Trypanosoma Brucei Research Articles

Strathclyde minor groove binders (S-MGBs) with activity against Acanthamoeba castellanii.

Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTm = 1 ± 0.1°C. Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.

In vitro Anti-trypanosomal Activity of Ethanolic and n-Hexane Extracts of Hymenocardia acida Stem Bark against Trypanosoma bruceibrucei and Trypanosoma congolense

Study’s Novelty/Excerpt The potential of Hymenocardia acida stem bark extracts as an anti-trypanosomal agent was assessed. The results indicate significant in vitro activity of both ethanolic and n-hexane extracts against Trypanosoma brucei brucei and Trypanosoma congolense. Hence, H. acida could be a valuable source for developing new, broad-spectrum trypanocidal drugs. Full Abstract The development of resistance by trypanosomes to existing trypanocidal drugs necessitates the need to search for safer and more effective alternative drugs with a broad spectrum of activity. Hence, this study was carried out to evaluate the in vitro anti-trypanosomal activity of ethanolic and n-hexane extracts of Hymenocardia acida stem bark against Trypanosoma congolense and Trypanosoma brucei brucei. The stem bark of H. acida was collected, identified, dried and then ground into fine powder. The powdered H. acida stem bark was extracted successively using n-hexane and ethanol to obtain n-hexane and ethanolic extracts, respectively. Phytochemical screening of the extracts was carried out using standard procedure. In vitro, anti-trypanosomal activity of different concentrations of the stem bark extracts (2.5 mg/mL to 40.0 mg/mL) were determined using motility assay with diminazeneaceturate (100 µg/mL) and phosphate buffered saline used as positive and negative controls, respectively. Results of the phytochemical screening revealed that flavonoids, alkaloids, saponins, and tannins were present in the ethanolic and n-hexane extracts. In vitro, the anti-trypanosomal activity of ethanolic and n-hexane extracts was observed against T. brucei brucei and T. congolense. Cessation of trypanosome motility was observed after 30 and 50 minutes of exposure to 2.5 mg/mL of ethanolic extract and n-hexane extract, respectively, for both T. brucei brucei and T. congolense. In conclusion, ethanolic and n-hexane extracts of H. acida exhibited anti-trypanosomal activity against Trypanosoma brucei brucei and Trypanosoma congolense. Hence, the plant could serve as a source of new trypanocidal drugs.

Molecular and Evolutionary Analysis of RNA-Protein Interactions in Telomerase Regulation.

Telomerase is an enzyme involved in the maintenance of telomeres. Telomere shortening due to the end-replication problem is a threat to the genome integrity of all eukaryotes. Telomerase inside cells depends on a myriad of protein-protein and RNA-protein interactions to properly assemble and regulate the function of the telomerase holoenzyme. These interactions are well studied in model eukaryotes, like humans, yeast, and the ciliated protozoan known as Tetrahymena thermophila. Emerging evidence also suggests that deep-branching eukaryotes, such as the parasitic protist Trypanosoma brucei require conserved and novel RNA-binding proteins for the assembly and function of their telomerase. In this review, we will discuss telomerase regulatory pathways in the context of telomerase-interacting proteins, with special attention paid to RNA-binding proteins. We will discuss these interactors on an evolutionary scale, from parasitic protists to humans, to provide a broader perspective on the extensive role that protein-protein and RNA-protein interactions play in regulating telomerase activity in eukaryotes.

A murine model of Trypanosoma brucei-induced myocarditis and cardiac dysfunction.

Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). Cardiac symptoms are commonly reported in HAT patients, and intracardiac parasites with accompanying myocarditis have been observed in both natural hosts and animal models of T. brucei infection. Despite the importance of T. brucei as a cause of cardiac dysfunction and the dramatic socioeconomic impact of African trypanosomiases in sub-Saharan Africa, there are currently no reproducible murine models of T. brucei-associated cardiomyopathy. We present the first clinically relevant, reproducible murine model of cardiac dysfunction in chronic T. brucei infection. Similar to humans, mice showed histological evidence of myocarditis and elevation of serum NT-proBNP with electrocardiographic abnormalities. Serum NT-proBNP levels were elevated prior to the development of severe ventricular dysfunction. On flow cytometry, myocarditis was associated with an increase of most myocardial immune cell populations, including multiple T cell and macrophage subsets, corroborating the notion that T. brucei-associated cardiac damage is an immune-mediated event. This novel mouse model represents a powerful and practical tool to investigate the pathogenesis of T. brucei-mediated heart damage and supports the development of therapeutic options for T. brucei-associated cardiac disease.

LRRC56 is an IFT cargo required for assembly of the distal dynein docking complex in Trypanosoma brucei.

Outer dynein arms (ODAs) are responsible for ciliary beating in eukaryotes. They are assembled in the cytoplasm and shipped by intraflagellar transport (IFT) before attachment to microtubule doublets via the docking complex. The LRRC56 protein has been proposed to contribute to ODAs maturation. Mutations or deletion of the LRRC56 gene lead to reduced ciliary motility in all species investigated so far, but with variable impact on dynein arm presence. Here, we investigated the role of LRRC56 in the protist Trypanosoma brucei, where its absence results in distal loss of ODAs, mostly in growing flagella. We show that LRRC56 is a transient cargo of IFT trains during flagellum construction and surprisingly, is required for efficient attachment of a subset of docking complex proteins present in the distal portion of the organelle. This relation is interdependent since the knockdown of the distal docking complex prevents LRRC56's association with the flagellum. Intriguingly, lrrc56-/- cells display shorter flagella whose maturation is delayed. Inhibition of cell division compensates for the distal ODAs absence thanks to the redistribution of the proximal docking complex, restoring ODAs attachment but not the flagellum length phenotype. This work reveals an unexpected connection between LRRC56 and the docking complex.

Aminopyridines in the development of drug candidates against protozoan neglected tropical diseases.

Neglected tropical diseases (NTDs) pose a major threat in tropical zones for impoverished populations. Difficulty of access, adverse effects or low efficacy limit the use of current therapeutic options. Therefore, development of new drugs against NTDs is a necessity. Compounds containing an aminopyridine (AP) moiety are of great interest for the design of new anti-NTD drugs due to their intrinsic properties compared with their closest chemical structures. Currently, over 40 compounds with an AP moiety are on the market, but none is used against NTDs despite active research on APs. The aim of this review is to present the medicinal chemistry work carried out with these scaffolds, against protozoan NTDs: Trypanosoma cruzi, Trypanosoma brucei or Leishmania spp.

Association between APOL 1 Risk Genotypes and Left Ventricular Hypertrophy among Sub-Saharan Africans in Trypanosoma Brucei Gambiense Endemic Rural Area

Purpose. The relationship between APOL1 variants and cardiovascular disease remains controversial, thus, this study assessed the association between APOL1 high-risk genotypes and left ventricular hypertrophy (LVH) among sub-Saharan African in T.b. gambiense endemic area. Methodology. We enrolled 238 subjects living in the region of Masimanimba, an endemic area of T.b.gambiense HAT. We evaluated the association between LVH on echocardiography and the status of APOL1 genes in participants with or without HAT. APOL1 high-risk genotype (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. 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 LVH. Odds ratios were provided with their 95% confidence intervals (Cis). Statistical significance was set at p < 0.05, based on 2-tailed test. Findings. The prevalence of LVH (31.5%) increased with age and was similar in HAT-infected and non-infected subjects (29.8% vs. 32.6%; p=0.376). The trend of a greater left ventricular mass in participants with LVH carrying APOL1 HRG compared to those with LRG was not statistically significant (141g/m2 vs. 125 g/m2; p = 0.253). The frequency of HRG among participants with LVH was similar between HAT-infected and non-infected (15.8% vs. 9.1%; p = 0.806). Age ≥ 38 years [OR 2.5 (95% CI: 1.4-4.5), p = 0.001], hypertension [OR 2.4 (95% CI: 1.1-5.3), p = 0.034], WHR > 0.5 [OR 2.0 (95% CI: 1.0-3.6), p = 0.018] and CKD [OR 1.7 (95% CI: 1.0-3.0), p = 0.049] were associated with LVH. In multivariable logistic regression analysis age ≥ 38 years was the only independent determinant of LVH [ORa 2.0 (95% CI: 1.1-3.8), p = 0.020]. Unique contribution to theory, practice and policy. An assessment of cardiovascular risk is essential for individuals with LVH carrying APOL1 HRG in order to benefit from early and appropriate medical intervention. Further larger prospective follow-up survey is required to assess the incidence of LVH in individuals with APOL1 HRG variants.

The Power of Molecular Dynamics Simulations and Their Applications to Discover Cysteine Protease Inhibitors.

A large family of enzymes with the function of hydrolyzing peptide bonds, called peptidases or cysteine proteases (CPs), are divided into three categories according to the peptide chain involved. CPs catalyze the hydrolysis of amide, ester, thiol ester, and thioester peptide bonds. They can be divided into several groups, such as papain-like (CA), viral chymotrypsin-like CPs (CB), papainlike endopeptidases of RNA viruses (CC), legumain-type caspases (CD), and showing active residues of His, Glu/Asp, Gln, Cys (CE). The catalytic mechanism of CPs is the essential cysteine residue present in the active site. These mechanisms are often studied through computational methods that provide new information about the catalytic mechanism and identify inhibitors. The role of computational methods during drug design and development stages is increasing. Methods in Computer-Aided Drug Design (CADD) accelerate the discovery process, increase the chances of selecting more promising molecules for experimental studies, and can identify critical mechanisms involved in the pathophysiology and molecular pathways of action. Molecular dynamics (MD) simulations are essential in any drug discovery program due to their high capacity for simulating a physiological environment capable of unveiling significant inhibition mechanisms of new compounds against target proteins, especially CPs. Here, a brief approach will be shown on MD simulations and how the studies were applied to identify inhibitors or critical information against cysteine protease from several microorganisms, such as Trypanosoma cruzi (cruzain), Trypanosoma brucei (rhodesain), Plasmodium spp. (falcipain), and SARS-CoV-2 (Mpro). We hope the readers will gain new insights and use our study as a guide for potential compound identifications using MD simulations.

Synthesis, structural characterization and antitrypanosomal activity of ferrocenyl-based imines with (2 or 4)-nitrophenyl-2-furan group

Two new metallocene complexes with formulation [Fe(η5-C5H5){(η5-C5H4)-NCH-2-C4H2O-(5-C6H4-2-NO2)}] (3) and [Fe(η5-C5H5){(η5-C5H4)-NCH-2-C4H2O-(5-C6H4-4-NO2)}] (4) have been synthesized and their antitrypanosomal activity has been evaluated. Complexes 3 and 4 were obtained by condensation reaction of ferrocenylamine (1) with respective 5-(2-nitrophenyl)-2-furancarboxaldehyde (2a) or 5-(4-nitrophenyl)-2-furancarboxaldehyde (2b). The complexes were fully characterized by spectroscopic techniques (FT-IR, 1H and 13C{1H} NMR, and HRMS), which confirm their correct obtainment. The crystal structures of 3 and 4 were also determined by single crystal X-ray diffraction. Furthermore, UV–visible studies revealed that compounds 3 and 4 exhibited suitable stability in DMSO:HEPES buffer solution (80:20) throughout 24 h. The in vitro antiparasitic activities of ferrocenyl imine derivatives 3 and 4 were assessed against two species of parasites, Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei). The obtained results revealed that compound 3 (EC50 = 0.44 μM) exhibited nearly a 40-fold greater efficacy as an anti-T. brucei agent compared to derivative 4 (EC50 = 16.0 μM). Moreover, compound 3 demonstrated superior potency when compared with its organometallic analogs (EC50 = 2.42–13.3 μM), which were previously reported by our research group. Interestingly, the EC50 value of 3 was found to be 8 times greater than that of nifurtimox (EC50 = 3.56 μM). The cytotoxicity of the ferrocenyl imines was also evaluated on the L6 rat skeletal myoblast cell line.

Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins.

Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.

Novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives and their antitrypanosomal activities against T.brucei

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the <1 µM range. We have demonstrated that substituted 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidines present promising antitrypanosomal hit molecules with potential for further preclinical development.

Synthesis and in vitro biological activity of chalcone derivatives as potential antiparasitic agents

Kinetoplastids are a group of flagellated protozoans including medically important parasites of the genus Trypanosoma and Leishmania. The corresponding diseases have afflicted humans for centuries. In an effort to combat kinetoplastid infections, a set of 21 chalcones was synthesized and evaluated for their in vitro anti-protozoal efficacy against Trypanosoma brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania infantum. To ensure safety, these compounds underwent a selectivity evaluation by assessing toxicity against a human lung fibroblast cell line. Compound K4 exhibited remarkable and selective trypanocidal activity against T. b. brucei with IC50 value of 0.31 ± 0.27 µM and T. b. rhodesiense with IC50 value of 0.96 ± 0.86 µM. Compound K9 also showed significant trypanocidal activity against T. b. brucei (IC50: 0.45 ± 0.14 µM) and T. b. rhodesiense (IC50: 0.93 ± 0.51 µM). In both compounds, electron withdrawing groups are appended to the styrenyl moiety.

Green-synthesized chitosan‑carbon dot nanocomposite as turn-on aptasensor for detection and quantification of Leishmania infantum parasite

Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on fluorescent probe based on an aptamer and Chitosan-CD nanocomposite. The CD used in this study were synthesized using Quercus cap extract and a microwave-assisted approach. The Chitosan-CD nanocomposite was optimized using several microscopic and spectroscopic techniques to possess a bright fluorescence emission before adding aptamer and totally quenched fluorescence after addition of aptamer. The designed probe was proficient in the detection and quantification Leishmania infantum parasite by selective targeting of poly(A) binding protein (PABP) on the surface of the parasite. The designed fluorescent biosensor with high sensitivity, excellent selectivity, and a limit of detection (LOD) of 94 cells/mL of the Leishmania infantum parasite as well as a linear response in the ranges of 188–750 cells/mL and 3000–6000 cells/mL (R2 ≥ 0.98 for both linear ranges). Additionally, the selectivity of the designed probe was evaluated in the presence of different pathogenic species such as Trypanosoma brucei parasite and Staphylococcus aureus bacteria, as well as LiIF2α and LiP2a and BSA proteins as interference substances. The results of this study shows that using Chitosan-CD nanocomposite is a great strategy for developing selective turn-on probes with extraordinary accuracy and sensitivity in identifying Leishmania infantum parasite, especially in the early stages of the disease, and it is promising for the future clinical applications.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

First person – Manu Ahmed

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Manu Ahmed is first author on ‘ Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure’, published in JCS. Manu conducted the research described in this article while a PhD student in Professor Sue Vaughan's lab at the Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK. He is now a postdoctoral fellow in the lab of Sumeda Nandadasa at the University of Massachusetts Chan Medical School, Worcester, MA, USA, investigating the molecular mechanisms of ciliopathies relating to cystic kidney disease and hydrocephalus.

Discovery of 1,3,4-Oxadiazole Derivatives as Broad-Spectrum Antiparasitic Agents.

Vector-borne parasitic diseases (VBPDs) pose a significant threat to public health on a global scale. Collectively, Human African Trypanosomiasis (HAT), Leishmaniasis, and Malaria threaten millions of people, particularly in developing countries. Climate change might alter the transmission and spread of VBPDs, leading to a global burden of these diseases. Thus, novel agents are urgently needed to expand therapeutic options and limit the spread of drug-resistant parasites. Herein, we report the development of broad-spectrum antiparasitic agents by screening a known library of antileishmanial and antimalarial compounds toward Trypanosoma brucei (T. brucei) and identifying a 1,3,4-oxadiazole derivative (19) as anti-T. brucei hit with predicted blood-brain barrier permeability. Subsequently, extensive structure-activity-relationship studies around the lipophilic tail of 19 led to a potent antitrypanosomal and antimalarial compound (27), with moderate potency also toward Leishmania infantum (L. infantum) and Leishmania tropica. In addition, we discovered a pan-active antiparasitic molecule (24), showing low-micromolar IC50s toward T. brucei and Leishmania spp. promastigotes and amastigotes, and nanomolar IC50 against Plasmodium falciparum, together with high selectivity for the parasites over mammalian cells (THP-1). Early ADME-toxicity assays were used to assess the safety profile of the compounds. Overall, we characterized 24 and 27, bearing the 1,3,4-oxadiazole privileged scaffold, as broad-spectrum low-toxicity agents for the treatment of VBPDs. An alkyne-substituted chemical probe (30) was synthesized and will be utilized in proteomics experiments aimed at deconvoluting the mechanism of action in the T. brucei parasite.

Synthesis and biological evaluation of orally active anti-Trypanosoma agents

In previous studies, we developed anti-trypanosome tubulin inhibitors with promising in vitro selectivity and activity against Human African Trypanosomiasis (HAT). However, for such agents, oral activity is crucial. This study focused on further optimizing these compounds to enhance their ligand efficiency, aiming to reduce bulkiness and hydrophobicity, which should improve solubility and, consequently, oral bioavailability. Using Trypanosoma brucei brucei cells as the parasite model and human normal kidney cells and mouse macrophage cells as the host model, we evaluated 30 new analogs synthesized through combinatorial chemistry. These analogs have fewer aromatic moieties and lower molecular weights than their predecessors. Several new analogs demonstrated IC50s in the low micromolar range, effectively inhibiting trypanosome cell growth without harming mammalian cells at the same concentration. We conducted a detailed structure–activity relationship (SAR) analysis and a docking study to assess the compounds’ binding affinity to trypanosome tubulin homolog. The results revealed a correlation between binding energy and anti-Trypanosoma activity. Importantly, compound 7 displayed significant oral activity, effectively inhibiting trypanosome cell proliferation in mice.

How are Trypanosoma brucei receptors protected from host antibody-mediated attack?

Trypanosoma brucei is the causal agent of African Trypanosomiasis in humans and other animals. It maintains a long-term infection through an antigenic variation based population survival strategy. To proliferate in a mammal, T. brucei acquires iron and haem through the receptor mediated uptake of host transferrin and haptoglobin-hemoglobin respectively. The receptors are exposed to host antibodies but this does not lead to clearance of the infection. Here we discuss how the trypanosome avoids this fate in the context of recent findings on the structure and cell biology of the receptors.

Transcriptome profiles of Trypanosoma brucei rhodesiense in Malawi reveal focus specific gene expression profiles associated with pathology.

Sleeping sickness caused by Trypanosoma brucei rhodesiense is a fatal disease and endemic in Southern and Eastern Africa. There is an urgent need to develop novel diagnostic and control tools to achieve elimination of rhodesiense sleeping sickness which might be achieved through a better understanding of trypanosome gene expression and genetics using endemic isolates. Here, we describe transcriptome profiles and population structure of endemic T. b. rhodesiense isolates in human blood in Malawi. Blood samples of r-HAT cases from Nkhotakota and Rumphi foci were collected in PaxGene tubes for RNA extraction before initiation of r-HAT treatment. 100 million reads were obtained per sample, reads were initially mapped to the human genome reference GRCh38 using HiSat2 and then the unmapped reads were mapped against Trypanosoma brucei reference transcriptome (TriTrypDB54_TbruceiTREU927) using HiSat2. Differential gene expression analysis was done using the DeSeq2 package in R. SNP calling from reads that were mapped to the T. brucei genome was done using GATK in order to identify T.b. rhodesiense population structure. 24 samples were collected from r-HAT cases of which 8 were from Rumphi and 16 from Nkhotakota foci. The isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. These parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a less symptomatic disease in Nkhotakota associated with the non-dividing stumpy form. Interestingly, the Malawi T.b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T.b. rhodesiense isolates. PCA analysis using SNPs called from the RNAseq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi. Our results suggest that the differences in disease presentation in the two foci is mainly driven by genetic differences in the parasites in the two major endemic foci of Rumphi and Nkhotakota rather than differences in the environment or host response.

Evaluation of &lt;i&gt;in vitro&lt;/i&gt; anti-trypanosomal activities of leaves, stem bark and root bark extracts of &lt;i&gt;Acacia nilotica&lt;/i&gt; (L) Willd ex Del., &lt;i&gt;Guiera senegalensis&lt;/i&gt; j. F. Gmel and &lt;i&gt;Ziziphus abyssinica&lt;/i&gt; Hochst ex A. rich

Currently, the control and treatment of African trypanosomiasis are limited by the number of chemotherapeutic drugs with associated side effects. Consequently, there is an urgent need for a non-toxic herbal treatment for African trypanosomiasis. Leaf, stem, and root bark extracts of Acacia nilotica L. and Guiera senegalensis J. F. Gmel and Ziziphus abyssinica Hochst ex A. Rich were sequentially extracted using hexane, ethyl acetate, methanol and water as solvents and evaluated for in vitro anti-trypanosomal activities against Trypanosoma brucei, and phytochemical contents. Results revealed that out of the 36 extracts, Methanol leaf extracts of G. senegalensis, aqueous leaf extract of G. senegalensis, methanol leaf extract of A. nilotica and methanol leaf extract of Z. abyssinica leaf extract (MIC 3.93±2.88, 10.98±3.21, 16.91±3.21 and 18.88±3.44 μg/ml respectively), gave the best in vitro anti-trypanosomal activity against T. b. brucei compare to the control. The quantitative phytochemical analysis of the 4 most trypanocidal plant extracts revealed the presence of alkaloids, flavonoids, terpene, quinines, saponins and tannins, with alkaloids and flavonoids having the highest concentrations (4.3.13±0.05 mg/100 g and 4.5±0.02 mg/100 g respectively) in the methanol leaf extract of G. senegalensis and quinone with the lowest concentration (0.1±0.07 mg/100 g).The methanol leaf extracts of G. senegalensis were found to have the most in vitro anti-trypanosomal activities (MIC of 3.93±2.88 ug/ml), possibly due to the high content of alkaloids and flavonoids. The results of this study revealed the potential of G. senegalensis for the treatment of African trypanosomiasis. Consequently, further studies are needed with this plant to evaluate its in vivo anti-trypanosomal potential, the structures of the bioactive compounds responsible for its activity, and its other medicinal properties.