. In addition to describing a new behavior in T. brucei, our findings enable future studies of how chemotaxis works in these pathogens, which will lead to deeper understanding of how they move through their hosts and may lead to new therapeutic or transmission-blocking strategies

Identification of Positive Chemotaxis in the Protozoan Pathogen Trypanosoma brucei.

To complete its infectious cycle, the protozoan parasite Trypanosoma brucei must navigate through diverse tissue environments in both its tsetse fly and mammalian hosts. This is hypothesized to be driven by yet unidentified chemotactic cues. Prior work has shown that parasites engaging in social motility in vitro alter their trajectory to avoid other groups of parasites, an example of negative chemotaxis. However, movement of T. brucei toward a stimulus, positive chemotaxis, has so far not been reported. Here, we show that upon encountering Escherichia coli, socially behaving T. brucei parasites exhibit positive chemotaxis, redirecting group movement toward the neighboring bacterial colony. This response occurs at a distance from the bacteria and involves active changes in parasite motility. By developing a quantitative chemotaxis assay, we show that the attractant is a soluble, diffusible signal dependent on actively growing E. coli Time-lapse and live video microscopy revealed that T. brucei chemotaxis involves changes in both group and single cell motility. Groups of parasites change direction of group movement and accelerate as they approach the source of attractant, and this correlates with increasingly constrained movement of individual cells within the group. Identification of positive chemotaxis in T. brucei opens new opportunities to study mechanisms of chemotaxis in these medically and economically important pathogens. This will lead to deeper insights into how these parasites interact with and navigate through their host environments.IMPORTANCE Almost all living things need to be able to move, whether it is toward desirable environments or away from danger. For vector-borne parasites, successful transmission and infection require that these organisms be able to sense where they are and use signals from their environment to direct where they go next, a process known as chemotaxis. Here, we show that Trypanosoma brucei, the deadly protozoan parasite that causes African sleeping sickness, can sense and move toward an attractive cue. To our knowledge, this is the first report of positive chemotaxis in these organisms. In addition to describing a new behavior in T. brucei, our findings enable future studies of how chemotaxis works in these pathogens, which will lead to deeper understanding of how they move through their hosts and may lead to new therapeutic or transmission-blocking strategies

Cell Surface Proteomics Provides Insight into Stage-Specific Remodeling of the Host-Parasite Interface in 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. Here, we use surface labeling, affinity purification, and proteomic analyses to describe cell surface proteomes from insect-stage and mammalian bloodstream-stage Trypanosoma brucei. The cell surface proteomes contain most previously characterized surface proteins. We additionally identify a substantial number of novel proteins, whose functions are unknown, indicating the parasite surface proteome is larger and more diverse than generally appreciated. We also show stage-specific expression for individual paralogs within several protein families, suggesting that fine-tuned remodeling of the parasite surface allows adaptation to diverse host environments, while still fulfilling universally essential cellular needs. Our surface proteome analyses complement existing transcriptomic, proteomic, and in silico analyses by highlighting proteins that are surface-exposed and thereby provide a major step forward in defining the host-parasite interface

Regulation of social motility.

<p><b>(A)</b> Three alternate models for regulation of social motility. (Left) Elevated cAMP at the flagellum tip (red) in response to regulation of tip-localized adenylate cyclase and the transition from early to late procyclics independently inhibit SoMo. (Middle) Elevated cAMP at the flagellum tip triggers the transition from early to late procyclics, which then inhibits SoMo. (Right) Development into late procyclics triggers elevated cAMP at the flagellum tip, which is then the inhibitory signal. <b>(B)</b> In addition to the known cAMP signaling systems that control SoMo (red), the trypanosome flagellum harbors several predicted signaling systems, e.g., ion transporters, kinases, additional ACs, and other receptor-like proteins [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005272#ppat.1005272.ref050" target="_blank">50</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005272#ppat.1005272.ref051" target="_blank">51</a>] whose functions await discovery.</p

Trypanosomes are social.

<p>Trypanosome cell–cell interactions operate in bloodstream and insect stage parasites. In the bloodstream, “long slender form” parasites (red) differentiate into growth-arrested “short stumpy forms” (purple) through a quorum sensing-mediated mechanism. Stumpy parasites are pre-adapted for the tsetse fly environment and the transition thus establishes transmission competence, while also limiting bloodstream parasitemia [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005272#ppat.1005272.ref052" target="_blank">52</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005272#ppat.1005272.ref053" target="_blank">53</a>]. Procyclic <i>T</i>. <i>brucei</i> (insect midgut stage, blue) undergo social motility when cultivated on semi-solid surfaces, using cell–cell signaling to promote collective motility across the surface and coordinating their movements in response to extracellular signals from nearby parasites. This leads to formation of radial projections that extend outward from the initial site of inoculation [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005272#ppat.1005272.ref008" target="_blank">8</a>]. These activities are hypothesized to support colonization and/or transit of tissue surfaces in the fly. Beyond their direct impact on understanding parasite development, recent studies of stumpy formation and social motility have provided insight into parasite signal transduction. See text for details.</p

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with <i>S. pyogenes</i> (Spy), <i>S. aureus</i> (Sau), and <i>C. jejuni</i> (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei.

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei.

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms