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Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle in live L. mexicana. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant™ DyeCycle™ Orange (DCO) is non-toxic and enables quantitative DNA imaging in live promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINF's cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei and many other cell types. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division. [ABSTRACT FROM AUTHOR]

Giardia doudenalis (lamblia, intestinalis) is a protozoan parasite that inhabits the lumen of the upper small intestine of vertebrates, causing chronic abdominal pains and severe diarrhea, symptoms of giardiasis, a persistent and recurrent infection. This characteristic is mainly due to the presence of membrane variant-specific surface proteins (VSPs) that give this parasite the ability to successively infect the host through antigenic variation. Using high-resolution scanning microscopy (HR-SM), we observed the presence, formation, and extension of tunneling-nanotube-like surface structures in Giardia, especially following parasite challenges with VSP antibodies. They were seen all over the parasite surface, both in vitro and in vivo, showing that G. duodenalis nanotube formation occurs in complex environments such as the gut. In addition, we also observed that some of these nanotubes displayed a periodic strangulation that produces 100 nm vesicles that seemed to be released in a process similar to that previously observed in Trypanosoma brucei. The presence of nanotube-like structures in G. duodenalis highlights yet another strategy of cellular communication utilized by these parasites, whether between themselves or with the host cell. [ABSTRACT FROM AUTHOR]

Eukaryotic cilia and flagella are essential for cell motility and sensory functions. Their biogenesis and maintenance rely on the intraflagellar transport (IFT). Several cargo adapters have been identified to aid IFT cargo transport, but how ciliary cargos are discharged from the IFT remains largely unknown. During our explorations of small GTPases ARL13 and ARL3 in Trypanosoma brucei, we found that ODA16, a known IFT cargo adapter present exclusively in motile cilia, is a specific effector of ARL3. In the cilia, active ARL3 GTPases bind to ODA16 and dissociate ODA16 from the IFT complex. Depletion of ARL3 GTPases stabilizes ODA16 interaction with the IFT, leading to ODA16 accumulation in cilia and defects in axonemal assembly. The interactions between human ODA16 homolog HsDAW1 and ARL GTPases are conserved, and these interactions are altered in HsDAW1 disease variants. These findings revealed a conserved function of ARL GTPases in IFT transport of motile ciliary components, and a mechanism of cargo unloading from the IFT. [ABSTRACT FROM AUTHOR]

Damage to the genetic material of the cell poses a universal threat to all forms of life. The DNA damage response is a coordinated cellular response to a DNA break, key to which is the phosphorylation signaling cascade. Identifying which proteins are phosphorylated is therefore crucial to understanding the mechanisms that underlie it. We have used stable isotopic labeling of amino acids in cell culture-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following double stranded DNA breaks, at two distinct loci in the genome of the single cell eukaryote Trypanosoma brucei. Here, we report on the T. brucei phosphoproteome following a single double-strand break at either a chromosome internal or subtelomeric locus, specifically the bloodstream form expression site. We detected >6500 phosphorylation sites, of which 211 form a core set of double-strand break responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we have identified two novel phosphorylation events on histone H2A and found that in response to a chromosome internal break, proteins are predominantly phosphorylated, while a greater proportion of proteins dephosphorylated following a DNA break at a subtelomeric bloodstream form expression site. Our data represent the first DNA damage phosphoproteome and provides novel insights into repair at distinct chromosomal contexts in T. brucei. [ABSTRACT FROM AUTHOR]

Background 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. Objectives 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. Methods 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. Results and discussion 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 Δ T m = 1 ± 0.1°C. Conclusions 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. [ABSTRACT FROM AUTHOR]

The skin is an anatomical reservoir for African trypanosomes, yet the prevalence of extravascular parasite carriage in the population at risk of gambiense Human African Trypanosomiasis (gHAT) remains unclear. Here, we conducted a prospective observational cohort study in the HAT foci of Forecariah and Boffa, Republic of Guinea. Of the 18,916 subjects serologically screened for gHAT, 96 were enrolled into our study. At enrolment and follow-up visits, participants underwent a dermatological examination and had blood samples and superficial skin snip biopsies taken for examination by molecular and immuno-histological methods. In seropositive individuals, dermatological symptoms were significantly more frequent as compared to seronegative controls. Trypanosoma brucei DNA was detected in the blood of 67% of confirmed cases (22/33) and 9% of unconfirmed seropositive individuals (3/32). However, parasites were detected in the extravascular dermis of up to 71% of confirmed cases (25/35) and 41% of unconfirmed seropositive individuals (13/32) by PCR and/or immuno-histochemistry. Six to twelve months after treatment, trypanosome detection in the skin dropped to 17% of confirmed cases (5/30), whereas up to 25% of unconfirmed, hence untreated, seropositive individuals (4/16) were still found positive. Dermal trypanosomes were observed in subjects from both transmission foci, however, the occurrence of pruritus and the PCR positivity rates were significantly higher in unconfirmed seropositive individuals in Forecariah. The lower sensitivity of superficial skin snip biopsies appeared critical for detecting trypanosomes in the basal dermis. These results are discussed in the context of the planned elimination of gHAT. Author summary: The skin is a reservoir for African trypanosomes. Here, we conducted a prospective study in Forecariah and Boffa, Guinea, to estimate the proportion of skin-dwelling parasites in the population. Of the 18,916 subjects screened for HAT, 96 were enrolled into our study. Participants underwent a dermatological examination and had blood samples and superficial skin biopsies taken for examination by molecular and immuno-histological methods. In individuals seropositive for HAT, dermatological symptoms were significantly more frequent. Trypanosome DNA was detected in the blood of 67% of confirmed cases and 9% of unconfirmed seropositive individuals. However, parasites were detected in the skin of up to 71% of confirmed cases and 41% of unconfirmed seropositive individuals. After treatment, trypanosome detection in the skin dropped to 17% of confirmed cases, whereas up to 25% of unconfirmed, hence untreated, seropositive individuals were still found positive. Dermal trypanosomes were observed in subjects from both regions; however, the occurrence of itching and the PCR positivity were significantly higher in unconfirmed seropositive individuals in Forecariah. The lower sensitivity of superficial skin biopsies appeared critical for detecting trypanosomes. These results are discussed in the context of the planned elimination of HAT. [ABSTRACT FROM AUTHOR]

Peptidyl-prolyl cis/trans isomerases (PPIases) are present in a wide variety of microorganisms, including protozoan parasites such as Trypanosoma cruzi, Trypanosoma brucei, Trichomonas vaginalis, Leishmania major, Leishmania donovani, Plasmodium falciparum, Plasmodium vivax, Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium hominis, all of which cause important neglected diseases. PPIases are classified as cyclophilins, FKBPs, or parvulins and play crucial roles in catalyzing the cis-trans isomerization of the peptide bond preceding a proline residue. This activity assists in correct protein folding. However, experimentally, the biological structure–function characterization of PPIases from these protozoan parasites has been poorly addressed. The recombinant production of these enzymes is highly relevant for this ongoing research. Thus, this review explores the structural diversity, functions, recombinant production, activity, and inhibition of protozoan PPIases. We also highlight their potential as biotechnological tools for the in vitro refolding of other recombinant proteins from these parasites. These applications are invaluable for the development of diagnostic and therapeutic tools. [ABSTRACT FROM AUTHOR]

The gRNA directed U-insertion and deletion editing of mitochondrial mRNAs that is essential in different life-cycle stages for the protozoan parasite Trypanosoma brucei is performed by three similar multiprotein catalytic complexes (CCs) that contain the requisite enzymes. These CCs also contain a common set of eight proteins that have no apparent direct catalytic function, including six that have an OB-fold domain. We show here that one of these OB-fold proteins, KREPA3 (A3), has structural homology to other editing proteins, is essential for editing, and is multifunctional. We investigated A3 function by analyzing the effects of single amino acid loss of function mutations, most of which were identified by screening bloodstream form (BF) parasites for loss of growth following random mutagenesis. Mutations in the zinc fingers (ZFs), an intrinsically disordered region (IDR), and several within or near the carboxy-terminal OB-fold domain variably impacted CC structural integrity and editing. Some mutations resulted in almost complete loss of CCs and its proteins and editing, whereas others retained CCs but had aberrant editing. All but a mutation which is near the OB-fold affected growth and editing in BF but not procyclic form (PF) parasites. These data indicate that multiple positions within A3 have essential functions that contribute to the structural integrity of CCs, the precision of editing and the developmental differences in editing between BF and PF stages.

Faithful mRNA decoding depends on the accuracy of aminoacyl-tRNA synthetases (ARSs). Aminoacyl-tRNA proofreading mechanisms have been well-described in bacteria, humans, and plants. However, our knowledge of translational fidelity in protozoans is limited. Trypanosoma brucei (Tb) is a eukaryotic, protozoan pathogen that causes Human African Trypanosomiasis, a fatal disease if untreated. Tb undergoes many physiological changes that are dictated by nutrient availability throughout its insect-mammal lifecycle. In the glucose-deprived insect vector, the tsetse fly, Tb use proline to make ATP via mitochondrial respiration. Alanine is one of the major by-products of proline consumption. We hypothesize that the elevated alanine pool challenges Tb prolyl-tRNA synthetase (ProRS), an ARS known to misactivate alanine in all three domains of life, resulting in high levels of misaminoacylated Ala-tRNAPro. Tb encodes two domains that are members of the INS superfamily of aminoacyl-tRNA deacylases. One homolog is appended to the N-terminus of Tb ProRS, and a second is the major domain of multi-aminoacyl-tRNA synthetase complex (MSC)-associated protein 3 (MCP3). Both ProRS and MCP3 are housed in the Tb MSC. Here, we purified Tb ProRS and MCP3 and observed robust Ala-tRNAPro deacylation activity from both enzymes in vitro. Size-exclusion chromatography multi-angle light scattering used to probe the oligomerization state of MCP3 revealed that although its unique N-terminal extension confers homodimerization in the absence of tRNA, the protein binds to tRNA as a monomer. Kinetic assays showed MCP3 alone has relaxed tRNA specificity and promiscuously hydrolyzes cognate Ala-tRNAAla; this activity is significantly reduced in the presence of Tb alanyl-tRNA synthetase, also housed in the MSC. Taken together, our results provide insight into translational fidelity mechanisms in Tb and lay the foundation for exploring MSC-associated proteins as novel drug targets. [ABSTRACT FROM AUTHOR]

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, the molecular docking model was used to investigate the antitrypanosomal inhibitory potential of Parkia biglobosa. Twenty-eight different compounds were identified as potential candidates; however, the returned binding energy (kcal/mol) suggested four suitable candidates (beta-sitosterol, epi-gallocatechin, epi-catechin-3-O-gallate, catechin and taraxerone). The antitrypanosomal activity of these compounds was investigated using a molecular docking assay against two proteins from T. brucei brucei, namely arginine kinase 3 (Accession No. EAN76668.1) and trans-sialidase (AlphaFold ID: AF-Q57YTZ-F1). Using the SwissAdme server (SwissADME), all herbal substances were screened in silico for drug-likeness according to the guidelines of Lipinsk, Egan and Veber. Molecular docking was performed to identify putative binding orientations and binding energies (BE) of drugs at the selected binding domains of trans-sialidase and arginine kinase 3 using Auto Dock Vina in the open-source Python Prescription 0.8 program. The returning binding energy (kcal/mol) of P. biglobosa's druglike compounds indicates that they bind well to the active sites of both proteins. However, additional investigation is needed to test P. biglobosa in vitro or in vivo to see if it may be used as an effective and less expensive drug because it is a more accessible plant. [ABSTRACT FROM AUTHOR]

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis. Author summary: Blood-stage trypanosome parasites have a specialised invagination on the cell surface named the flagellar pocket (FP), where invariant essential nutrient receptors are located. The pocket houses diverse proteins, including a transferrin receptor (TfR), which facilitates uptake of host transferrin-bound iron for survival. Several FP proteins, including TfR, are linked to complex sugar molecules (carbohydrates), the functions of which are not well understood. Complex carbohydrates are made by enzymes called glycosyltransferases (GTs) and previously we partially inhibited complex carbohydrate synthesis by deletion of either TbGT8 or TbGT10. However, mutant parasites lacking either one of these enzymes survived, suggesting functional redundancy. Here, we created a parasite mutant that lacks both TbGT8 and TbG10 to understand the combined effect of losing both enzymes. The mutant parasites showed a decreased ability to uptake tomato lectin, a protein that specifically binds to these sugar conjugates in the FP, indicating a reduction in carbohydrate complexity. Despite reduced complexity in the sugar structures attached to TfR, its critical function in transferrin/iron uptake remained effective. Furthermore, the mutants remained viable in culture and in animal models, challenging previous assumptions about the necessity and function of these carbohydrate conjugates. Our findings imply a greater flexibility and redundancy in the carbohydrate complex roles than previously appreciated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human African trypanosomiasis (HAT), or sleeping sickness, continues to be a major threat to human health in 36 countries throughout sub-Saharan Africa with up to 60 million people at risk. Over the last decade, there have been several advances in this area, some of which are discussed in this overview. Due to the concerted efforts of several bodies, including better identification and treatment of cases and improved tsetse fly vector control, the number of cases of HAT has declined dramatically. The clinical heterogeneity of HAT has also been increasingly recognized, and the disease, while usually fatal if untreated or inadequately treated, does not always have a uniformly fatal outcome. Improved methods of HAT diagnosis have now been developed including rapid diagnostic tests. Novel drug treatment of HAT has also been developed, notably nifurtimox–eflornithine combination therapy (NECT) for late-stage Trypanosoma brucei gambiense , oral fexinidazole for early and the early component of the late-stage of T.b. gambiense , and the new oral compounds of the oxaborole group, which have shown considerable promise in field trials. Advances in HAT neuropathogenesis have been steady, though largely incremental, with a particular focus on the role of the blood–brain barrier in parasite entry into the central nervous system and the relevant importance of both innate and adaptive immunity. While the World Health Organization goal of elimination of HAT as a public health problem by 2020 has probably been achieved, it remains to be seen whether the second more ambitious goal of interruption of transmission of HAT by 2030 will be attained. [ABSTRACT FROM AUTHOR]

The use of plants in traditional medicine is increasingly gaining ground in modern medicine because phytochemical components of most secondary metabolites can be used to treat a wide range of diseases. The anti-trypanosomal effects of ethanolic extracts of Justicia secunda leaf were investigated in albino rats. Thirty albino rats were used for the study and they were divided into six groups of five rats each. Group I was uninfected untreated (control), group II was infected untreated, group III was infected and treated with diminazene aceturate (DA), while groups IV, V and VI were infected and treated with J. secunda at 100mg/kg, 200mg/kg and 400mg/kg body weights, respectively. The parasite clearance time did not show any significant (p>0.05) difference in the extract-treated groups. However, relapse of infection occurred on day 28 post-treatment (PT) in 100mg/kg treated group and day 63 PT in 200mg/kg and 400mg/kg treated groups. The red cell parameters (PCV, Hb and RBC counts) were significantly (p<0.05) decreased in the infected groups, but improved in the groups treated with 200mg/kg and 400mg/kg to a level comparable to the uninfected untreated (control). The mean total white cell counts (TWBC) count was significantly (p<0.05) higher in the infected untreated group. The extract-treated groups did not show any variation (p>0.05) and were comparable with the uninfected untreated control. In conclusion, the ethanolic extract of J. secunda leaf exhibited dose-dependent anti-trypanosomal activity in T. brucei-infected rats and was able to ameliorate and conserve anaemia as shown in the improved haematological parameters and reduced risk of relapse. [ABSTRACT FROM AUTHOR]

A concise set of 4‐substituted pyrazolo[3,4‐d]pyrimidine carboriboside analogues was synthesized in a divergent fashion via a central intermediate. An oxaziridine reagent allowed the synthesis of a crucial hydrazine glycon intermediate, which was transformed through a base‐build‐up strategy into the desired intermediate using a commercially available pyrimidine building block. Reaction with a variety of nucleophiles allowed late‐stage diversification. All analogues were evaluated for activity against a representative panel of Trypanosomatida. A methoxy‐substituted analogue displayed single‐digit micromolar activity against two subspecies of Trypanosoma brucei in cellular assays. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pentamidine and melarsoprol are primary drugs used to treat the lethal human sleeping sickness caused by the parasite Trypanosoma brucei. Cross-resistance to these two drugs has recently been linked to aquaglyceroporin 2 of the trypanosome (TbAQP2). TbAQP2 is the first member of the aquaporin family described as capable of drug transport; however, the underlying mechanism remains unclear. Here, we present cryo-electron microscopy structures of TbAQP2 bound to pentamidine or melarsoprol. Our structural studies, together with the molecular dynamic simulations, reveal the mechanisms shaping substrate specificity and drug permeation. Multiple amino acids in TbAQP2, near the extracellular entrance and inside the pore, create an expanded conducting tunnel, sterically and energetically allowing the permeation of pentamidine and melarsoprol. Our study elucidates the mechanism of drug transport by TbAQP2, providing valuable insights to inform the design of drugs against trypanosomiasis. Pentamidine and melarsoprol are drugs used to treat sleeping sickness caused by Trypanosoma brucei. Here, authors present cryo-EM structures of TbAQP2 with molecular dynamic simulations, revealing mechanisms shaping substrate specificity and drug permeation. [ABSTRACT FROM AUTHOR]

Background: 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. Methodology: 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. Results: 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. Conclusion: 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. Author summary: A better understanding of Trypanosoma brucei rhodesiense gene expression profiles and population structure using endemic isolate may speed up the search for novel diagnostic and control tools for rhodesiense sleeping sickness. Here, we analysed T. b. rhodesiense transcriptome profiles from endemic isolated from peripheral blood in Nkhotakota and Rumphi foci in Malawi. In Nkhotakota focus, T. b. rhodesiense transcripts were enriched for cell cycle arrest and stumpy marker whereas in Rumphi focus, the isolates were enriched for antigenic variation and folate biosynthesis biological pathways. Furthermore, we also found that T. b. rhodesiense population structure in Nkhotakota focus is different from Rumphi focus. The differences in trypanosome gene expression profiles and population structure are consistent with a less severe and acute sleeping sickness clinical profiles in Nkhotakota and Rumphi foci respectively and suggest that the differences in pathology in the two foci are mainly due to differences in the parasite rather than in the host response. [ABSTRACT FROM AUTHOR]

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 μM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 μM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme. [ABSTRACT FROM AUTHOR]

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Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an ‘all in one’ solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

Kinetoplastid organisms, including Trypanosoma brucei, are a significant health burden in many tropical and semitropical countries. Much of their metabolism is poorly understood. To better study kinetoplastid metabolism, chemical probes that inhibit kinetoplastid enzymes are needed. To discover chemical probes, we have developed a high-throughput flow cytometry screening assay that simultaneously measures multiple glycolysis-relevant metabolites in live T. brucei bloodstream form parasites. We transfected parasites with biosensors that measure glucose, ATP, or glycosomal pH. The glucose and ATP sensors were FRET biosensors, while the pH sensor was a GFP-based biosensor. The pH sensor exhibited a different fluorescent profile from the FRET sensors, allowing us to simultaneously measure pH and either glucose or ATP. Cell viability was measured in tandem with the biosensors using thiazole red. We pooled sensor cell lines, loaded them onto plates containing a compound library, and then analyzed them by flow cytometry. The library was analyzed twice, once with the pooled pH and glucose sensor cell lines and once with the pH and ATP sensor cell lines. Multiplexing sensors provided some internal validation of active compounds and gave potential clues for each compound's target(s). We demonstrated this using the glycolytic inhibitor 2-deoxyglucose and the alternative oxidase inhibitor salicylhydroxamic acid. Individual biosensor-based assays exhibited a Z′-factor value acceptable for high-throughput screening, including when multiplexed. We tested assay performance in a pilot screen of 14,976 compounds from the Life Chemicals Compound Library. We obtained hit rates from 0.2 to 0.4% depending on the biosensor, with many compounds impacting multiple sensors. We rescreened 44 hits, and 28 (64%) showed repeatable activity for one or more sensors. One compound exhibited EC50 values in the low micromolar range against two sensors. We expect this method will enable the discovery of glycolytic chemical probes to improve metabolic studies in kinetoplastid parasites.

In Trypanosoma brucei, gene expression is primarily regulated posttranscriptionally making RNA metabolism critical. T. brucei has an epitranscriptome containing modified RNA bases. Yet, the identity of the enzymes catalyzing modified RNA base addition and the functions of the enzymes and modifications remain unclear. Homology searches indicate the presence of numerous T. brucei cytosine RNA methyltransferase homologs. One such homolog, TbNop2 was studied in detail. TbNop2 contains the six highly conserved motifs found in cytosine RNA methyltransferases and is evolutionarily related to the Nop2 protein family required for rRNA modification and processing. RNAi experiments targeting TbNop2 resulted in reduced levels of TbNop2 RNA and protein, and a cessation of parasite growth. Next generation sequencing of bisulfite-treated RNA (BS-seq) detected the presence of two methylation sites in the large rRNA; yet TbNop2 RNAi did not result in a significant reduction of methylation. However, TbNop2 RNAi resulted in the retention of 28S internal transcribed spacer RNAs, indicating a role for TbNop2 in rRNA processing. [ABSTRACT FROM AUTHOR]

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of β-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy. [ABSTRACT FROM AUTHOR]

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat. Author summary: In the blood of their human and animal hosts, single-celled parasites of the species Trypanosoma brucei need to hide from the body's defense system to survive. These parasites have a special armor made of proteins (known as VSGs) that changes regularly, helping them stay undetected by the immune system. This armor also covers other important proteins (ISGs) that the parasites need in order to interact with their host, but must hide to avoid being attacked. However, this protective layer makes it hard for the parasites to reach outside their shield. In our study, we looked at these hidden proteins in detail and discovered that they can change shape. Some can stretch out beyond their protective layer when necessary, while others stay concealed inside. This ability to change shape lets these otherwise hidden proteins interact with their surroundings without getting caught by the immune system. Our findings provide further evidence for a multi-layered defense strategy in these parasites. [ABSTRACT FROM AUTHOR]

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degroncontrolled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages. [ABSTRACT FROM AUTHOR]

Background: The severe late stage Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei rhodesiense (T.b.r) is characterized by damage to the blood brain barrier, severe brain inflammation, oxidative stress and organ damage. Melarsoprol (MelB) is currently the only treatment available for this disease. MelB use is limited by its lethal neurotoxicity due to post-treatment reactive encephalopathy. This study sought to assess the potential of Ginkgo biloba (GB), a potent anti-inflammatory and antioxidant, to protect the integrity of the blood brain barrier and ameliorate detrimental inflammatory and oxidative events due to T.b.r in mice treated with MelB. Methodology: Group one constituted the control; group two was infected with T.b.r; group three was infected with T.b.r and treated with 2.2 mg/kg melarsoprol for 10 days; group four was infected with T.b.r and administered with GB 80 mg/kg for 30 days; group five was given GB 80mg/kg for two weeks before infection with T.b.r, and continued thereafter and group six was infected with T.b.r, administered with GB and treated with MelB. Results: Co-administration of MelB and GB improved the survival rate of infected mice. When administered separately, MelB and GB protected the integrity of the blood brain barrier and improved neurological function in infected mice. Furthermore, the administration of MelB and GB prevented T.b.r-induced microcytic hypochromic anaemia and thrombocytopenia, as well as T.b.r-driven downregulation of total WBCs. Glutathione analysis showed that co-administration of MelB and GB prevented T.b.r-induced oxidative stress in the brain, spleen, heart and lungs. Notably, GB averted peroxidation and oxidant damage by ameliorating T.b.r and MelB-driven elevation of malondialdehyde (MDA) in the brain, kidney and liver. In fact, the co-administered group for the liver, registered the lowest MDA levels for infected mice. T.b.r-driven elevation of serum TNF-α, IFN-γ, uric acid and urea was abrogated by MelB and GB. Co-administration of MelB and GB was most effective in stabilizing TNFα levels. GB attenuated T.b.r and MelB-driven up-regulation of nitrite. Conclusion: Utilization of GB as an adjuvant therapy may ameliorate detrimental effects caused by T.b.r infection and MelB toxicity during late stage HAT. Author summary: Human African Trypanosomiasis (HAT) is a lethal disease caused by Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense (T.b.r) protozoan parasites. The only treatment available for the late, CNS stage due to T.b.r, is melarsoprol (MelB). Notably, MelB has an arsenic moiety, that makes it very neurotoxic, causing fatal post-treatment reactive encephalopathy in about 5% of those treated. This level of drug-induced mortality is unacceptable in this day and age. The question is, how can we block T.b.r-induced organ damage and safely administer MelB? In the late stage HAT, parasites wreak havoc by inducing severe oxidative stress, inflammation and breach of the blood brain barrier (bbb); while on the other hand, MelB is driving lethal encephalopathy. We used a HAT mouse model and sought to elucidate the potential of a standardized GB extract–a potent antioxidant anti-inflammatory, to alleviate T.b.r-driven damage to the bbb, induction of oxidative stress and inflammation; while ameliorating MelB-driven neurotoxic effects. According to the results, GB protected from lethal parasite induced organ damage, as well as MelB toxicity, providing a novel approach for development of an adjuvant therapy. [ABSTRACT FROM AUTHOR]

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events. [ABSTRACT FROM AUTHOR]

Leishmaniasis, an infectious disease caused by pathogenic Leishmania parasites, affects millions of people in developing countries, and its re-emergence in developed countries, particularly in Europe, poses a growing public health concern. The limitations of current treatments and the absence of effective vaccines necessitate the development of novel therapeutics. In this study, we focused on identifying small molecule inhibitors which prevents the interaction between peroxin 5 (PEX5) and peroxisomal targeting signal 1 (PTS1), pivotal for kinetoplastid parasite survival. The Leishmania donovani PEX5, containing a C-terminal tetratricopeptide repeat (TPR) domain, was expressed and purified, followed by the quantification of kinetic parameters of PEX5-PTS1 interactions. A fluorescence polarization-based high-throughput screening assay was developed and small molecules inhibiting the LdPEX5-PTS1 interaction were discovered through the screening of a library of 51,406 compounds. Based on the confirmatory assay, nine compounds showed half maximal inhibitory concentration (IC50) values ranging from 3.89 to 24.50 µM. In silico docking using a homology model of LdPEX5 elucidated that the molecular interactions between LdPEX5 and the inhibitors share amino acids critical for PTS1 binding. Notably, compound P20 showed potent activity against the growth of L. donovani promastigotes, L. major promastigotes, and Trypanosoma brucei blood stream form, with IC50 values of 12.16, 19.21, and 3.06 μM, respectively. The findings underscore the potential of targeting LdPEX5-PTS1 interactions with small molecule inhibitors as a promising strategy for the discovery of new anti-parasitic compounds. [ABSTRACT FROM AUTHOR]

Introduction: 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. Objectives: 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. Methods: 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. Results: 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). Conclusion: 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. [ABSTRACT FROM AUTHOR]

2,6-Diaryl-4H-tetrahydro-thiopyran-4-ones and corresponding sulfoxide and sulfone derivatives were designed to lower the major toxicity of their parent anti-kinetoplatidal diarylideneacetones through a prodrug effect. Novel diastereoselective methodologies were developed and generalized from diarylideneacetones and 2,6-diaryl-4H-tetrahydro-thiopyran-4-ones to allow the introduction of a wide substitution profile and to prepare the related S-oxides. The in vitro biological activity and selectivity of diarylideneacetones, 2,6-diaryl-4H-tetrahydro-thiopyran-4-ones, and their S-sulfoxide and sulfone metabolites were evaluated against Trypanosoma brucei brucei, Trypanosoma cruzi, and various Leishmania species in comparison with their cytotoxicity against human fibroblasts hMRC-5. The data revealed that the sulfides, sulfoxides, and sulfones, in which the Michael acceptor sites are temporarily masked, are less toxic against mammal cells while the anti-trypanosomal potency was maintained against T. b. brucei, T. cruzi, L. infantum, and L. donovani, thus confirming the validity of the prodrug strategy. The mechanism of action is proposed to be due to the involvement of diarylideneacetones in cascades of redox reactions involving the trypanothione system. After Michael addition of the dithiol to the double bonds, resulting in an elongated polymer, the latter—upon S-oxidation, followed by syn-eliminations—fragments, under continuous release of reactive oxygen species and sulfenic/sulfonic species, causing the death of the trypanosomal parasites in the micromolar or submicromolar range with high selectivity indexes. [ABSTRACT FROM AUTHOR]

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-β-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-β-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola. [ABSTRACT FROM AUTHOR]

Adaptation to a change of environment is an essential process for survival, in particular for parasitic organisms exposed to a wide range of hosts. Such adaptations include rapid control of gene expression through the formation of membraneless organelles composed of poly-A RNA and proteins. The African trypanosome Trypanosoma brucei is exquisitely sensitive to well-defined environmental stimuli that trigger cellular adaptations through differentiation events that characterise its complex life cycle. The parasite has been shown to form stress granules in vitro, and it has been proposed that such a stress response could have been repurposed to enable differentiation and facilitate parasite transmission. Therefore, we explored the composition and positional dynamics of membraneless granules formed in response to starvation stress and during differentiation in the mammalian host between the replicative slender and transmission-adapted stumpy forms. We find that T. brucei differentiation does not reflect the default response to environmental stress. Instead, the developmental response of the parasites involves a specific and programmed hierarchy of membraneless granule assembly, with distinct components and regulation by protein kinases such as TbDYRK, that are required for the parasite to successfully progress through its life cycle development and prepare for transmission. The African trypanosome Trypanosoma brucei has been shown to form stress granules in vitro that might be repurposed to enable differentiation and facilitate parasite transmission. Here, Cayla et al. show that differentiation between slender and stumpy forms does involve membrane-less granules that are different from nutritional stress granules. [ABSTRACT FROM AUTHOR]

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes. Trypanosoma brucei DOT1A and DOT1B methylate H3K76 without H2B-ubiquitin. Based on structural and enzymatic data, Frisbie et al. reveal a mechanism of how these enzymes cooperatively and efficiently tri-methylate H3K76 in a ubiquitin-independent way. [ABSTRACT FROM AUTHOR]

This study determined the in vitro trypanocidal activity of lemon grass(Cymbopogon citratus) methanolic extract against Trypanosoma brucei brucei and its bio infectivity in an in vivo study using male albino rat as the experimental animal. Five treatment levels were applied (0.002mg/ml, 0.02mg/ml, 0.2mg/ml, 2.0mg/ml and 0.0mg/ml). Trypanosome was inactive at all treatment levels of lemongrass. PCV was statistically the same at all exposure times whereas the control mice had significantly lower PCV than all treated mice apart from the control, all other treatments gave similar PCV results, but the highest grand mean value (51.87%) was obtained at 0.02mg/ml while week 4 recorded the highest value (51.53%). All treatment levels had equal effects on the mice's rectal temperature, but exposure time had significant effects on the temperature parameter where the grand mean temperature readings significantly varied from 35.55oC in week 2 to 37.47oC in week 5. The weight of mice varied significantly with both treatment (F=16.01, p<0.05) and time (F=5.10, p<0.05) factors. The highest mean weight recorded was 38.37±2.46g at 2.0mg/ml treatment in week 5 thus, the weight of mice increased as lemon grass treatment concentration and exposure time increased. Treatment concentrations significantly affected mice PCV and weight where the control level had the lowest values for these physical parameters. Also, exposure time significantly affected both the temperature and weight of mice. A general fluctuation but decline in feed intake was observed in mice at different treatment levels of methanolic extracts of lemon grass from weeks 1-7. A strong positive significant correlation exists between PCV and feed intake (r= -0.930, p<0.05) as well as between rectal temperature and weight of mice (r= -0.948, p<0.05). PCV and weight had a strong positive correlation though insignificant (r= 0.728, p>0.05). Therefore, lemon grass extract showed trypanocidal efficacy in the control of trypanosomiasis in Africa. [ABSTRACT FROM AUTHOR]

Human African Trypanosomiasis is a serious public health concern, and new chemical therapeutic agents need to be developed to combat this disease. Rhodesain (RhD) inhibitors have shown promising results in medicinal chemistry, specifically against Trypanosoma brucei. These inhibitors target the cysteine protease RhD, which is essential for the survival of T. brucei. However, as the pharmaceutical industry lacks interest in these inhibitors, the development of drugs based on them is challenging. In this review, we showed the impact of RhD inhibitors on medicinal chemistry in the past 10 years (2013–2022), particularly against T. brucei, showing interesting RhD-based inhibitors, including peptidomimetic inhibitors such as Michael acceptors, cyanide groups, 3-bromoisoxazole, benzodiazepine, among others, as well as non-peptidyl inhibitors. Peptidomimetic inhibitors (5–7, 9–15, and 17) exhibited the highest potency with respect to dissociation constant (Ki) values for rhodesain, demonstrating promising activity against T. brucei targeting rhodesain. Thus, we explored recent advancements and perspectives in the context of RhD for potential treatments in sleeping sickness, highlighting its relevance in drug discovery applications. [ABSTRACT FROM AUTHOR]

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment. Sleeping sickness caused by African trypanosome parasites induces a chronic, and potentially lethal, infection in humans. Here, the authors uncover a conserved protein, Q586B2, playing an important regulatory role in Trypanosomatid infection establishment. [ABSTRACT FROM AUTHOR]

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. Author summary: Trypanosoma brucei is a unicellular parasite causing sleeping sickness in humans and nagana disease in cattle. The parasite invades the bloodstream and cerebrospinal fluid and only recently, it has been shown to infect additional tissues such as skin, adipose tissue, or lungs. While the glucose-based metabolism of the bloodstream form is well understood, the parasite's metabolism in these secondary tissues has not been sufficiently explored, despite its importance for drug development. One possibility is the use of gluconeogenesis since the mammalian-infective stages can use glycerol as a carbon and ATP source. First, enzymes involved in gluconeogenesis have to be identified, then it can be tested if the pathway is advantageous for the survival of the parasite. We generated mutants in four different enzymes potentially involved in this metabolic pathway. Surprisingly, the flux in gluconeogenesis was maintained in all cell lines tested, implying that another non-canonical enzyme participates in the production of glucose from glycerol in these parasites. [ABSTRACT FROM AUTHOR]

Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein–Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle. [ABSTRACT FROM AUTHOR]

Tubulin polyglutamylation, catalysed by members of the tubulin tyrosine ligase-like (TTLL) protein family, is an evolutionarily highly conserved mechanism involved in the regulation of microtubule dynamics and function in eukaryotes. In the protozoan parasite Trypanosoma brucei, the microtubule cytoskeleton is essential for cell motility and maintaining cell shape. In a previous study, we showed that T. brucei TTLL6A and TTLL12B are required to regulate microtubule dynamics at the posterior cell pole. Here, using gene deletion, we show that the polyglutamylase TTLL1 is essential for the integrity of the highly organised microtubule structure at the cell pole, with a phenotype distinct from that observed in TTLL6Aand TTLL12B-depleted cells. Reduced polyglutamylation in TTLL1-deficient cells also leads to increased levels in tubulin tyrosination, providing new evidence for an interplay between the tubulin tyrosination and detyrosination cycle and polyglutamylation. We also show that TTLL1 acts differentially on specific microtubule doublets of the flagellar axoneme, although the absence of TTLL1 appears to have no measurable effect on cell motility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human African trypanosomosis (HAT) which is also known as sleeping sickness is caused by Trypanosoma brucei gambiense that is endemic in western and central Africa and T. b. rhodesiense that is endemic in eastern and southern Africa. Drugs used for treatment against HAT first stage have limited effectiveness, and the second stage drugs have been reported to be toxic, expensive, and have time‐consuming administration, and parasitic resistance has developed against these drugs. The aim of this study was to evaluate the anti‐trypanosomal activity of nitrofurantoin‐triazole hybrids against T. b. gambiense and T. b. rhodesiense parasites in vitro. This study screened 19 synthesized nitrofurantoin‐triazole (NFT) hybrids on two strains of human trypanosomes, and cytotoxicity was evaluated on Madin‐Darby bovine kidney (MDBK) cells. The findings in this study showed that an increase in the chain length and the number of carbon atoms in some n‐alkyl hybrids influenced the increase in anti‐trypanosomal activity against T. b. gambiense and T. b. rhodesiense. The short‐chain n‐alkyl hybrids showed decreased activity compared to the long‐chain n‐alkyl hybrids, with increased activity against both T. b. gambiense and T. b. rhodesiense. Incorporation of additional electron‐donating substituents in some NFT hybrids showed increased anti‐trypanosomal activity than to electron‐withdrawing substituents in NFT hybrids. All 19 NFT hybrids tested displayed better anti‐trypanosomal activity against T. b. gambiense than T. b. rhodesiense. The NFT hybrid no. 16 was among the best performing hybrids against both T. b. gambiense (0.08 ± 0.04 μM) and T. b.rhodesiense (0.11 ± 0.06 μM), and its activity might be influenced by the introduction of fluorine in the para‐position on the benzyl ring. Remarkably, the NFT hybrids in this study displayed weak to moderate cytotoxicity on MDBK cells. All of the NFT hybrids in this study had selectivity index values ranging from 18 to greater than 915, meaning that they were up to 10–100 times fold selective in their anti‐trypanosomal activity. The synthesized NFT hybrids showed strong selectivity >10 to T. b. gambiense and T. b. rhodesiense, which indicates that they qualify from the initial selection criteria for potential hit drugs. [ABSTRACT FROM AUTHOR]

Faithful mRNA decoding depends on the accuracy of aminoacyl-tRNA synthetases (ARSs). Aminoacyl-tRNA proofreading mechanisms have been well-described in bacteria, humans, and plants. However, our knowledge of translational fidelity in protozoans is limited. Trypanosoma brucei (Tb) is a eukaryotic, protozoan pathogen that causes Human African Trypanosomiasis, a fatal disease if untreated. Tb undergoes many physiological changes that are dictated by nutrient availability throughout its insect-mammal lifecycle. In the glucose-deprived insect vector, the tsetse fly, Tb use proline to make ATP via mitochondrial respiration. Alanine is one of the major by-products of proline consumption. We hypothesize that the elevated alanine pool challenges Tb prolyl-tRNA synthetase (ProRS), an ARS known to misactivate alanine in all three domains of life, resulting in high levels of misaminoacylated Ala-tRNAPro. Tb encodes two domains that are members of the INS superfamily of aminoacyl-tRNA deacylases. One homolog is appended to the N-terminus of Tb ProRS, and a second is the major domain of multi-aminoacyl-tRNA synthetase complex (MSC)-associated protein 3 (MCP3). Both ProRS and MCP3 are housed in the Tb MSC. Here, we purified Tb ProRS and MCP3 and observed robust Ala-tRNAPro deacylation activity from both enzymes in vitro. Size-exclusion chromatography multi-angle light scattering used to probe the oligomerization state of MCP3 revealed that although its unique N-terminal extension confers homodimerization in the absence of tRNA, the protein binds to tRNA as a monomer. Kinetic assays showed MCP3 alone has relaxed tRNA specificity and promiscuously hydrolyzes cognate Ala-tRNAAla; this activity is significantly reduced in the presence of Tb alanyl-tRNA synthetase, also housed in the MSC. Taken together, our results provide insight into translational fidelity mechanisms in Tb and lay the foundation for exploring MSC-associated proteins as novel drug targets.

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.

We report 4 cases of human African trypanosomiasis that occurred in Ethiopia in 2022, thirty years after the last previously reported case in the country. Two of 4 patients died before medicine became available. We identified the infecting parasite as Trypanosoma brucei rhodesiense. Those cases imply human African trypanosomiasis has reemerged.

Suramin is one of the oldest drugs in use today. It is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense, and it is also used for surra in camels caused by Trypanosoma evansi. Yet despite one hundred years of use, suramin's mode of action is not fully understood. Suramin is a polypharmacological molecule that inhibits diverse proteins. Here we demonstrate that a DNA helicase of the pontin/ruvB-like 1 family, termed T. brucei RuvBL1, is involved in suramin resistance in African trypanosomes. Bloodstream-form T. b. rhodesiense under long-term selection for suramin resistance acquired a homozygous point mutation, isoleucine-312 to valine, close to the ATP binding site of T. brucei RuvBL1. The introduction of this missense mutation, by reverse genetics, into drug-sensitive trypanosomes significantly decreased their sensitivity to suramin. Intriguingly, the corresponding residue of T. evansi RuvBL1 was found mutated in a suramin-resistant field isolate, in that case to a leucine. RuvBL1 (Tb927.4.1270) is predicted to build a heterohexameric complex with RuvBL2 (Tb927.4.2000). RNAi-mediated silencing of gene expression of either T. brucei RuvBL1 or RuvBL2 caused cell death within 72 h. At 36 h after induction of RNAi, bloodstream-form trypanosomes exhibited a cytokinesis defect resulting in the accumulation of cells with two nuclei and two or more kinetoplasts. Taken together, these data indicate that RuvBL1 DNA helicase is involved in suramin action in African trypanosomes.