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African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (μCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: In the 20th century, epidemics of human African trypanosomiasis (HAT) ravaged communities in a number of African countries. The latest surge in disease transmission was recorded in the late 1990s, with more than 35,000 cases reported annually in 1997 and 1998. In 2013, after more than a decade of sustained control efforts and steady progress, the World Health Assembly resolved to target the elimination of HAT as a public health problem by 2020. We report here on recent progress towards this goal., Methodology/principal Findings: With 992 and 663 cases reported in 2019 and 2020 respectively, the first global target was amply achieved (i.e. fewer than 2,000 HAT cases/year). Areas at moderate or higher risk of HAT, where more than 1 case/10,000 people/year are reported, shrunk to 120,000 km2 for the five-year period 2016-2020. This reduction of 83% from the 2000-2004 baseline (i.e. 709,000 km2) is slightly below the target (i.e. 90% reduction). As a result, the second global target for HAT elimination as a public health problem cannot be considered fully achieved yet. The number of health facilities able to diagnose and treat HAT expanded (+9.6% compared to a 2019 survey), thus reinforcing the capacity for passive detection and improving epidemiological knowledge of the disease. Active surveillance for gambiense HAT was sustained. In particular, 2.8 million people were actively screened in 2019 and 1.6 million in 2020, the decrease in 2020 being mainly caused by COVID-19-related restrictions. Togo and Côte d'Ivoire were the first countries to be validated for achieving elimination of HAT as a public health problem at the national level; applications from three additional countries are under review by the World Health Organization (WHO)., Conclusions/significance: The steady progress towards the elimination of HAT is a testament to the power of multi-stakeholder commitment and coordination. At the end of 2020, the World Health Assembly endorsed a new road map for 2021-2030 that set new bold targets for neglected tropical diseases. While rhodesiense HAT remains among the diseases targeted for elimination as a public health problem, gambiense HAT is targeted for elimination of transmission. The goal for gambiense HAT is expected to be particularly arduous, as it might be hindered by cryptic reservoirs and a number of other challenges (e.g. further integration of HAT surveillance and control into national health systems, availability of skilled health care workers, development of more effective and adapted tools, and funding for and coordination of elimination efforts)., Competing Interests: The authors have declared that no competing interests exist.

Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Trypanosoma brucei is responsible for lethal diseases in humans and cattle in Sub-Saharan Africa. These extracellular parasites extravasate from the blood circulation into several tissues. The importance of the vasculature in tissue tropism is poorly understood. Using intravital imaging and bioluminescence, we observe that gonadal white adipose tissue and pancreas are the two main parasite reservoirs. We show that reservoir establishment happens before vascular permeability is compromised, suggesting that extravasation is an active mechanism. Blocking endothelial surface adhesion molecules (E-selectin, P-selectins, or ICAM2) significantly reduces extravascular parasite density in all organs and delays host lethality. Remarkably, blocking CD36 has a specific effect on adipose tissue tropism that is sufficient to delay lethality, suggesting that establishment of the adipose tissue reservoir is necessary for parasite virulence. This work demonstrates the importance of the vasculature in a T. brucei infection and identifies organ-specific adhesion molecules as key players for tissue tropism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). In the mammalian host, the parasite lives entirely extracellularly, in both the blood and interstitial spaces in tissues. Although most T. brucei research has focused on the biology of blood- and central nervous system (CNS)-resident parasites, a number of recent studies have highlighted parasite reservoirs in the dermis and adipose tissue, leading to a renewed interest in tissue-resident parasite populations. In light of this renewed interest, work describing tissue-resident parasites can serve as a valuable resource to inform future investigations of tissue-resident T. brucei. Here, we review this body of literature, which describes infections in humans, natural hosts, and experimental animal models, providing a wealth of information on the distribution and biology of extravascular parasites, the corresponding immune response in each tissue, and resulting host pathology. We discuss the implications of these studies and future questions in the study of extravascular T. brucei., Competing Interests: The authors have declared that no competing interests exist.

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host's circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia., Competing Interests: SS, JL, IS, HZ, CR, TM, BM, ME No competing interests declared, (© 2021, Schuster et al.)

Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Infections with Trypanosoma brucei sp. are established after the injection of metacyclic trypomastigotes into the skin dermis by the tsetse fly vector. The parasites then gain access to the local lymphatic vessels to infect the local draining lymph nodes and disseminate systemically via the bloodstream. Macrophages are considered to play an important role in host protection during the early stage of systemic trypanosome infections. Macrophages are abundant in the skin dermis, but relatively little is known of their impact on susceptibility to intradermal (ID) trypanosome infections. We show that although dermal injection of colony stimulating factor 1 (CSF1) increased the local abundance of macrophages in the skin, this did not affect susceptibility to ID T. brucei infection. However, bacterial LPS-stimulation in the dermis prior to ID trypanosome infection significantly reduced disease susceptibility. In vitro assays showed that LPS-stimulated macrophage-like RAW264.7 cells had enhanced cytotoxicity towards T. brucei, implying that dermal LPS-treatment may similarly enhance the ability of dermal macrophages to eliminate ID injected T. brucei parasites in the skin. A thorough understanding of the factors that reduce susceptibility to ID injected T. brucei infections may lead to the development of novel strategies to help reduce the transmission of African trypanosomes.

Trypanosoma brucei is a protozoan parasite that causes important human and livestock diseases in sub-Saharan Africa. By overexpressing a single RNA-binding protein, RBP6, in non-infectious procyclics trypanosomes, we previously recapitulated in vitro the events occurring in the tsetse fly vector, namely the development of epimastigotes and infectious, quiescent metacyclic parasites. To identify genes involved in this developmental progression, we individually targeted 86 transcripts by RNAi in the RBP6 overexpression cell line and assessed the loss-of-function phenotypes on repositioning the kinetoplast, an organelle that contains the mitochondrial genome, the expression of BARP or brucei alanine rich protein, a marker for epimastigotes, and metacyclic variant surface glycoprotein. This screen identified 22 genes that positively or negatively regulate the stepwise progression towards infectivity at different stages. Two previously uncharacterized putative nucleic acid binding proteins emerged as potent regulators, namely the cold shock domain-containing proteins CSD1 and CSD2. RNA-Seq data from a selected group of cell lines further revealed that the components of gene expression regulatory networks identified in this study affected the abundance of a subset of transcripts in very similar fashion. Finally, our data suggest a considerable overlap between the genes that regulate the formation of stumpy bloodstream form trypanosomes and the genes that govern the development of metacyclic form parasites.

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., (Copyright © 2021 Vélez-Ramírez et al.)

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, and Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, the agents of Sleeping sickness (Human African Trypanosomiasis, HAT), as well as Trypanosoma brucei brucei, the agent of the cattle disease nagana, contain cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes are the cysteine proteases from the Clan CA, the Cathepsin L-like cruzipain and rhodesain, and the Cathepsin B-like enzymes, which have essential roles in the parasites and thus are potential targets for chemotherapy. In addition, several other proteases, present in one or both parasites, have been characterized, and some of them are also promising candidates for the developing of new drugs. Recently, new inhibitors, with good selectivity for the parasite proteasomes, have been described and are very promising as lead compounds for the development of new therapies for these neglected diseases. This article is part of a Special Issue entitled: "Play and interplay of proteases in health and disease"., (Copyright © 2020 Elsevier B.V. All rights reserved.)

DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum , which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei , which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.

Trypanosomiases are diseases caused by parasitic protozoan trypanosomes of the genus Trypanosoma . In humans, this includes Chagas disease and African trypanosomiasis. There are few therapeutic options, and there is low efficacy to clinical treatment. Therefore, the search for new drugs for the trypanosomiasis is urgent. This review describes studies of the trypanocidal properties of essential oils, an important group of natural products widely found in several tropical countries. Seventy-seven plants were selected from literature for the trypanocidal activity of their essential oils. The main chemical constituents and mechanisms of action are also discussed. In vitro and in vivo experimental data show the therapeutic potential of these natural products for the treatment of infections caused by species of Trypanosoma .

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Trypanosoma brucei possesses a highly complex RNA editing system that uses guide RNAs to direct the insertion and deletion of uridines in mitochondrial mRNAs. These changes extensively alter the target mRNAs and can more than double them in length. Recently, analyses showed that several of the edited genes possess the capacity to encode two different protein products. The overlapped reading frames can be accessed through alternative RNA editing that shifts the translated reading frame. In this study, we analyzed the editing patterns of three putative dual-coding genes, ribosomal protein S12 (RPS12), the 5' editing domain of NADH dehydrogenase subunit 7 (ND7 5'), and C-rich region 3 (CR3). We found evidence that alternatively 5'-edited ND7 5' and CR3 transcripts are present in the transcriptome, providing evidence for the use of dual ORFs in these transcripts. Moreover, we found that CR3 has a complex set of editing pathways that vary substantially between cell lines. These findings suggest that alternative editing can work to introduce genetic variation in a system that selects against nucleotide mutations., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei , but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei ., (© 2020 Pham and Li.)

Cultivation of pleomorphic Trypanosoma brucei strains was introduced in 1996 when matrix dependence of growth of natural isolates was recognized. Semisolid agarose or liquid methylcellulose are currently used and here we provide optimized protocols for these culture methods and for transfection of pleomorphic strains. Although more laborious than standard liquid culture, culture of native pleomorphic strains is important for a number of research questions including differentiation, virulence, tissue tropism, and regulated metabolism. Some subclones of pleomorphic strains have acquired matrix independence upon passage in culture but maintained a pleomorphic phenotype. It appears that matrix dependence and pleomorphism are not tightly linked traits, yet phenotypes have to be verified before choosing one of these subclones for given experiments. Based on direct comparisons, we give recommendations for pleomorphic strain selection and culture conditions that guarantee truly pleomorphic and differentiation competent Trypanosoma brucei.

Although CRIg was originally identified as a macrophage receptor for binding complement C3b/iC3b in vitro, recent studies reveal that CRIg functions as a pattern recognition receptor in vivo for Kupffer cells (KCs) to directly bind bacterial pathogens in a complement-independent manner. This raises the critical question of whether CRIg captures circulating pathogens through interactions with complement in vivo under flow conditions. Furthermore, the role of CRIg during parasitic infection is unknown. Taking advantage of intravital microscopy and using African trypanosomes as a model, we studied the role of CRIg in intravascular clearance of bloodborne parasites. Complement C3 is required for intravascular clearance of African trypanosomes by KCs, preventing the early mortality of infected mice. Moreover, antibodies are essential for complement-mediated capture of circulating parasites by KCs. Interestingly, reduced antibody production was observed in the absence of complement C3 during infection. We further demonstrate that CRIg but not CR3 is critically involved in KC-mediated capture of circulating parasites, accounting for parasitemia control and host survival. Of note, CRIg cannot directly catch circulating parasites and antibody-induced complement activation is indispensable for CRIg-mediated parasite capture. Thus, we provide evidence that CRIg, by interacting with complement in vivo, plays an essential role in intravascular clearance of bloodborne parasites. Targeting CRIg may be considered as a therapeutic strategy., Competing Interests: The authors declare no competing interest.

Trypanosoma brucei parasites successfully evade the host immune system by periodically switching the dense coat of variant surface glycoprotein (VSG) at the cell surface. Each parasite expresses VSGs in a monoallelic fashion that is tightly regulated. The consequences of exposing multiple VSGs during an infection, in terms of antibody response and disease severity, remain unknown. In this study, we overexpressed a high-mobility group box protein, TDP1, which was sufficient to open the chromatin of silent VSG expression sites, to disrupt VSG monoallelic expression, and to generate viable and healthy parasites with a mixed VSG coat. Mice infected with these parasites mounted a multi-VSG antibody response, which rapidly reduced parasitemia. Consequently, we observed prolonged survival in which nearly 90% of the mice survived a 30-d period of infection with undetectable parasitemia. Immunodeficient RAG2 knock-out mice were unable to control infection with TDP1-overexpressing parasites, showing that the adaptive immune response is critical to reducing disease severity. This study shows that simultaneous exposure of multiple VSGs is highly detrimental to the parasite, even at the very early stages of infection, suggesting that drugs that disrupt VSG monoallelic expression could be used to treat trypanosomiasis., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes., Competing Interests: The authors have declared that no competing interests exist.

Experiments on infections caused by trypanosomes are widely performed in Swiss white mice through various inoculation routes. To better understand the effect of route of trypanosome inoculation on disease outcomes in this model, we characterised the virulence of two isolates, Trypanosoma brucei KETRI 2710 and T. congolense KETRI 2765 in Swiss white mice. For each of the isolates, five routes of parasite inoculation, namely intraperitoneal (IP), subcutaneous (SC), intramuscular (IM) intradermal (ID) and intravenous (IV) were compared using groups (n = 6) of mice, with each mouse receiving 1x104 trypanosomes. We subsequently assessed impact of the routes on disease indices that included pre-patent period (PP), parasitaemia levels, Packed Cell Volume (PCV), bodyweight changes and survival time. Pre-patent period for IP inoculated mice was a mean ± SE of 3.8 ± 0.2 and 6.5 ± 0.0 for the T brucei and T. congolense isolates respectively; the PP for mice groups inoculated using other routes were not significantly different(p> 0.05) irrespective of route of inoculation and species of trypanosomes. With ID and IP routes, parasitaemia was significantly higher in T. brucei and significantly lower in T. congolense infected mice and the progression to peak parasitaemia routes showed no significant different between the routes of either species of trypanosome. The IM and ID routes in T. congolense inoculations, and IP and IV in T. b. brucei induced the fastest and slowest parasitaemia progressions respectively. There were significant differences in rates of reduction of PCV with time post infection in mice infected by the two species and which was more pronounced in sc and ip injected mice. No significant differences in mice body weight changes and survivorship was observed between the routes of inoculation. Inoculation route therefore appears to be a critical determinant of pathogenicity of Trypanosoma congolense and Trypanosoma brucei brucei in murine mouse model of African trypanosomiasis., Competing Interests: The authors have declared that no competing interests exist.

Equine trypanosomosis is a complex of infectious diseases called dourine, nagana and surra. It is caused by several species of the genus Trypanosoma that are transmitted cyclically by tsetse flies, mechanically by other haematophagous flies, or sexually. Trypanosoma congolense (subgenus Nannomonas) and T. vivax (subgenus Dutonella) are genetically and morphologically distinct from T. brucei, T. equiperdum and T. evansi (subgenus Trypanozoon). It remains controversial whether the three latter taxa should be considered distinct species. Recent outbreaks of surra and dourine in Europe illustrate the risk and consequences of importation of equine trypanosomosis with infected animals into non-endemic countries. Knowledge on the epidemiological situation is fragmentary since many endemic countries do not report the diseases to the World Organisation for Animal Health, OIE. Other major obstacles to the control of equine trypanosomosis are the lack of vaccines, the inability of drugs to cure the neurological stage of the disease, the inconsistent case definition and the limitations of current diagnostics. Especially in view of the ever-increasing movement of horses around the globe, there is not only the obvious need for reliable curative and prophylactic drugs but also for accurate diagnostic tests and algorithms. Unfortunately, clinical signs are not pathognomonic, parasitological tests are not sufficiently sensitive, serological tests miss sensitivity or specificity, and molecular tests cannot distinguish the taxa within the Trypanozoon subgenus. To address the limitations of the current diagnostics for equine trypanosomosis, we recommend studies into improved molecular and serological tests with the highest possible sensitivity and specificity. We realise that this is an ambitious goal, but it is dictated by needs at the point of care. However, depending on available treatment options, it may not always be necessary to identify which trypanosome taxon is responsible for a given infection.

African trypanosomiasis remains a lethal disease to both humans and livestock. The disease persists due to limited drug availability, toxicity and drug resistance, hence the need for a better understanding of the parasite's biology and provision of alternative forms of therapy. In this study, the in vitro effects of phenolic acids were assessed for their trypanocidal activities against Trypanosoma brucei brucei. The effect of the phenolic acids on Trypanosoma brucei brucei was determined by the alamarBlue assay. The cell cycle effects were determined by flow cytometry and parasite morphological analysis was done by microscopy. Effect on cell proliferation was determined by growth kinetic analysis. Reverse Transcriptase quantitative Polymerase Chain Reaction was used to determine expression of iron dependent enzymes and iron distribution determined by atomic absorption spectroscopy. Gallic acid gave an IC50 of 14.2±1.5 μM. Deferoxamine, gallic acid and diminazene aceturate showed a dose dependent effect on the cell viability and the mitochondrion membrane integrity. Gallic acid, deferoxamine and diminazene aceturate caused loss of kinetoplast in 22%, 26% and 82% of trypanosomes respectively and less than 10% increase in the number of trypanosomes in S phase was observed. Gallic acid caused a 0.6 fold decrease, 50 fold increase and 7 fold increase in the expression levels of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes respectively while treatment with deferoxamine and diminazene aceturate also showed differential expressions of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes. The data suggests that gallic acid possibly exerts its effect on T. brucei via iron chelation leading to structural and morphological changes and arrest of the cell cycle. These together provide information on the cell biology of the parasite under iron starved conditions and provide leads into alternative therapeutic approaches in the treatment of African trypanosomiasis., Competing Interests: The authors have declared that no competing interests exist.

Human African trypanosomiasis (HAT), known as sleeping sickness and caused by Trypanosoma brucei , is threatening low-income populations in sub-Saharan African countries with 61 million people at risk of infection. In order to discover new natural products against HAT, thirty-seven Vietnamese essential oils (EOs) were screened for their activity in vitro on Trypanosoma brucei brucei ( Tbb ) and cytotoxicity on mammalian cells (WI38, J774). Based on the selectivity indices (SIs), the more active and selective EOs were analyzed by gas chromatography. The anti-trypanosomal activity and cytotoxicity of some major compounds (isolated or commercial) were also determined. Our results showed for the first time the selective anti-trypanosomal effect of four EOs, extracted from three Zingiberaceae species ( Curcuma longa , Curcuma zedoaria , and Zingiber officinale ) and one Lauraceae species ( Litsea cubeba ) with IC 50 values of 3.17 ± 0.72, 2.51 ± 1.08, 3.10 ± 0.08, and 2.67 ± 1.12 nL/mL respectively and SI > 10. Identified compounds accounted for more than 85% for each of them. Among the five major components of Curcuma longa EO, curlone is the most promising anti-trypanosomal candidate with an IC 50 of 1.38 ± 0.45 µg/mL and SIs of 31.7 and 18.2 compared to WI38 and J774 respectively.

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans , showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4'-methyl ether ( 4 ), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans , while the purified compounds had low activity against P. falciparum . Anti-trypanosomal activity (EC 50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol ( 10 ) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

New anti-infective agents are urgently needed to fight microbial resistance. Methicillin-resistant Staphylococcus aureus (MRSA) strains are particularly responsible for complicated pathologies that are difficult to treat due to their virulence and the formation of persistent biofilms forming a complex protecting shell. Parasitic infections caused by Trypanosoma brucei and Leishmania mexicana are also of global concern, because of the mortality due to the low number of safe and effective treatments. Female inflorescences of hop produce specialized metabolites known for their antimicrobial effects but underexploited to fight against drug-resistant microorganisms. In this study, we assessed the antimicrobial potential of phenolic compounds against MRSA clinical isolates, T. brucei and L. mexicana . By fractionation process, we purified the major prenylated chalcones and acylphloroglucinols, which were quantified by UHPLC-UV in different plant parts, showing their higher content in the active flowers extract. Their potent antibacterial action (MIC < 1 µg/mL for the most active compound) was demonstrated against MRSA strains, through kill curves, post-antibiotic effects, anti-biofilm assays and synergy studies with antibiotics. An antiparasitic activity was also shown for some purified compounds, particularly on T. brucei (IC 50 < 1 to 11 µg/mL). Their cytotoxic activity was assessed both on cancer and non-cancer human cell lines., Competing Interests: The authors declare no conflict of interest.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco&#95;Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco&#95;Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco&#95;Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco&#95;Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco&#95;Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco&#95;Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco&#95;Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes., Competing Interests: The authors have declared that no competing interests exist.

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.

In this review we describe how Trypanosoma brucei brucei, a rodent pathogenic strain of African trypanosomes, can invade the nervous system, first by localization to the choroid plexus, the circumventricular organs (CVOs) and peripheral ganglia, which have fenestrated vessels, followed by crossing of the blood-brain barrier (BBB) into the white matter, hypothalamus, thalamus and basal ganglia. White blood cells (WBCs) pave the way for the trypanosome neuroinvasion. Experiments with immune deficient mice show that the invasion of WBCs is initiated by the toll-like receptor 9, followed by an augmentation phase that depends on the cytokine IFN-γ and the chemokine CXCL10. Nitric oxide (NO) derived from iNOS then prevents a break-down of the BBB and non-regulated passage of cells. This chain of events is relevant for design of better diagnostic tools to distinguish the different stages of the disease as well as for better understanding of the pathogenesis of the nervous system dysfunctions, which include circadian rhythm changes with sleep pattern disruption, pain syndromes, movement disorders and mental disturbances including dementia., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Especially in tropical and developing countries, the clinically relevant protozoa Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (sleeping sickness) and Leishmania species (leishmaniasis) stand out and infect millions of people worldwide leading to critical social-economic implications. Low-income populations are mainly affected by these three illnesses that are neglected by the pharmaceutical industry. Current anti-trypanosomatid drugs present variable efficacy with remarkable side effects that almost lead to treatment discontinuation, justifying a continuous search for alternative compounds that interfere with essential and specific parasite pathways. In this scenario, the triggering of trypanosomatid cell death machinery emerges as a promising approach, although the exact mechanisms involved in unicellular eukaryotes are still unclear as well as the controversial biological importance of programmed cell death (PCD). In this review, the mechanisms of autophagy, apoptosis-like cell death and necrosis found in pathogenic trypanosomatids are discussed, as well as their roles in successful infection. Based on the published genomic and proteomic maps, the panel of trypanosomatid cell death molecules was constructed under different experimental conditions. The lack of PCD molecular regulators and executioners in these parasites up to now has led to cell death being classified as an unregulated process or incidental necrosis, despite all morphological evidence published. In this context, the participation of metacaspases in PCD was also not described, and these proteases play a crucial role in proliferation and differentiation processes. On the other hand, autophagic phenotype has been described in trypanosomatids under a great variety of stress conditions (drugs, starvation, among others) suggesting that this process is involved in the turnover of damaged structures in the protozoa and is not a cell death pathway. Death mechanisms of pathogenic trypanosomatids may be involved in pathogenesis, and the identification of parasite-specific regulators could represent a rational and attractive alternative target for drug development for these neglected diseases.

In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 ( Tb PRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking Tb PRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in Tb PRMT1-depleted cells, and a large number of Tb PRMT1-interacting proteins, thereby highlighting potential Tb PRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with Tb PRMT1, prompting analysis of the response of Tb PRMT1-depleted cells to nutrient deprivation. Indeed, depletion of Tb PRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that Tb PRMT1 itself binds nucleic acids in vitro and in vivo , a feature completely novel to protein arginine methyltransferases. IMPORTANCE Trypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), Tb PRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation., (Copyright © 2018 Kafková et al.)

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination., Competing Interests: The authors have declared that no competing interests exist.

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM&#95;Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM&#95;Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM&#95;Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM&#95;Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM&#95;Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM&#95;Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues., Competing Interests: The authors have declared that no competing interests exist.

The flagellated parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT). By a mechanism not well understood yet, trypanosomes enter the central nervous system (CNS), invade the brain parenchyma, and cause a fatal encephalopathy if is not treated. Trypanosomes are fast dividing organisms that, without any immune response, would kill the host in a short time. However, infected individuals survive either 6-12 months or more than 3 years for the acute and chronic forms, respectively. Thus, only when the brain defense collapses a lethal encephalopathy will occur. Here, we evaluated interactions between trypanosomes and microglial cells, which are the primary immune effector cells within the CNS. Using co-cultures of primary microglia and parasites, we found clear evidences of trypanosome phagocytosis by microglial cells. Microglia activation was also evident; analysis of its ultrastructure showed changes that have been reported in activated microglia undergoing oxidative stress caused by infections or degenerative diseases. Accordingly, an increase of the nitric oxide production was detected in supernatants of microglia/parasite co-cultures. Altogether, our results demonstrate that microglial cells respond to the presence of the parasite, leading to parasite's engulfment and elimination.

An understanding of the genes and mechanisms regulating environmental stress in crops is critical for boosting agricultural yield and safeguarding food security. Under adverse conditions, response pathways are activated for tolerance or resistance. In multiple species, the alternative oxidase ( AOX ) genes encode proteins which help in this process. Recently, this gene family has been extensively investigated in the vital crop plants, wheat, barley and rice. Cumulatively, these three species and/or their wild ancestors contain the genes for AOX1a , AOX1c , AOX1e , and AOX1d , and common patterns in the protein isoforms have been documented. Here, we add more information on these trends by emphasizing motifs that could affect expression, and by utilizing the most recent discoveries from the AOX isoform in Trypanosoma brucei to highlight clade-dependent biases. The new perspectives may have implications on how the AOX gene family has evolved and functions in monocots. The common or divergent amino acid substitutions between these grasses and the parasite are noted, and the potential effects of these changes are discussed. There is the hope that the insights gained will inform the way future AOX research is performed in monocots, in order to optimize crop production for food, feed, and fuel.

Unlike most other model eukaryotes, Trypanosoma brucei and its relatives have a single mitochondrion with a single-unit mitochondrial genome that is termed kinetoplast DNA (kDNA). Replication of the kDNA is coordinated with the cell cycle. During binary mitochondrial fission and prior to cytokinesis, the replicated kDNA has to be faithfully segregated to the daughter organelles. This process depends on the tripartite attachment complex (TAC) that physically links the kDNA across the two mitochondrial membranes with the basal body of the flagellum. Thus, the TAC couples segregation of the replicated kDNA with segregation of the basal bodies of the old and the new flagellum. In this Review, we provide an overview of the role of the TAC in kDNA inheritance in T. brucei We focus on recent advances regarding the molecular composition of the TAC, and discuss how the TAC is assembled and how its subunits are targeted to their respective TAC subdomains. Finally, we will contrast the segregation of the single-unit kDNA in trypanosomes to mitochondrial genome inheritance in yeast and mammals, both of which have numerous mitochondria that each contain multiple genomes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Trypanosomes of African wild ungulates transmitted by tsetse flies can cause human and livestock diseases. However, trypanosome diversity in wild tsetse flies remains greatly underestimated. We employed FFLB (fluorescent fragment length barcoding) for surveys of trypanosomes in tsetse flies (3086) from the Gorongosa National Park (GNP) and Niassa National Reserve (NNR) in Mozambique (MZ), identified as Glossina morsitans morsitans (GNP/NNR=77.6%/90.5%) and Glossina pallidipes (22.4%/9.5%). Trypanosomes were microscopically detected in 8.3% of tsetse guts. FFLB of gut samples revealed (GNP/NNR): Trypanosoma congolense of Savannah (27%/63%), Kilifi (16.7%/29.7%) and Forest (1.0%/0.3%) genetic groups; T. simiae Tsavo (36.5%/6.1%); T. simiae (22.2%/17.7%); T. godfreyi (18.2%/7.0%); subgenus Trypanozoon (20.2%/25.7%); T. vivax/T. vivax-like (1.5%/5.2%); T. suis/T. suis-like (9.4%/11.9%). Tsetse proboscises exhibited similar species composition, but most prevalent species were (GNP/NNR): T. simiae (21.9%/28%), T. b. brucei (19.2%/31.7%), and T. vivax/T. vivax-like (19.2%/28.6%). Flies harboring mixtures of trypanosomes were common (~ 64%), and combinations of more than four trypanosomes were especially abundant in the pristine NNR. The non-pathogenic T. theileri was found in 2.5% while FFLB profiles of unknown species were detected in 19% of flies examined. This is the first report on molecular diversity of tsetse flies and their trypanosomes in MZ; all trypanosomes pathogenic for ungulates were detected, but no human pathogens were detected. Overall, two species of tsetse flies harbor 12 species/genotypes of trypanosomes. This notable species richness was likely uncovered because flies were captured in wildlife reserves and surveyed using the method of FFLB able to identify, with high sensitivity and accuracy, known and novel trypanosomes. Our findings importantly improve the knowledge on trypanosome diversity in tsetse flies, revealed the greatest species richness so far reported in tsetse fly of any African country, and indicate the existence of a hidden trypanosome diversity to be discovered in African wildlife protected areas., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and 'switching' to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact.

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.

Messenger RNA is modified by the addition of a 5' methylated cap structure, which protects the transcript and recruits protein complexes that mediate RNA processing and/or the initiation of translation. Two genes encoding mRNA cap methyltransferases have been identified in T. brucei: TbCMT1 and TbCGM1. Here we analysed the impact of TbCMT1 gene deletion on bloodstream form T. brucei cells. TbCMT1 was dispensable for parasite proliferation in in vitro culture. However, significantly decreased parasitemia was observed in mice inoculated with TbCMT1 null and conditional null cell lines. Using RNA-Seq, we observed that several cysteine peptidase mRNAs were downregulated in TbCMT1 null cells lines. The cysteine peptidase Cathepsin-L was also shown to be reduced at the protein level in TbCMT1 null cell lines. Our data suggest that TbCMT1 is not essential to bloodstream form T. brucei growth in vitro or in vivo but that it contributes significantly to parasite virulence in vivo., Competing Interests: The authors have declared that no competing interests exist.

The sleeping sickness parasite Trypanosoma brucei has a complex life cycle, alternating between a mammalian host and the tsetse fly vector. A tightly controlled developmental programme ensures parasite transmission between hosts as well as survival within them and involves strict regulation of mitochondrial activities. In the glucose-rich bloodstream, the replicative 'slender' stage is thought to produce ATP exclusively via glycolysis and uses the mitochondrial F1FO-ATP synthase as an ATP hydrolysis-driven proton pump to generate the mitochondrial membrane potential (ΔΨm). The 'procyclic' stage in the glucose-poor tsetse midgut depends on mitochondrial catabolism of amino acids for energy production, which involves oxidative phosphorylation with ATP production via the F1FO-ATP synthase. Both modes of the F1FO enzyme critically depend on FO subunit a, which is encoded in the parasite's mitochondrial DNA (kinetoplast or kDNA). Comparatively little is known about mitochondrial function and the role of kDNA in non-replicative 'stumpy' bloodstream forms, a developmental stage essential for disease transmission. Here we show that the L262P mutation in the nuclear-encoded F1 subunit γ that permits survival of 'slender' bloodstream forms lacking kDNA ('akinetoplastic' forms), via FO-independent generation of ΔΨm, also permits their differentiation into stumpy forms. However, these akinetoplastic stumpy cells lack a ΔΨm and have a reduced lifespan in vitro and in mice, which significantly alters the within-host dynamics of the parasite. We further show that generation of ΔΨm in stumpy parasites and their ability to use α-ketoglutarate to sustain viability depend on F1-ATPase activity. Surprisingly, however, loss of ΔΨm does not reduce stumpy life span. We conclude that the L262P γ subunit mutation does not enable FO-independent generation of ΔΨm in stumpy cells, most likely as a consequence of mitochondrial ATP production in these cells. In addition, kDNA-encoded genes other than FO subunit a are important for stumpy form viability., Competing Interests: The authors have declared that no competing interests exist.

Trypanosomatid parasites are causative agents of neglected human diseases. Their lineage diverged early from the common eukaryotic ancestor, and they evolved singular mechanisms of gene expression that are crucial for their survival. Studies on unusual and essential molecular pathways lead to new drug targets. In this respect, assays to analyze transcriptional activity will provide useful information to identify essential and specific factors. However, the current methods are laborious and do not provide global and accurate measures. For this purpose, a previously reported radiolabeling in vitro nascent mRNA methodology was used to establish an alternative fluorescent-based assay that is able to precisely quantify nascent mRNA using both flow cytometry and a high-content image system. The method allowed accurate and global measurements in Trypanosoma brucei, a representative species of trypanosomatid parasites. We obtained data demonstrating that approximately 70% of parasites from a population under normal growth conditions displayed mRNA transcriptional activity, whilst the treatment with α-amanitin (75 µg/ml) inhibited the polymerase II activity. The adaptation of the method also allowed the analyses of the transcriptional activity during the cell cycle. Therefore, the methodology described herein contributes to obtaining precise measurements of transcriptional rates using multiparametric analysis. This alternative method can facilitate investigations of genetic and biochemical processes in trypanosome parasites and consequently provide additional information related to new treatment or prophylaxis strategies involving these important human parasites., (© 2018 International Society for Advancement of Cytometry.)

Sleeping sickness is a neglected tropical disease caused by Trypanosoma brucei parasites, affecting the poorest communities in sub-Saharan Africa. The great efforts done by the scientific community, local governments, and non-governmental organizations (NGOs) via active patients' screening, vector control, and introduction of improved treatment regimens have significantly contributed to the reduction of human African trypanosomiasis (HAT) incidence during the last 15 years. Consequently, the WHO has announced the objective of HAT elimination as a public health problem by 2020. Studies at both parasite and host levels have improved our understanding of the parasite biology and the mechanisms of parasite interaction with its mammalian host. In this review, the impact that 'omics studies have had on sleeping sickness by revealing novel properties of parasite's subcellular organelles are summarized, by highlighting changes induced in the host during the infection and by proposing potential disease markers and therapeutic targets., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote., Competing Interests: The authors have declared that no competing interests exist.

As part of our research for new leads against human African trypanosomiasis (HAT), we report on a 3D-QSAR study for antitrypanosomal activity and cytotoxicity of aminosteroid-type alkaloids recently isolated from the African medicinal plant Holarrhena africana A. DC. (Apocynaceae), some of which are strong trypanocides against Trypanosoma brucei rhodesiense ( Tbr ), with low toxicity against mammalian cells. Fully optimized 3D molecular models of seventeen congeneric Holarrhena alkaloids were subjected to a comparative molecular field analysis (CoMFA). CoMFA models were obtained for both, the anti- Tbr and cytotoxic activity data. Model performance was assessed in terms of statistical characteristics ( R ², Q ², and P ² for partial least squares (PLS) regression, internal cross-validation (leave-one-out), and external predictions (test set), respectively, as well as the corresponding standard deviation error in prediction (SDEP) and F -values). With R ² = 0.99, Q ² = 0.83 and P ² = 0.79 for anti- Tbr activity and R ² = 0.94, Q ² = 0.64, P ² = 0.59 for cytotoxicity against L6 rat skeletal myoblasts, both models were of good internal and external predictive power. The regression coefficients of the models representing the most prominent steric and electrostatic effects on anti- Tbr and for L6 cytotoxic activity were translated into contour maps and analyzed visually, allowing suggestions for possible modification of the aminosteroids to further increase the antitrypanosomal potency and selectivity. Very interestingly, the 3D-QSAR model established with the Holarrhena alkaloids also applied to the antitrypanosomal activity of two aminocycloartane-type compounds recently isolated by our group from Buxus sempervirens L. (Buxaceae), which indicates that these structurally similar natural products share a common structure⁻activity relationship (SAR) and, possibly, mechanism of action with the Holarrhena steroids. This 3D-QSAR study has thus resulted in plausible structural explanations of the antitrypanosomal activity and selectivity of aminosteroid- and aminocycloartane-type alkaloids as an interesting new class of trypanocides and may represent a starting point for lead optimization.