Bloodstream Trypanosomes Research Articles

The Janus-faced functions of Apolipoproteins L in membrane dynamics

The functions of human Apolipoproteins L (APOLs) are poorly understood, but involve diverse activities like lysis of bloodstream trypanosomes and intracellular bacteria, modulation of viral infection and induction of apoptosis, autophagy, and chronic kidney disease. Based on recent work, I propose that the basic function of APOLs is the control of membrane dynamics, at least in the Golgi and mitochondrion. Together with neuronal calcium sensor-1 (NCS1) and calneuron-1 (CALN1), APOL3 controls the activity of phosphatidylinositol-4-kinase-IIIB (PI4KB), involved in both Golgi and mitochondrion membrane fission. Whereas secreted APOL1 induces African trypanosome lysis through membrane permeabilization of the parasite mitochondrion, intracellular APOL1 conditions non-muscular myosin-2A (NM2A)-mediated transfer of PI4KB and APOL3 from the Golgi to the mitochondrion under conditions interfering with PI4KB-APOL3 interaction, such as APOL1 C-terminal variant expression or virus-induced inflammatory signalling. APOL3 controls mitophagy through complementary interactions with the membrane fission factor PI4KB and the membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). In mice, the basic APOL1 and APOL3 activities could be exerted by mAPOL9 and mAPOL8, respectively. Perspectives regarding the mechanism and treatment of APOL1-related kidney disease are discussed, as well as speculations on additional APOLs functions, such as APOL6 involvement in adipocyte membrane dynamics through interaction with myosin-10 (MYH10).

Selenosugars targeting the infective stage of Trypanosoma brucei with high selectivity

Earlier evidences showed that diglycosyl diselenides are active against the infective stage of African trypanosomes (top hits IC50 0.5 and 1.5 μM) but poorly selective (selectivity index <10). Here we extended the study to 33 new seleno-glycoconjugates with the aim to improve potency and selectivity. Three selenoglycosides and three glycosyl selenenylsulfides displayed IC50 against bloodstream Trypanosoma brucei in the sub-μM range (IC50 0.35–0.77 μM) and four of them showed an improved selectivity (selectivity index >38-folds vs. murine and human macrohages). For the glycosyl selenylsulfides, the anti-trypanosomal activity was not significantly influenced by the nature of the moiety attached to the sulfur atom. Except for a quinoline-, and to a minor extent a nitro-derivative, the most selective hits induced a rapid (within 60 min) and marked perturbation of the LMWT-redox homeostasis. The formation of selenenylsulfide glycoconjugates with free thiols has been identified as a potential mechanism involved in this process.

A METHOD FOR ISOLATING AND SEQUENCING TRYPANOSOME CELLS TO INVESTIGATE SPECIES ASSOCIATIONS IN MULTIPLE MORPHOTYPE INFECTIONS.

Trypanosome infections containing multiple morphologies have been described from all classes of vertebrates, including mammals, birds, non-avian reptiles, amphibians, and fish. These mixed infections make it challenging to evaluate trypanosome diversity, as it is not immediately clear whether the forms present in the bloodstream represent different species or a single pleomorphic species. Amphibians are common hosts for trypanosomes and are often infected by multiple trypanosome morphologies in the bloodstream. Based on morphological observations and life cycle studies, many authors have considered multiple trypanosome morphotypes found infecting the same frogs to be a single pleomorphic species. However, molecular evidence supporting pleomorphic trypanosome species in amphibians is lacking, primarily because linking sequence data to bloodstream trypanosome morphology in mixed infections is extremely challenging. Here we present methods to isolate individual trypanosome cells of 6 morphotypes from frog blood for nested PCR of the 18S rRNA and gGAPDH genes. Single trypanosome cells were isolated by dilution and 3 DNA extraction methods, and 5 nested PCR primer regimes were utilized to optimize amplification from very low starting concentrations. The success rates of extraction methods ranged from 29 to 50% with the use of a Direct PCR kit having the highest success rate. Although the success rate varied in the different combinations of extraction methods and primer regimes, multiple individuals of all 6 trypanosome morphotypes were sequenced for both genes in a novel way that links sequence data to cell morphology by observing isolated cells with a microscope before PCR amplification. All 6 morphologically distinguishable morphotypes coinfecting a frog were genetically distinct. The only other recent molecular study on amphibian trypanosomes also found genetic differences between morphotypes in multiple infections. Together these studies suggest that the occurrence of pleomorphism may be overestimated in amphibian trypanosomes. The methods presented here offer a promising solution to characterize trypanosome diversity within multiple morphotype infections.

The indole motif is essential for the antitrypanosomal activity of N5-substituted paullones.

Severe infections with potentially fatal outcomes are caused by parasites from the genera Trypanosoma and Leishmania (class Kinetoplastea). The diseases affect people of remote areas in the tropics and subtropics with limited access to adequate health care. Besides insufficient diagnostics, treatment options are limited, with tenuous developments in recent years. Therefore, new antitrypanosomal antiinfectives are required to fight these maladies. In the presented approach, new compounds were developed and tested on the target trypanothione synthetase (TryS). This enzyme is crucial to the kinetoplastids' unique trypanothione-based thiol redox metabolism and thus for pathogen survival. Preceding studies have shown that N5-substituted paullones display antitrypanosomal activity as well as TryS inhibition. Herein, this compound class was further examined regarding the structure-activity relationships (SAR). Diverse benzazepinone derivatives were designed and tested in cell-based assays on bloodstream Trypanosoma brucei brucei (T. b. brucei) and intracellular amastigotes of Leishmania infantum (L. infantum) as well as in enzyme-based assays on L. infantum TryS (LiTryS) and T. b. brucei TryS (TbTryS). While an exchange of just the substituent in the 9-position of paullones led to potent inhibitors on LiTryS and T. b. brucei parasites, new compounds lacking the indole moiety showed a total loss of activity in both assays. Conclusively, the indole as part of the paullone structure is pivotal for keeping the TryS inhibitory and antitrypanosomal activity of this substance class.

The developmental hierarchy and scarcity of replicative slender trypanosomes in blood challenges their role in infection maintenance

The development of Trypanosoma brucei in its mammalian host is marked by a distinct morphological change as replicative "slender" forms differentiate into cell cycle arrested "stumpy" forms in a quorum-sensing-dependent manner. Although stumpy forms dominate chronic infections at the population level, the proportion of replicative parasites at the individual cell level and the irreversibility of arrest in the bloodstream are unclear. Here, we experimentally demonstrate that developmental cell cycle arrest is definitively irreversible in acute and chronic infections in mice. Furthermore, analysis of replicative capacity and single-cell transcriptome profiling reveal a temporal hierarchy, whereby cell cycle arrest and appearance of a reversible stumpy-like transcriptome precede irreversible commitment and morphological change. Unexpectedly, we show that proliferating parasites are exceptionally scarce in the blood after infections are established. This challenges the ability of bloodstream trypanosomes to sustain infection by proliferation or antigenic variation, these parasites instead being overwhelmingly adapted for transmission.

New multifunctional Ru(II) organometallic compounds show activity against Trypanosoma brucei and Leishmania infantum

Human African trypanosomiasis (sleeping sickness) and leishmaniasis are prevalent zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Leishmania spp). Additionally, both are co-endemic in certain regions of the world. Only a small number of old drugs exist for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these trypanosomatid parasites by including in a single molecule 1,1′-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: 8-hydroxyquinoline derivatives (8HQs) and polypyridyl ligands (NN). Three [Ru(8HQs)(dppf)(NN)](PF6) compounds were synthesized and fully characterized. They showed in vitro activity on bloodstream Trypanosoma brucei (IC50 140–310 nM) and on Leishmania infantum promastigotes (IC50 3.0–4.8 μM). The compounds showed good selectivity towards T. brucei in respect to J774 murine macrophages as mammalian cell model (SI 15–38). Changing hexafluorophosphate counterion by chloride led to a three-fold increase in activity on both parasites and to a two to three-fold increase in selectivity towards the pathogens. The compounds affect in vitro at least the targets of the individual bioactive moieties included in the new chemical entities: DNA and generation of ROS. The compounds are stable in solution and are more lipophilic than the free bioactive ligands. No clear correlation between lipophilicity, interaction with DNA or generation of ROS and activity was detected, which agrees with their overall similar anti-trypanosoma potency and selectivity. These compounds are promising candidates for further drug development.

Organocatalytic Synthesis and Anti‐Trypanosomal Activity Evaluation of L‐Pentofuranose‐Mimetic Iminosugars

AbstractA series of L‐lyxo and L‐xylo‐ pentofuranose‐mimetic iminosugars and derivatives were efficiently synthesized in a stereoselective manner, through a concise strategy which involves an organocatalyzed Mannich reaction of an imine and a protected dioxanone as key step, using D‐proline as catalyst. A preliminary evaluation of their activity as potential anti‐trypanosomal agents was studied. One of the synthesized compounds showed an interesting activity against bloodstream Trypanosoma brucei brucei (EC50=3.8±1.0 μM).

Iboga Inspired N-Indolylethyl-Substituted Isoquinuclidines as a Bioactive Scaffold: Chemoenzymatic Synthesis and Characterization as GDNF Releasers and Antitrypanosoma Agents.

The first stage of the drug discovery process involves the identification of small compounds with biological activity. Iboga alkaloids are monoterpene indole alkaloids (MIAs) containing a fused isoquinuclidine-tetrahydroazepine ring. Both the natural products and the iboga-inspired synthetic analogs have shown a wide variety of biological activities. Herein, we describe the chemoenzymatic preparation of a small library of novel N-indolylethyl-substituted isoquinuclidines as iboga-inspired compounds, using toluene as a starting material and an imine Diels–Alder reaction as the key step in the synthesis. The new iboga series was investigated for its potential to promote the release of glial cell line-derived neurotrophic factor (GDNF) by C6 glioma cells, and to inhibit the growth of infective trypanosomes. GDNF is a neurotrophic factor widely recognized by its crucial role in development, survival, maintenance, and protection of dopaminergic neuronal circuitries affected in several neurological and psychiatric pathologies. Four compounds of the series showed promising activity as GDNF releasers, and a leading structure (compound 11) was identified for further studies. The same four compounds impaired the growth of bloodstream Trypanosoma brucei brucei (EC50 1–8 μM) and two of them (compounds 6 and 14) showed a good selectivity index.

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.

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome.

The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.

The mitochondrial peroxiredoxin displays distinct roles in different developmental stages of African trypanosomes

Hydroperoxide reduction in African trypanosomes relies on 2-Cys-peroxiredoxins (Prxs) and glutathione peroxidase-type enzymes (Pxs) which both obtain their reducing equivalents from the trypanothione/tryparedoxin couple and thus act as tryparedoxin peroxidases. While the cytosolic forms of the peroxidases are essential, the mitochondrial mPrx and Px III appear dispensable in bloodstream Trypanosoma brucei. This led to the suggestion that in this developmental stage which is characterized by a mitochondrion that lacks an active respiratory chain, only one of the two peroxidases might be required. Here we show that bloodstream cells in which the Px III gene is deleted and mPrx is down-regulated by RNA interference, proliferate as the parental cells indicating that both mitochondrial peroxidases are dispensable. However, when we raised the culture temperature to 39 °C, mPrx-depleted cells died indicating that under conditions mimicking a fever situation in the mammalian host, the protein becomes essential. In contrast, depletion of mPrx in insect stage procyclic T. brucei causes a proliferation defect under standard conditions at 27 °C, in the absence of any stress. In the absence of mPrx, a tryparedoxin-coupled roGFP2 biosensor expressed in the mitochondrial matrix is unable to respond to antimycin A treatment. Thus mPrx reduces mitochondrial H2O2 with the generation of trypanothione disulfide and acts as peroxidase. However, mPrx-depleted procyclic cells neither display any alteration in the cytosolic or mitochondrial trypanothione redox state nor increased sensitivity towards exogenous oxidative stressors suggesting that the peroxidase activity is not the crucial physiological function. After prolonged mPrx-depletion, the cells almost stop proliferation and display a highly elongated shape and diminished MitoTracker Red staining. In contrast to the situation in the mammalian bloodstream T. brucei and Leishmania, mPrx appears to play a constitutive role for the morphology, mitochondrial function and proliferation of the insect stage of African trypanosomes.

SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes

SUMOylation is a post‐translational modification that positively regulates monoallelic expression of the trypanosome variant surface glycoprotein (VSG). The presence of a highly SUMOylated focus associated with the nuclear body, where the VSG gene is transcribed, further suggests an important role of SUMOylation in regulating VSG expression. Here, we show that SNF2PH, a SUMOylated plant homeodomain (PH)‐transcription factor, is upregulated in the bloodstream form of the parasite and enriched at the active VSG telomere. SUMOylation promotes the recruitment of SNF2PH to the VSG promoter, where it is required to maintain RNA polymerase I and thus to regulate VSG transcript levels. Further, ectopic overexpression of SNF2PH in insect forms, but not of a mutant lacking the PH domain, induces the expression of bloodstream stage‐specific surface proteins. These data suggest that SNF2PH SUMOylation positively regulates VSG monoallelic transcription, while the PH domain is required for the expression of bloodstream‐specific surface proteins. Thus, SNF2PH functions as a positive activator, linking expression of infective form surface proteins and VSG regulation, thereby acting as a major regulator of pathogenicity.

The Trypanosomal Transferrin Receptor of Trypanosoma Brucei-A Review.

Iron is an essential element for life. Its uptake and utility requires a careful balancing with its toxic capacity, with mammals evolving a safe and bio-viable means of its transport and storage. This transport and storage is also utilized as part of the iron-sequestration arsenal employed by the mammalian hosts’ ‘nutritional immunity’ against parasites. Interestingly, a key element of iron transport, i.e., serum transferrin (Tf), is an essential growth factor for parasitic haemo-protozoans of the genus Trypanosoma. These are major mammalian parasites causing the diseases human African trypanosomosis (HAT) and animal trypanosomosis (AT). Using components of their well-characterized immune evasion system, bloodstream Trypanosoma brucei parasites adapt and scavenge for the mammalian host serum transferrin within their broad host range. The expression site associated genes (ESAG6 and 7) are utilized to construct a heterodimeric serum Tf binding complex which, within its niche in the flagellar pocket, and coupled to the trypanosomes’ fast endocytic rate, allows receptor-mediated acquisition of essential iron from their environment. This review summarizes current knowledge of the trypanosomal transferrin receptor (TfR), with emphasis on the structure and function of the receptor, both in physiological conditions as well as in conditions where the iron supply to parasites is being limited. Potential applications using current knowledge of the parasite receptor are also briefly discussed, primarily focused on potential therapeutic interventions.

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions.

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_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_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_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_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_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.

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl-phosphatidylinositol (GPI)-anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI-anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI-anchored surface proteins. We proposed a two-step model for VSG turnover involving release of VSG-containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.

Serological tests for gambiense human African trypanosomiasis detect antibodies in cattle

BackgroundSerological tests for gambiense human African trypanosomiasis (gHAT) detect antibodies to antigens on the cell surface of bloodstream trypanosomes. As trypanosomes that cause animal African trypanosomiasis (AAT) also express related antigens, we have evaluated two rapid diagnostic tests (RDTs) on cattle in trypanosomiasis endemic and non-endemic regions, to determine whether gHAT serological tests could also be used to screen for AAT.MethodsTwo RDTs, 1G RDT, made with native antigens, and p2G RDT, made with recombinant antigens, were tested on 121 cattle in a trypanosomiasis-free region, and on 312 cattle from a rhodesiense HAT and AAT endemic region. A subset of samples from the endemic region were also tested with two immune trypanolysis (TL) tests. The sensitivity of the tests was estimated by evaluating the result of the RDT on samples that were positive by both microscopy and internal transcribed spacer (ITS) PCR, whilst specificity was the result of the RDT on samples that were negative by ITS PCR and microscopy, and others from the non-endemic region.ResultsThe specificity of the p2G RDT on cattle from the non-endemic region was 97.5% (95% CI: 93.0–99.2%), compared to only 57.9% (95% CI: 48.9–66.3%) for 1G RDT. The specificities of 1G RDT, p2G RDT and TL on endemic control cattle were 14.6% (95% CI: 9.7–21.5%), 22.6% (95% CI: 16.4–30.3%) and 68.3% (95% CI: 59.6–75.9%), respectively. The sensitivities of the tests on trypanosome positive samples were 85.1% (95% CI: 79.1–89.7%), 89.1% (95% CI: 83.7–93.0%) and 59.3% (95% CI: 51.8–66.4%), respectively. Among the same samples, 51.7% were positive by both TL and the 1G RDT.ConclusionsThese serological tests detect cross-reacting antibodies in cattle. The p2G RDT based on recombinant antigens had a high specificity in a non-endemic region, while the 1G RDT had a lower specificity, suggesting cross-reactivity with other pathogens.

Stress-Induced Protein S-Glutathionylation and S-Trypanothionylation in African Trypanosomes-A Quantitative Redox Proteome and Thiol Analysis.

Aims: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation.Results: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification.Innovation: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides.Conclusion: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517–533.

Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes.

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin—thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0–2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.

Novel Effects of Lapatinib Revealed in the African Trypanosome by Using Hypothesis-Generating Proteomics and Chemical Biology Strategies.

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei Lapatinib, a human epidermal growth factor receptor (EGFR) inhibitor, can cure 25% of trypanosome-infected mice, although the parasite lacks EGFR-like tyrosine kinases. Four trypanosome protein kinases associate with lapatinib, suggesting that the drug may be a multitargeted inhibitor of phosphoprotein signaling in the bloodstream trypanosome. Phosphoprotein signaling pathways in T. brucei have diverged significantly from those in humans. As a first step in the evaluation of the polypharmacology of lapatinib in T. brucei, we performed a proteome-wide phosphopeptide analysis before and after drug addition to cells. Lapatinib caused dephosphorylation of Ser/Thr sites on proteins predicted to be involved in scaffolding, gene expression, and intracellular vesicle trafficking. To explore the perturbation of phosphotyrosine (pTyr)-dependent signaling by lapatinib, proteins in lapatinib-susceptible pTyr complexes were identified by affinity chromatography; they included BILBO-1, MORN, and paraflagellar rod (PFR) proteins PFR1 and PFR2. These data led us to hypothesize that lapatinib disrupts PFR functions and/or endocytosis in the trypanosome. In direct chemical biology tests of these speculations, lapatinib-treated trypanosomes (i) lost segments of the PFR inside the flagellum, (ii) were inhibited in the endocytosis of transferrin, and (iii) changed morphology from long and slender to rounded. Thus, our hypothesis-generating phosphoproteomics strategy predicted novel physiological pathways perturbed by lapatinib, which were verified experimentally. General implications of this workflow for identifying signaling pathways perturbed by drug hits discovered in phenotypic screens are discussed.

Discovery of a Carbazole-Derived Lead Drug for Human African Trypanosomiasis.

The protozoan parasite Trypanosoma brucei causes the fatal illness human African trypanosomiasis (HAT). Standard of care medications currently used to treat HAT have severe limitations, and there is a need to find new chemical entities that are active against infections of T. brucei. Following a “drug repurposing” approach, we tested anti-trypanosomal effects of carbazole-derived compounds called “Curaxins”. In vitro screening of 26 compounds revealed 22 with nanomolar potency against axenically cultured bloodstream trypanosomes. In a murine model of HAT, oral administration of compound 1 cured the disease. These studies established 1 as a lead for development of drugs against HAT. Pharmacological time-course studies revealed the primary effect of 1 to be concurrent inhibition of mitosis coupled with aberrant licensing of S-phase entry. Consequently, polyploid trypanosomes containing 8C equivalent of DNA per nucleus and three or four kinetoplasts were produced. These effects of 1 on the trypanosome are reminiscent of “mitotic slippage” or endoreplication observed in some other eukaryotes.