FAP20 is required for flagellum assembly in Trypanosoma brucei.

Trypanosoma brucei is a human and animal pathogen that depends on flagellar motility for transmission and infection. The trypanosome flagellum is built around a canonical "9+2" axoneme, containing nine doublet microtubules (DMTs) surrounding two singlet microtubules. Each DMT contains a 13-protofilament A-tubule and a 10-protofilament B-tubule, connected to the A-tubule by a conserved, non-tubulin inner junction (IJ) filament made up of alternating PACRG and FAP20 subunits. Here we investigate FAP20 in procyclic form T. brucei. A FAP20-NeonGreen fusion protein localized to the axoneme as expected. Surprisingly, FAP20 knockdown led to a catastrophic failure in flagellum assembly and concomitant lethal cell division defect. This differs from other organisms, where FAP20 is required for normal flagellum motility, but generally dispensable for flagellum assembly and viability. Transmission electron microscopy demonstrates failed flagellum assembly in FAP20 mutants is associated with a range of DMT defects and defective assembly of the paraflagellar rod, a lineage-specific flagellum filament that attaches to DMT 4-7 in trypanosomes. Our studies reveal a lineage-specific requirement for FAP20 in trypanosomes, offering insight into adaptations for flagellum stability and motility in these parasites and highlighting pathogen versus host differences that might be considered for therapeutic intervention in trypanosome diseases.

Reconnecting to Internal Sensations and Experiences (RISE): An online, multi-session intervention improves interoceptive sensibility for military personnel.

OBJECTIVE: This preregistered randomized controlled trial tested the effects of a four-session, online interoceptive awareness intervention relative to an active comparator, matched for time and attention on interoception and suicidal ideation. METHOD: Participants (N = 195; 69% male; mean age = 37) were active duty service members (62%) and veterans (38%) who completed measures of interoceptive sensibility, interoceptive accuracy, and suicidal ideation at baseline. They were randomized to either the interoceptive awareness intervention, Reconnecting to Internal Sensations and Experiences (RISE), or the comparator, Healthy Habits. Participants completed the assessment battery again at posttest as well as a 1 and 3-month follow-up. RESULTS: RISE was rated as acceptable and demonstrated excellent feasibility per completion rates (85% completed all four modules). RISE improved the majority of interoceptive sensibility domains assessed (noticing body sensations, not worrying about sensations of pain or discomfort, emotional awareness, self-regulation, body listening, and body trust), and most of these gains remained at 1 and 3-month follow-ups. There were no differences between conditions on suicidal ideation, perhaps due to the low levels of ideation reported, or interoceptive accuracy. CONCLUSIONS: RISE is a disseminable, cost-effective, and transdiagnostic intervention that improves interoceptive sensibility up to 3 months.

FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.

Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.

Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells.

Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked "density scissors" connect with one another to form a "scissors stack network (SSN)" plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.

APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific Proteins in Trypanosoma brucei.

Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 "proximity proteome" having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCE Sleeping sickness is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need for approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei and has allowed identification of proximity proteomes for different flagellar subdomains that cannot be purified. This capacity opens the possibility to study the composition and function of other compartments. We expect this approach may be extended to other eukaryotic pathogens and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilium function is impaired.

Right place, right time: Environmental sensing and signal transduction directs cellular differentiation and motility in Trypanosoma brucei.

Trypanosoma brucei and other African trypanosomes are vector-borne parasites that cause substantial human suffering across sub-Saharan Africa. The T. brucei life cycle is punctuated by numerous developmental stages, each occurring in a specific environmental niche and characterized by a unique morphology, metabolism, surface protein coat, and gene expression profile. The environmental cues and signaling pathways that drive transitions between these stages remain incompletely understood. Recent studies have started to fill this gap in knowledge. Likewise, several new studies have expanded our understanding of parasite movement through specific tissues and the parasite's ability to alter movement in response to external cues. Life cycle stage differentiation and motility are intimately integrated phenomena, as parasites must be at the right place (i.e., within a specific environmental milieu) at the right time (i.e., when they are appropriately staged and preadapted for perceiving and responding to signals) in order to complete their life cycle. In this review, we highlight some of the recent work that has transformed our understanding of signaling events that control parasite differentiation and motility. Increased knowledge of T. brucei environmental sensing and signal transduction advances our understanding of parasite biology and may direct prospective chemotherapeutic and transmission blockade strategies that are critical to eradication efforts.

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.

Cryo electron tomography with volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei.

The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella and cilia. However, high-resolution 3D axoneme structures are unavailable for organisms among the Excavates, which include pathogens of medical and economic importance. Here we report cryo electron tomography structures of the 96-nm repeat from Trypanosoma brucei, a protozoan parasite in the Excavate lineage that causes African trypanosomiasis. We examined bloodstream and procyclic life cycle stages, and a knockdown lacking DRC11/CMF22 of the nexin dynein regulatory complex (NDRC). Sub-tomogram averaging yields a resolution of 21.8 Å for the 96-nm repeat. We discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins (MIPs). We establish that DRC11/CMF22 is required for the NDRC proximal lobe that binds the adjacent doublet microtubule. We propose that lineage-specific elaboration of axoneme structure in T. brucei reflects adaptations to support unique motility needs in diverse host environments.

Flagellar cAMP signaling controls trypanosome progression through host tissues.

The unicellular parasite Trypanosoma brucei is transmitted between mammals by tsetse flies. Following the discovery that flagellar phosphodiesterase PDEB1 is required for trypanosomes to move in response to signals in vitro (social motility), we investigated its role in tsetse flies. Here we show that PDEB1 knockout parasites exhibit subtle changes in movement, reminiscent of bacterial chemotaxis mutants. Infecting flies with the knockout, followed by live confocal microscopy of fluorescent parasites within dual-labelled insect tissues, shows that PDEB1 is important for traversal of the peritrophic matrix, which separates the midgut lumen from the ectoperitrophic space. Without PDEB1, parasites are trapped in the lumen and cannot progress through the cycle. This demonstrates that the peritrophic matrix is a barrier that must be actively overcome and that the parasite's flagellar cAMP signaling pathway facilitates this. Migration may depend on perception of chemotactic cues, which could stem from co-infecting parasites and/or the insect host.

Motility-based label-free detection of parasites in bodily fluids using holographic speckle analysis and deep learning.

Parasitic infections constitute a major global public health issue. Existing screening methods that are based on manual microscopic examination often struggle to provide sufficient volumetric throughput and sensitivity to facilitate early diagnosis. Here, we demonstrate a motility-based label-free computational imaging platform to rapidly detect motile parasites in optically dense bodily fluids by utilizing the locomotion of the parasites as a specific biomarker and endogenous contrast mechanism. Based on this principle, a cost-effective and mobile instrument, which rapidly screens ~3.2 mL of fluid sample in three dimensions, was built to automatically detect and count motile microorganisms using their holographic time-lapse speckle patterns. We demonstrate the capabilities of our platform by detecting trypanosomes, which are motile protozoan parasites, with various species that cause deadly diseases affecting millions of people worldwide. Using a holographic speckle analysis algorithm combined with deep learning-based classification, we demonstrate sensitive and label-free detection of trypanosomes within spiked whole blood and artificial cerebrospinal fluid (CSF) samples, achieving a limit of detection of ten trypanosomes per mL of whole blood (~five-fold better than the current state-of-the-art parasitological method) and three trypanosomes per mL of CSF. We further demonstrate that this platform can be applied to detect other motile parasites by imaging Trichomonas vaginalis, the causative agent of trichomoniasis, which affects 275 million people worldwide. With its cost-effective, portable design and rapid screening time, this unique platform has the potential to be applied for sensitive and timely diagnosis of neglected tropical diseases caused by motile parasites and other parasitic infections in resource-limited regions.

Parasite motility is critical for virulence of African trypanosomes.

African trypanosomes, Trypanosoma brucei spp., are lethal pathogens that cause substantial human suffering and limit economic development in some of the world's most impoverished regions. The name Trypanosoma ("auger cell") derives from the parasite's distinctive motility, which is driven by a single flagellum. However, despite decades of study, a requirement for trypanosome motility in mammalian host infection has not been established. LC1 is a conserved dynein subunit required for flagellar motility. Prior studies with a conditional RNAi-based LC1 mutant, RNAi-K/R, revealed that parasites with defective motility could infect mice. However, RNAi-K/R retained residual expression of wild-type LC1 and residual motility, thus precluding definitive interpretation. To overcome these limitations, here we generate constitutive mutants in which both LC1 alleles are replaced with mutant versions. These double knock-in mutants show reduced motility compared to RNAi-K/R and are viable in culture, but are unable to maintain bloodstream infection in mice. The virulence defect is independent of infection route but dependent on an intact host immune system. By comparing different mutants, we also reveal a critical dependence on the LC1 N-terminus for motility and virulence. Our findings demonstrate that trypanosome motility is critical for establishment and maintenance of bloodstream infection, implicating dynein-dependent flagellar motility as a potential drug target.

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.

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.

Cyclic AMP Regulates Social Behavior in African Trypanosomes.

UNLABELLED: The protozoan parasite Trypanosoma brucei engages in surface-induced social behavior, termed social motility, characterized by single cells assembling into multicellular groups that coordinate their movements in response to extracellular signals. Social motility requires sensing and responding to extracellular signals, but the underlying mechanisms are unknown. Here we report that T. brucei social motility depends on cyclic AMP (cAMP) signaling systems in the parasite's flagellum (synonymous with cilium). Pharmacological inhibition of cAMP-specific phosphodiesterase (PDE) completely blocks social motility without impacting the viability or motility of individual cells. Using a fluorescence resonance energy transfer (FRET)-based sensor to monitor cAMP dynamics in live cells, we demonstrate that this block in social motility correlates with an increase in intracellular cAMP levels. RNA interference (RNAi) knockdown of the flagellar PDEB1 phenocopies pharmacological PDE inhibition, demonstrating that PDEB1 is required for social motility. Using parasites expressing distinct fluorescent proteins to monitor individuals in a genetically heterogeneous community, we found that the social motility defect of PDEB1 knockdowns is complemented by wild-type parasites in trans. Therefore, PDEB1 knockdown cells are competent for social motility but appear to lack a necessary factor that can be provided by wild-type cells. The combined data demonstrate that the role of cyclic nucleotides in regulating microbial social behavior extends to African trypanosomes and provide an example of transcomplementation in parasitic protozoa. IMPORTANCE: In bacteria, studies of cell-cell communication and social behavior have profoundly influenced our understanding of microbial physiology, signaling, and pathogenesis. In contrast, mechanisms underlying social behavior in protozoan parasites are mostly unknown. Here we show that social behavior in the protozoan parasite Trypanosoma brucei is governed by cyclic-AMP signaling systems in the flagellum, with intriguing parallels to signaling systems that control bacterial social behavior. We also generated a T. brucei social behavior mutant and found that the mutant phenotype is complemented by wild-type cells grown in the same culture. Our findings open new avenues for dissecting social behavior and signaling in protozoan parasites and illustrate the capacity of these organisms to influence each other's behavior in mixed communities.

Insect stage-specific adenylate cyclases regulate social motility in African trypanosomes.

Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed "social motility," based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies.

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei.

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

Motility and more: the flagellum of Trypanosoma brucei.

Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host-parasite interactions.