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.
Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.
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.
The current pharmacopeia to treat the lethal human and animal diseases caused by the protozoan parasite Trypanosoma brucei remains limited. The parasite's ability to undergo antigenic variation represents a considerable barrier to vaccine development, making the identification of new drug targets extremely important. Recent studies have demonstrated that fatty acid synthesis is important for growth and virulence of Trypanosoma brucei brucei, suggesting this pathway may have therapeutic potential. The first committed step of fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC), which is a known target of (-)-epigallocatechin-3-gallate (EGCG), an active polyphenol compound found in green tea. EGCG exerts its effects on ACC through activation of AMP-dependent protein kinase, which phosphorylates and inhibits ACC. We found that EGCG inhibited TbACC activity with an EC50 of 37 μM and 55 μM for bloodstream form and procyclic form lysates, respectively. Treatment with 100 μM EGCG induced a 4.7- and 1.7- fold increase in TbACC phosphorylation in bloodstream form and procyclic lysates. EGCG also inhibited the growth of bloodstream and procyclic parasites in culture, with a 48 h EC50 of 33 μM and 27 μM, respectively, which is greater than the EGCG plasma levels typically achievable in humans through oral dosing. Daily intraperitoneal administration of EGCG did not reduce the virulence of an acute mouse model of T. b. brucei infection. These data suggest a reduced potential for EGCG to treat T. brucei infections, but suggest that EGCG may prove to be useful as a tool to probe ACC regulation.
Trypanosoma brucei is a eukaryotic parasite that causes African sleeping sickness. T. brucei is transmitted by the blood-sucking tsetse fly. In order to adapt to its two very different hosts, T. brucei must sense the host environment and alter its metabolism to maximize utilization of host resources and minimize expenditure of its own resources. One key nutrient class is represented by fatty acids, which the parasite can either take from the host or make themselves. Our work describes a novel environmental regulatory pathway for fatty acid synthesis where the parasite turns off fatty acid synthesis when environmental lipids are abundant and turns on synthesis when the lipid supply is scarce. This pathway was observed in the tsetse midgut form but not the mammalian bloodstream form. However, pharmacological activation of this pathway in the bloodstream form to turn fatty acid synthesis off may be a promising new avenue for sleeping sickness drug discovery.
25Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne 26 disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and 27pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into 28 subdomains, each having specialized composition and function. Overall flagellum proteome has been 29 well-studied, but a critical gap in knowledge is the protein composition of individual flagellum 30 subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition 31 of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused 32 APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed 33 along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification 34 of biotinylated proteins yields a DRC1 "proximity proteome" showing good overlap with proteomes 35 obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that 36 is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is 37 excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of 38 biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. 39Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is 40 enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our 41 combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can 42 be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily 43 purified. 44 IMPORTANCE 45Sleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. 46The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different 48 specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is 49 need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that 50 APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and 51 has allowed identification of proximity proteomes for different subdomains. This capacity opens the 52 possibility to study the composition and function of other compartments. We further expect that this 53 approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a 54 model organism to study ciliopathies, heritable human diseases in which cilia function is impaired. 55 INTRODUCTION 56Trypanosoma brucei is a flagellated parasite that is transmitted between mammalian hosts by a 57 hematophagous vector, the tsetse fly, ...
Trypanosoma brucei, a protozoan parasite transmitted by the tsetse fly, invades the bloodstream and CNS of humans, causing African sleeping sickness. In addition to antigenic variation of its surface glycoprotein coat, T. brucei employs a second immune evasion tactic: upregulation of endocytosis to clear lytic cell surface immune complexes, a strategy likely involving membrane turnover and fatty acid synthesis (FAS). Acetyl‐CoA carboxylase (ACC) catalyzes the first committed step of FAS and is a regulatory control point. Previous work showed that ACC RNA interference (RNAi) in T. brucei bloodstream forms (BFs) reduced virulence in mice, but had no effect on growth in culture. We hypothesized that ACC is required in BFs for immune evasion via endocytosis upregulation. We examined endocytosis in BF ACC RNAi cells and observed a 90% reduction in fluid phase and receptor‐mediated endocytosis upon ACC RNAi. ACC RNAi also caused a 30% delay in the clearance of surface‐bound antibodies and 40% increase in the susceptibility to complement‐mediated lysis. Because endocytosis upregulation may increase demand for FAS, we examined if ACC is regulated in response to changes in lipid supply. In BFs, we found no change in ACC expression, activity, or phosphorylation upon growth in low or high lipid media. Also, ACC RNAi had similar effects on endocytosis, antibody clearance, and complement‐mediated lysis under low and high lipid conditions. In contrast, insect form cells exhibited a 2X increase in ACC protein and enzymatic activity in low lipid media. Phosphorylation of insect form ACC increased 3X in high lipid media and decreased by 80% in low lipid media. We propose a model whereby T. brucei ACC is up‐regulated in the mammalian host to support increased endocytosis and immune evasion, while in the insect host, ACC is regulated in response to the environmental lipid supply. Grant Funding Source: Supported by NIH R15 AI081207
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.