A nuclear orthologue of the dNTP triphosphohydrolase SAMHD1 controls dNTP homeostasis and genomic stability in Trypanosoma brucei.

Maintenance of dNTPs pools in Trypanosoma brucei is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle which is essential for the preservation of genomic stability. Both the salvage and de novo pathways participate in the provision of pyrimidine dNTPs while purine dNTPs are made available solely through salvage. In order to identify enzymes involved in degradation here we have characterized the role of a trypanosomal SAMHD1 orthologue denominated TbHD82. Our results show that TbHD82 is a nuclear enzyme in both procyclic and bloodstream forms of T. brucei. Knockout forms exhibit a hypermutator phenotype, cell cycle perturbations and an activation of the DNA repair response. Furthermore, dNTP quantification of TbHD82 null mutant cells revealed perturbations in nucleotide metabolism with a substantial accumulation of dATP, dCTP and dTTP. We propose that this HD domain-containing protein present in kinetoplastids plays an essential role acting as a sentinel of genomic fidelity by modulating the unnecessary and detrimental accumulation of dNTPs.

KLIF-associated cytoskeletal proteins in Trypanosoma brucei regulate cytokinesis by promoting cleavage furrow positioning and ingression.

Cytokinesis in the early divergent protozoan Trypanosoma brucei occurs from the anterior cell tip of the new-flagellum daughter toward the nascent posterior end of the old-flagellum daughter of a dividing biflagellated cell. The cleavage furrow ingresses unidirectionally along the preformed cell division fold and is regulated by an orphan kinesin named kinesin localized to the ingressing furrow (KLIF) that localizes to the leading edge of the ingressing furrow. Little is known about how furrow ingression is controlled by KLIF and whether KLIF interacts with and cooperates with other cytokinesis regulatory proteins to promote furrow ingression. Here, we investigated the roles of KLIF in cleavage furrow ingression and identified a cohort of KLIF-associated cytoskeletal proteins as essential cytokinesis regulators. By genetic complementation, we demonstrated the requirement of the kinesin motor activity, but not the putative tropomyosin domain, of KLIF in promoting furrow ingression. We further showed that depletion of KLIF impaired the resolution of the nascent posterior of the old-flagellar daughter cell, thereby stalking cleavage furrow ingression at late stages of cytokinesis. Through proximity biotinylation, we identified a subset of cytoskeleton-associated proteins (CAPs) as KLIF-proximal proteins, and functional characterization of these cytoskeletal proteins revealed the essential roles of CAP46 and CAP52 in positioning the cleavage furrow and the crucial roles of CAP42 and CAP50 in promoting cleavage furrow ingression. Together, these results identified multiple cytoskeletal proteins as cytokinesis regulators and uncovered their essential and distinct roles in cytokinesis.

Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing.

Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative 'slender' to transmissible 'stumpy' bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.

Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro.

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

A specific basal body linker protein provides the connection function for basal body inheritance in trypanosomes.

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1-G2 tether and S-M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement-loss of orthogonal orientation and ability to separate and move apart-are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.

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.

CEP164C regulates flagellum length in stable flagella.

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to "lock" at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.

Isolation and Antitrypanosomal Characterization of Furoquinoline and Oxylipin from Zanthoxylum zanthoxyloides.

In the absence of vaccines, there is a need for alternative sources of effective chemotherapy for African trypanosomiasis (AT). The increasing rate of resistance and toxicity of commercially available antitrypanosomal drugs also necessitates an investigation into the mode of action of new antitrypanosomals for AT. In this study, furoquinoline 4, 7, 8-trimethoxyfuro (2, 3-b) quinoline (compound 1) and oxylipin 9-oxo-10, 12-octadecadienoic acid (compound 2) were isolated from the plant species Zanthoxylum zanthoxyloides (Lam) Zepern and Timler (root), and their in vitro efficacy and mechanisms of action investigated in Trypanosomabrucei (T. brucei), the species responsible for AT. Both compounds resulted in a selectively significant growth inhibition of T. brucei (compound 1, half-maximal effective concentration EC50 = 1.7 µM, selectivity indices SI = 74.9; compound 2, EC50 = 1.2 µM, SI = 107.3). With regards to effect on the cell cycle phases of T. brucei, only compound 1 significantly arrested the second growth-mitotic (G2-M) phase progression even though G2-M and DNA replication (S) phase arrest resulted in the overall reduction of T. brucei cells in G0-G1 for both compounds. Moreover, both compounds resulted in the aggregation and distortion of the elongated slender morphology of T. brucei. Analysis of antioxidant potential revealed that at their minimum and maximum concentrations, the compounds exhibited significant oxidative activities in T. brucei (compound 1, 22.7 µM Trolox equivalent (TE), 221.2 µM TE; compound 2, 15.0 µM TE, 297.7 µM TE). Analysis of growth kinetics also showed that compound 1 exhibited a relatively consistent growth inhibition of T. brucei at different concentrations as compared to compound 2. The results suggest that compounds 1 and 2 are promising antitrypanosomals with the potential for further development into novel AT chemotherapy.

The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria.

tRNAs universally carry a CCA nucleotide triplet at their 3'-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

Light Microscopy in Trypanosomes: Use of Fluorescent Proteins and Tags.

Fluorescence microscopy enables the localization of proteins to specific structures within a cell which have either been fused to a fluorescence protein or detected by immunofluorescence. Here, we describe the various procedures that can be used to prepare both the procyclic form and bloodstream form of the human pathogen Trypanosoma brucei for fluorescence microscopy. The choice of procedure to be used is determined by various parameters, including protein characteristics and the scientific question being investigated.

Motility Analysis of Trypanosomatids.

Motility analysis of microswimmers has long been limited to a few model cell types and broadly restricted by technical challenges of high-resolution in vivo microscopy. Recently, interdisciplinary interest in detailed analysis of the motile behavior of various species has gained momentum. Here we describe a basic protocol for motility analysis of an important, highly diverse group of eukaryotic flagellate microswimmers, using high spatiotemporal resolution videomicroscopy. Further, we provide a special, time-dependent tomographic approach for the proof of rotational locomotion of periodically oscillating microswimmers, using the same data. Taken together, the methods describe part of an integrative approach to generate decisive information on three-dimensional in vivo motility from standard two-dimensional videomicroscopy data.

A Scalable Purification Method for Mitochondria from Trypanosoma brucei.

Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.

Measurement of Energy States of the Trypanosomatid Mitochondrion.

The evaluation of mitochondrial functionality is critical to interpret most biological data at the (eukaryotic) cellular level. For example, metabolism, cell cycle, epigenetic regulation, cell death mechanisms, autophagy, differentiation, and response redox imbalance are dependent on the mitochondrial state. In case of parasitic organisms, such as trypanosomatids, it is very often important to have information on mitochondrial functionality in order to assess the mechanisms of actions of drugs being proposed for therapy. In this chapter we present a set of methods that together allow to evaluate with some precision the mitochondrial functionality in Trypanosoma cruzi and Trypanosoma brucei. We discuss how to determine O2 consumption, mitochondrial inner membrane potential, ATP production, and the endogenous production of reactive oxygen species.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids.

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigens.

Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface-expressed variant surface glycoprotein (VSG), while ensuring that only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated in the catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signaled to guide the appropriate reaction or to integrate switching into parasite growth is unknown. Here, we show that the loss of ATR, a DNA damage-signaling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localized damage. Furthermore, ATR loss leads to the increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with the altered localization of RNA polymerase I and VEX1. This work shows that ATR acts in antigenic variation both through DNA damage signaling and surface antigen expression control.

High-speed multifocal plane fluorescence microscopy for three-dimensional visualisation of beating flagella.

Analysis of flagellum and cilium beating in three dimensions (3D) is important for understanding cell motility, and using fluorescence microscopy to do so would be extremely powerful. Here, high-speed multifocal plane fluorescence microscopy, where the light path is split to visualise multiple focal planes simultaneously, was used to reconstruct Trypanosoma brucei and Leishmania mexicana movement in 3D. These species are uniflagellate unicellular parasites for which motility is vital. It was possible to use either a fluorescent stain or a genetically-encoded fluorescent protein to visualise flagellum and cell movement at 200 Hz frame rates. This addressed two open questions regarding Trypanosoma and Leishmania flagellum beating, which contributes to their swimming behaviours: 1) how planar is the L. mexicana flagellum beat, and 2) what is the nature of flagellum beating during T. brucei 'tumbling'? We showed that L. mexicana has notable deviations from a planar flagellum beat, and that during tumbling the T. brucei flagellum bends the cell and beats only in the distal portion to achieve cell reorientation. This demonstrates high-speed multifocal plane fluorescence microscopy as a powerful tool for the analysis of beating flagella.

The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei.

Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. bruceiIMPORTANCETrypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.

Non-equivalence in old- and new-flagellum daughter cells of a proliferative division in Trypanosoma brucei.

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.

Antileishmanial and antitrypanosomal activity of symmetrical dibenzyl-substituted α,ß-unsaturated carbonyl-based compounds.

Background: Human African Trypanosomiasis (HAT) and leishmaniasis are two of the most neglected challenging tropical diseases, caused by the kinetoplastid parasites Trypanosoma and Leishmania species, respectively. For both of these complex disease spectra, treatment options are limited and threatened by drug resistance, justifying urgent new drug discovery efforts. Purpose: In the present study we investigated the antitrypanosomal and antileishmanial activity of a series of 21 symmetrical α,ß-unsaturated carbonyl-based compounds, each featuring two 3-methoxybenzene attached to a central cyclohexanone, tetrahydro-4-pyranone scaffold or 4-piperidone ring. Structure-activity relationships were explored with respect to substitution on positions 3, 4 and 6 of the terminal 3-methoxybenzyl groups, and seven types of central ring. Results: Compounds 3a, 3o, 3s and 3t, showed broad anti-kinetoplastid activity against all species and strains tested. Conclusion: Compound 3o featuring N-methyl-4-piperidone was found to be the most potent analog and therefore can serve as a potential lead for the development of new drug candidates for trypanosomiasis and leishmaniasis.

A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation.

The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single-cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, time-lapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of the responses of the parasite to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.