Trypanosoma Brucei Research Articles

APOL 1 Risk Genotype is associated with Albuminuria in Sub-Saharan African without Hypertension: A Case Study of Trypanosoma Brucei Gambiense Endemic Area

Purpose. An association between APOL1 risk genotypes (HRG) and hypertension has been reported in African Americans with chronic kidney diseases (CKD). However, such data from African populations living in a Human African Trypanosomiasis (HAT) endemic area remain limited.This study assessed the association between APOL1 high-risk genotypes (HRG) and hypertension among sub-Saharan African in T.b. gambiense endemic area.
 Methodology. This cross-sectional study enrolled 94 HAT-infected and 144 non–infected participants in Masimanimba, the Democratic Republic of the Congo, from April 2019 to April 2021. We evaluated the association between APOL1 HRG and hypertension in HAT-infected and non–infected participants. APOL1 HRG was defined as the presence of two risk variants (G1/G1, G2/G2, or G1/G2), and a low-risk genotype (LRG) with the presence of 0 or 1 single variant. The elevated albuminuria was defined as urinary albumin/creatinine ratio ≥ 30 mg/g. Student’s and Pearson's Chi2 tests or Fisher’s exact test were used to compare means and proportions. The Wilcoxon/Mann–Whitney test was used to compare medians. A multivariate logistic regression model was used to identify independent determinants of hypertension. Odds ratios were provided with their 95% confidence intervals (Cis). Statistical significance was set at p < 0.05, based on 2-tailed test.
 Findings. APOL1 sequence analysis revealed that 3 of 21 (14.3%) hypertensive participants carried HRG (G1G1) and 7 of 103 (6.8%) non-hypertensive carried HRG (G1G1, G2G2, and G1G2) (p=0.371). The frequency of APOL1 HRG among hypertensive participants was 14.3% in both HAT- infected and uninfected individuals. Ten of 21 (47.6%) hypertensive individuals with elevated albuminuria had a higher incidence of CKD (100% vs. 0%; p < 0.001) and HRG (30% vs. 0%; p = 0.09) than 11 (52.3%) without albuminuria who carried LRG. Of 103 non-hypertensive subjects, 43 (41.7%) with elevated albuminuria had a higher frequency of HRG (16.3% vs. 0%; p = 0.002) and CKD (100% vs. 1.7%; p<0.001) compared with 60 of 103 (58.3%) without albuminuria who carried LRG.
 Unique contributor to theory, policy and practice: APOL1 HRG was associated with albuminuria and CKD, regardless of the hypertension status in T.b. gambiense HAT endemic area. However, further prospective cohort studies are required to confirm these results. The High-risk subjects will benefit from early preventive measures in low-income countries.

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463–645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

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.

An In vitro and in silico investigation of the antitrypanosomal activities of the stem bark extracts of Anopyxis klaineana (Pierre) Engl

African Trypanosomiasis caused by trypanosome parasites continues to be a major neglected health problem, particularly in developing countries. Current treatments are marked by serious side effects, low effectiveness, high toxicity, and drug resistance prompting the need to develop novel, safe, effective, and alternative antitrypanosomal compounds. Anopyxis klaineana is an ethnomedicinal plant used in West Africa to treat many ailments including protozoan diseases. In this study, we investigated the antitrypanosomal potential of stem bark extracts of A. klaineana through in vitro and in silico approaches. A. klaineana extracts were tested for their antitrypanosomal activities against Trypanosoma brucei parasite in vitro using Alamar blue assay. In addition, the antioxidant and cytotoxic activities were determined. LC-ESI-QTOF-MS was used to identify potential bioactive compounds present in the A. klaineana extracts. Bioactive compounds identified were subjected to molecular docking studies against Trypanosoma brucei's trypanothione reductase (TR) and Uridine Diphosphate Galactose 4′-Epimerase (UDP). The A. klaineana extracts (methanol, hexane, chloroform, and ethyl acetate) exhibited potential anti-trypanosomal activities with IC50 values of 21.25 ± 0.755,4.35 ± 0.166,2.57 ± 0.153 and 22.92 ± 2.321 μg/mL respectively. Moreover, the methanolic crude extracts showed moderate cytotoxicity against HepG2 and PNT2 cells, with IC50 values of 68.0 ± 2.05 and 78.7 ± 2.63 μg/mL respectively. LC-MS analysis revealed the presence of 24 bioactive compounds with 5 being druglike. Risperidone, Ranolazine, Dihydro-7-Desacetyldeoxygedunin, 6 beta-Hydroxytriamcinolone acetonide, and Dimethylmatairesinol were identified as novel potential inhibitors of TR and UDP with binding affinities of −10.4, −7.9, −8.7, −8.4 and −7.1 kcal/mol respectively against TR and −10.8, −8.4, −8.4, −7.6 and −8.1 respectively against UDP. This study indicates that A. klaineana has potential antitrypanosomal properties and therefore may have the potential to be developed as a therapeutic intervention for treating African trypanosomiasis.

Target-Based 6-5 Fused Ring Heterocyclic Scaffolds Display Broad Antiparasitic Potency In Vitro

Malaria, leishmaniasis, and African trypanosomiasis are protozoan diseases that constitute major global health problems, especially in developing countries; however, the development of drug resistance coupled with the toxicity of current treatments has hindered their management. The involvement of certain enzymes (dihydrofolate reductase [DHFR]) or proteins (potassium channels) in the pathogenesis of these protozoan diseases is undeniable. In this study, a series of three DHFR inhibitors (6-5 fused heterocyclic derivatives X, Y, and Z) and one K+ channel blocker (E4031) were screened for their inhibitory effects on Leishmania donovani, Plasmodium falciparum, and Trypanosoma brucei. A resazurin assay was used to assess the antitrypanosomal and antileishmanial activities of the test compounds, whereas the antiplasmodial activity was evaluated through the SYBR Green I test. Moreover, the cytotoxicities of the test compounds were evaluated in Vero, Raw 264.7, and HepG-2 cells using a resazurin-based test, while their pharmacokinetic properties were predicted using the online tool, pkCSM. As a result, compound Y exhibited selective (selectivity index range: from 2.69 to &gt;61.4; Vero, Raw 264.7, and HepG-2 cells) and broad-spectrum antiprotozoal activity against L. donovani promastigotes (IC50: 12.4 µM), amastigotes (IC50: 4.28 µM), P. falciparum (IC50: 0.028 µM), and T. brucei brucei (IC50: 0.81 µM). In addition, compound X inhibited the growth of P. falciparum (IC50: 0.0052 µM) and T. brucei brucei (IC50: 6.49 µM). In silico screening of the active antiprotozoal compounds revealed positive drug likeness scores, as none of the criteria for Lipinski’s rule were violated by these compounds. However, in-depth pharmacokinetic and mechanistic studies are warranted to support the discovery of novel antiprotozoal agents against malaria, leishmaniasis, and African trypanosomiasis by repurposing K+ channel blockers and DHFR inhibitors.

A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense.

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunoreactive proteins in experimental bovine infections, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission.

Novel quinoline derivatives with broad-spectrum antiprotozoal activities.

Several quinoline derivatives incorporating arylnitro and aminochalcone moieties were synthesized and evaluated in vitro against a broad panel of trypanosomatid protozoan parasites responsible for sleeping sickness (Trypanosoma brucei rhodesiense), nagana (Trypanosoma brucei brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania infantum). Several of the compounds demonstrated significant antiprotozoal activity. Specifically, compounds 2c, 2d, and 4i displayed submicromolar activity against T. b. rhodesiense with half-maximal effective concentration (EC50) values of 0.68, 0.8, and 0.19 µM, respectively, and with a high selectivity relative to human lung fibroblasts and mouse primary macrophages (∼100-fold). Compounds 2d and 4i also showed considerable activity against T. b. brucei with EC50 values of 1.4 and 0.4 µM, respectively.

Tractable Quinolone Hydrazides Exhibiting Sub-Micromolar and Broad Spectrum Antitrypanosomal Activities.

Nagana and Human African Trypanosomiasis (HAT), caused by (sub)species of Trypanosoma, are diseases that impede human and animal health, and economic growth in Africa. The few drugs available have drawbacks including suboptimal efficacy, adverse effects, drug resistance, and difficult routes of administration. New drugs are needed. A series of 20 novel quinolone compounds with affordable synthetic routes was made and evaluated in vitro against Trypanosoma brucei and HEK293 cells. Of the 20 compounds, 12 had sub-micromolar potencies against the parasite (EC50 values=0.051-0.57 μM), and most were non-toxic to HEK293 cells (CC50 values>5 μM). Two of the most potent compounds presented sub-micromolar activities against other trypanosome (sub)species (T. cruzi and T. b. rhodesiense). Although aqueous solubility is poor, both compounds possess good logD values (2-3), and either robust or poor microsomal stability profiles. These varying attributes will be addressed in future reports.

First comprehensive untargeted metabolomics study of suramin-treated Trypanosoma brucei: an integrated data analysis workflow from multifactor data modelling to functional analysis.

Human African trypanosomiasis, commonly known as sleeping sickness, is a vector-borne parasitic disease prevalent in sub-Saharan Africa and transmitted by the tsetse fly. Suramin, a medication with a long history of clinical use, has demonstrated varied modes of action against Trypanosoma brucei. This study employs a comprehensive workflow to investigate the metabolic effects of suramin on T. brucei, utilizing a multimodal metabolomics approach. The primary aim of this study is to comprehensively analyze the metabolic impact of suramin on T. brucei using a combined liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) approach. Statistical analyses, encompassing multivariate analysis and pathway enrichment analysis, are applied to elucidate significant variations and metabolic changes resulting from suramin treatment. A detailed methodology involving the integration of high-resolution data from LC-MS and NMR techniques is presented. The study conducts a thorough analysis of metabolite profiles in both suramin-treated and control T. brucei brucei samples. Statistical techniques, including ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA), ANOVA 2 analysis, and bootstrap tests, are employed to discern the effects of suramin treatment on the metabolomics outcomes. Our investigation reveals substantial differences in metabolic profiles between the control and suramin-treated groups. ASCA and PCA analysis confirm distinct separation between these groups in both MS-negative and NMR analyses. Furthermore, ANOVA 2 analysis and bootstrap tests confirmed the significance of treatment, time, and interaction effects on the metabolomics outcomes. Functional analysis of the data from LC-MS highlighted the impact of treatment on amino-acid, and amino-sugar and nucleotide-sugar metabolism, while time effects were observed on carbon intermediary metabolism (notably glycolysis and di- and tricarboxylic acids of the succinate production pathway and tricarboxylic acid (TCA) cycle). Through the integration of LC-MS and NMR techniques coupled with advanced statistical analyses, this study identifies distinctive metabolic signatures and pathways associated with suramin treatment in T. brucei. These findings contribute to a deeper understanding of the pharmacological impact of suramin and have the potential to inform the development of more efficacious therapeutic strategies against African trypanosomiasis.

The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies.

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

A kinesin-13 family kinesin in Trypanosoma brucei regulates cytokinesis and cytoskeleton morphogenesis by promoting microtubule bundling.

The early branching eukaryote Trypanosoma brucei divides uni-directionally along the longitudinal cell axis from the cell anterior toward the cell posterior, and the cleavage furrow ingresses along the cell division plane between the new and the old flagella of a dividing bi-flagellated cell. Regulation of cytokinesis in T. brucei involves actomyosin-independent machineries and trypanosome-specific signaling pathways, but the molecular mechanisms underlying cell division plane positioning remain poorly understood. Here we report a kinesin-13 family protein, KIN13-5, that functions downstream of FPRC in the cytokinesis regulatory pathway and determines cell division plane placement. KIN13-5 localizes to multiple cytoskeletal structures, interacts with FPRC, and depends on FPRC for localization to the site of cytokinesis initiation. Knockdown of KIN13-5 causes loss of microtubule bundling at both ends of the cell division plane, leading to mis-placement of the cleavage furrow and unequal cytokinesis, and at the posterior cell tip, causing the formation of a blunt posterior. In vitro biochemical assays demonstrate that KIN13-5 bundles microtubules, providing mechanistic insights into the role of KIN13-5 in cytokinesis and posterior morphogenesis. Altogether, KIN13-5 promotes microtubule bundle formation to ensure cleavage furrow placement and to maintain posterior cytoskeleton morphology in T. brucei.

Structural analysis of the Trypanosoma brucei EIF4E6/EIF4G5 complex reveals details of the interaction between unusual eIF4F subunits

Recognition of the mRNA 5′ end is a critical step needed for translation initiation. This step is performed by the cap binding protein eIF4E, which joins the larger eIF4G subunit to form the eIF4F complex. Trypanosomatids have a minimum of five different eIF4F-like complexes formed through specific but not well-defined interactions between four different eIF4E and five eIF4G homologues. The EIF4E6/EIF4G5 complex has been linked with the stage-specific translation of mRNAs encoding the major Trypanosoma brucei virulence factors. Here, to better define the molecular basis for the TbEIF4E6/TbEIF4G5 interaction, we describe the identification of the peptide interacting with TbEIF4E6 in the region comprising residues 79–166 of TbEIF4G5. The TbEIF4E6-TbEIF4G5_79-116 complex reconstituted with recombinant proteins is highly stable even in the absence of cap-4. The crystal structure of the complex was subsequently solved, revealing extensive interacting surfaces. Comparative analyses highlight the conservation of the overall structural arrangement of different eIF4E/eIF4G complexes. However, highly different interacting surfaces are formed with distinct binding contacts occurring both in the canonical and noncanonical elements within eIF4G and the respective eIF4E counterpart. These specific pairs of complementary interacting surfaces are likely responsible for the selective association needed for the formation of distinct eIF4F complexes in trypanosomatids.

Unique Interactions of the Small Translocases of the Mitochondrial Inner Membrane (Tims) in Trypanosoma brucei.

The infectious agent for African trypanosomiasis, Trypanosoma brucei, possesses a unique and essential translocase of the mitochondrial inner membrane, known as the TbTIM17 complex. TbTim17 associates with six small TbTims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction patterns of these smaller TbTims with each other and TbTim17 are not clear. Through yeast two-hybrid (Y2H) and co-immunoprecipitation analyses, we demonstrate that all six small TbTims interact with each other. Stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. However, TbTim10 shows weaker associations with TbTim13, which has a stronger connection with TbTim17. Each of the small TbTims also interacts strongly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial for maintaining the steady-state levels of the TbTIM17 complex. Further analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except for TbTim13, is present in ~70 kDa complexes, possibly existing in heterohexameric forms. In contrast, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionates with TbTim17. Altogether, our results demonstrate that, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific to T. brucei.

A yeast-based high-throughput screen identifies inhibitors of trypanosomatid HRG heme transporters with potent leishmanicidal and trypanocidal activity

ObjectivesNew drugs are required to treat neglected diseases caused by trypanosomatid parasites such as Leishmania, Trypanosoma brucei and Trypanosoma cruzi. An Achilles’ heel of these parasites is their heme auxotrophy; they have an absolute dependence on scavenging this molecule from the host, and trypanosomatid HRG heme transporters (TrypHRG) play an important role in this process. As these proteins are essential for the parasites and have low similarity with their human orthologue, they have been proposed as attractive therapeutic targets. Here, we have developed two yeast-based assays that allow an inexpensive high-throughput screening of TrypHRG inhibitors within a cellular context. MethodsWe first assessed that Leishmania major, Leishmania donovani and T. brucei HRG proteins were heterologously expressed in the digestive vacuole membrane of a mutant heme auxotrophic yeast strain. Here, TrypHRG imports hemoglobinderived heme into the cytosol, allowing mutant yeast to grow in the presence of low hemoglobin concentrations and promoting the activity of hemeproteins such as catalase, which was used as a reporter of cytosolic heme levels. ResultsIn the presence of a TrypHRG inhibitor, both catalase activity (test 1) and yeast growth (test 2) were diminished, being easily monitored. The assays were then tested on a pilot scale for HTS purposes using a collection of repurposing drugs and food antioxidants. Some of the TrypHRG inhibitors identified in yeast presented strong trypanocidal and leishmanicidal activity in the submicromolar range, proving the potential of this approach. ConclusionsCumulatively, it was shown that the inhibition bioassays developed were robust and applicable to large-scale HTS.

Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites

RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB, and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first published draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131, and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major.Key points• A four-subunit TFIIIC complex is present in T. brucei and L. major• TbTau95 associates with tRNA and U2 snRNA genes• Putative interacting partners of Tau95 might include some RNAP II regulators

Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location-specific mitochondrial targeting signals and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, targeting signals of multi-pass Tims are less defined. Here, we report the characterization of the targeting signals of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy, we located at least two internal targeting signals (ITS): (i) within TM1 (31-50 AAs) and (ii) TM4 + loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the ITS for a multipass inner membrane protein in a divergent eukaryote, like T. brucei.IMPORTANCEAfrican trypanosomiasis (AT) is a deadly disease in human and domestic animals, caused by the parasitic protozoan Trypanosoma brucei. Therefore, AT is not only a concern for human health but also for economic development in the vast area of sub-Saharan Africa. T. brucei possesses a single mitochondrion per cell that imports hundreds of nuclear-encoded mitochondrial proteins for its functions. T. brucei Tim17 (TbTim17), an essential component of the TbTIM17 complex, is a nuclear-encoded protein; thus, it is necessary to be imported from the cytosol to form the TbTIM17 complex. Here, we demonstrated that the internal targeting signals within the transmembrane 1 (TM1) and TM4 with loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block TbTim17 function and parasite growth.

Experimental genetic crosses in tsetse flies of the livestock pathogen Trypanosoma congolense savannah

BackgroundIn tropical Africa animal trypanosomiasis is a disease that has severe impacts on the health and productivity of livestock in tsetse fly-infested regions. Trypanosoma congolense savannah (TCS) is one of the main causative agents and is widely distributed across the sub-Saharan tsetse belt. Population genetics analysis has shown that TCS is genetically heterogeneous and there is evidence for genetic exchange, but to date Trypanosoma brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, with meiosis and production of haploid gametes. In T. brucei sex occurs in the fly salivary glands, so by analogy, sex in TCS should occur in the proboscis, where the corresponding portion of the developmental cycle takes place. Here we test this prediction using genetically modified red and green fluorescent clones of TCS.MethodsThree fly-transmissible strains of TCS were transfected with genes for red or green fluorescent protein, linked to a gene for resistance to the antibiotic hygromycin, and experimental crosses were set up by co-transmitting red and green fluorescent lines in different combinations via tsetse flies, Glossina pallidipes. To test whether sex occurred in vitro, co-cultures of attached epimastigotes of one red and one green fluorescent TCS strain were set up and sampled at intervals for 28 days.ResultsAll interclonal crosses of genetically modified trypanosomes produced hybrids containing both red and green fluorescent proteins, but yellow fluorescent hybrids were only present among trypanosomes from the fly proboscis, not from the midgut or proventriculus. It was not possible to identify the precise life cycle stage that undergoes mating, but it is probably attached epimastigotes in the food canal of the proboscis. Yellow hybrids were seen as early as 14 days post-infection. One intraclonal cross in tsetse and in vitro co-cultures of epimastigotes also produced yellow hybrids in small numbers. The hybrid nature of the yellow fluorescent trypanosomes observed was not confirmed by genetic analysis.ConclusionsDespite absence of genetic characterisation of hybrid trypanosomes, the fact that these were produced only in the proboscis and in several independent crosses suggests that they are products of mating rather than cell fusion. The three-way strain compatibility observed is similar to that demonstrated previously for T. brucei, indicating that a simple two mating type system does not apply for either trypanosome species.Graphical

Unwrap RAP1's Mystery at Kinetoplastid Telomeres.

Although located at the chromosome end, telomeres are an essential chromosome component that helps maintain genome integrity and chromosome stability from protozoa to mammals. The role of telomere proteins in chromosome end protection is conserved, where they suppress various DNA damage response machineries and block nucleolytic degradation of the natural chromosome ends, although the detailed underlying mechanisms are not identical. In addition, the specialized telomere structure exerts a repressive epigenetic effect on expression of genes located at subtelomeres in a number of eukaryotic organisms. This so-called telomeric silencing also affects virulence of a number of microbial pathogens that undergo antigenic variation/phenotypic switching. Telomere proteins, particularly the RAP1 homologs, have been shown to be a key player for telomeric silencing. RAP1 homologs also suppress the expression of Telomere Repeat-containing RNA (TERRA), which is linked to their roles in telomere stability maintenance. The functions of RAP1s in suppressing telomere recombination are largely conserved from kinetoplastids to mammals. However, the underlying mechanisms of RAP1-mediated telomeric silencing have many species-specific features. In this review, I will focus on Trypanosoma brucei RAP1's functions in suppressing telomeric/subtelomeric DNA recombination and in the regulation of monoallelic expression of subtelomere-located major surface antigen genes. Common and unique mechanisms will be compared among RAP1 homologs, and their implications will be discussed.

A Review on Exploring the Phytochemical and Pharmacological Significance of Indigofera astragalina

Indigofera astragalina, a versatile medicinal plant, has gained significant attention because of its rich phytochemical composition and diverse therapeutic properties. I. astragalina contains sterols, phenolics, alkaloids, and flavonoids, making it ideal for a variety of therapeutic purposes. In this review, an in-depth look at the phytoconstituents and their biological activity is presented. The anti-microbial properties of the plant extracts have been demonstrated against drug-resistant Enterococcus and Staphylococcus aureus strains resistant to vancomycin and methicillin. Moreover, the plant exhibits moderate to potent anti-protozoal activity against Trypanosoma brucei and weak anti-protozoal activity against Plasmodium falciparum. In various cancer cell lines, astragalin extracts have demonstrated significant cytotoxic effects, suggesting their potential as anti-cancer agents. Due to its high phenolic content, the plant has an antioxidant effect, which has contributed to its traditional use for managing oxidative stress. In addition, I. astragalina is packed with essential amino acids, minerals (such as iron, manganese, and zinc), and vitamins. The comprehensive analysis of I. astragalina emphasizes its important role in drug discovery, nutrition, and healthcare, as well as its tremendous therapeutic potential.