Trypanosoma Brucei Research Articles

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Unwrap RAP1's Mystery at Kinetoplastid Telomeres.

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

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

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

Trypanosoma brucei gambiense group 2 experimental in vivo life cycle: from procyclic to bloodstream form.

Trypanosoma brucei gambiense (Tbg) group 2 is a subgroup of trypanosomes able to infect humans and is found in West and Central Africa. Unlike other agents causing sleeping sickness, such as Tbg group 1 and Trypanosoma brucei rhodesiense, Tbg2 lacks the typical molecular markers associated with resistance to human serum. Only 36 strains of Tbg2 have been documented, and therefore, very limited research has been conducted despite their zoonotic nature. Some of these strains are only available in their procyclic form, which hinders human serum resistance assays and mechanistic studies. Furthermore, the understanding of Tbg2's potential to infect tsetse flies and mammalian hosts is limited. In this study, 165 Glossina palpalis gambiensis flies were experimentally infected with procyclic Tbg2 parasites. It was found that 35 days post-infection, 43 flies out of the 80 still alive were found to be Tbg2 PCR-positive in the saliva. These flies were able to infect 3 out of the 4 mice used for blood-feeding. Dissection revealed that only six flies in fact carried mature infections in their midguts and salivary glands. Importantly, a single fly with a mature infection was sufficient to infect a mammalian host. This Tbg2 transmission success confirms that Tbg2 strains can establish in tsetse flies and infect mammalian hosts. This study describes an effective in vivo protocol for transforming Tbg2 from procyclic to bloodstream form, reproducing the complete Tbg2 cycle from G. p. gambiensis to mice. These findings provide valuable insights into Tbg2's host infectivity, and will facilitate further research on mechanisms of human serum resistance.

Analysis of the Leishmania mexicana promastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration.

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.

Effects of seasonal variation on phytochemicals contributing to the antimalarial and antitrypanosomal activities of Breonadia salicina using a metabolomic approach

This study involves the investigation of various plant parts of Breonadia salicina (Vahl) Hepper and J.R.I. Wood across multiple consecutive seasons. It aims to delve into the phytochemistry of these different plant parts and establish connections between the findings and their biological activities. This comprehensive approach employs metabolomics techniques, with the ultimate goal of exploring the potential for drug development. Samples were collected in Fondwe, a village in Limpopo (South Africa), based on local reports of the efficacy of this plant used by traditional healers in the area. The antimalarial and antitrypanosomal activities of samples collected over the seasons were determined with the parasite lactate dehydrogenase (pLDH) and specific Trypanosoma brucei assays, respectively. Consequently, a total of 24 compounds were tentatively identified through ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Chemical profiles of the different plant parts of Breonadia salicina collected in different seasons produced contrasting metabolic profiles. Chemometric analysis of the UPLC-QTOF-MS data enabled us to determine the chemical variability of the crude stem bark, root and leaf extracts (n = 48) collected over four consecutive seasons by evaluating the metabolomics fingerprinting of the samples using an untargeted approach. Principal component analysis (PCA), hierarchical cluster analysis (HCA), and partial least squares discriminant analysis (PLS-DA) indicated the existence of two key clusters that are linked to the root, stem bark, and leaves. The stem and root chemistry differed from that of the leaves. Seasonal variations were noted in each plant part, with autumn and winter samples closely grouped compared to spring and summer samples in the methanol leaf extracts. Biochemometric analysis could not relate specific compounds to the antimalarial and antitrypanosomal activities of the active extracts, underscoring the intricate interactions among the secondary metabolites. This study further confirms the optimal plant parts to collect in each season for the most effective antimalarial and antitrypanosomal activities.

Morphometric changes in the gonads of the West African dwarf bucks experimentally infected with Trypanosoma vivax and Trypanosoma brucei and response due to diminazine aceturate treatment

Trypanosomosis, one of the major diseases affecting livestock production in Africa has been associated with reproductive disorders. The disease has not been extensively studied in goats especially the West African Dwarf (WAD) goat. The morphology of the gonads of WAD bucks experimentally infected with Trypanosoma (T.) vivax and Trypanosoma brucei and treatment with Diminazine aceturate were investigated in this study. The study lasted a period of 20 weeks, the first 3 weeks were used to acclimatize the animal to their new environment. The remaining 16 weeks were used to carry out the experiment. Two species of trypanosome were used for the experiment; T. brucei (NITR CT/28- Federe strain) and T. vivax (A field strain obtained from an abattoir). The experiment was divided into three stages tagged Pre-infection (A), infection (B) and treatment (C) while the animals were also divided into control group and infected groups- Trypanosoma brucei and Trypanosoma vivax groups. Biometric parameters that include testicular weight, height and others testicular and epididymal indices were measured according to standard techniques. Data were summarized with mean± SEM and analysed using ANOVA. There were no significant changes in body weight between the control group and treated groups with either T. brucei or T. vivax. The little reduction in scrotal circumference during infection and subsequent increase during post treatment were not significant (P > 0.05) compared to the control. The Testicular weight, testicular length and epididymal weight were not significantly affected during infection with both T. brucei and T. vivax. Tubular diameter and epithelia height of the seminiferous tubules were significantly (P≤0.05) lower in the T. brucei and T. vivax infected groups compared to the control. On the other hand, the epididymal tubular diameter and epididymal epithelia height were not significantly (P>00.05) affected by the treatment. The study showed that the fertility impairment sequel to trypanosomosis is possibly due to alteration in the biometric parameter of the testis.

Tetrahydropyridinylidene ammonium salts with arylalkyl and diarylalkyl substitution and their antiprotozoal activity

Tetrahydropyridin-4-ylidene salts with benzyl and dibenzyl substitution showed good antiprotozoal activity. This paper reports the synthesis of analogues with longer side chains. They were investigated for their antiprotozoal activities as well as for their cytotoxicity using microplate assays. The most active compounds showed activity against Trypanosoma brucei rhodesiense in concentrations < 0.06 µM. A series of compounds was active against Plasmodium falciparum NF54 in low nanomolar concentration and exhibited outstanding selectivity. The influence of substitution pattern and chain length on the antiprotozoal potencies were analyzed and structure–activity relationships were given. New compounds were characterized by FT-IR, HRMS, and NMR spectroscopy.Graphical abstract

FLAgellum Member 8 modulates extravascular distribution of African trypanosomes.

In the mammalian host, the biology of tissue-dwelling Trypanosoma brucei parasites is not completely understood, especially the mechanisms involved in their extravascular colonization. The trypanosome flagellum is an essential organelle in multiple aspects of the parasites' development. The flagellar protein termed FLAgellar Member 8 (FLAM8) acts as a docking platform for a pool of cyclic AMP response protein 3 (CARP3) that is involved in signaling. FLAM8 exhibits a stage-specific distribution suggesting specific functions in the mammalian and vector stages of the parasite. Analyses of knockdown and knockout trypanosomes in their mammalian forms demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes can establish and maintain an infection in the blood circulation and differentiate into insect transmissible forms. However, quantitative bioluminescence imaging and gene expression analysis revealed that FLAM8-null parasites exhibit a significantly impaired dissemination in the extravascular compartment, that is restored by the addition of a single rescue copy of FLAM8. In vitro trans-endothelial migration assays revealed significant defects in trypanosomes lacking FLAM8. FLAM8 is the first flagellar component shown to modulate T. brucei distribution in the host tissues, possibly through sensing functions, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches, especially in the skin.

The evolving spectrum of human African trypanosomiasis.

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.

Synthetic arrest of Man5GlcNAc2-PP-Dol increases procyclin mRNA level and induces cell death in the bloodstream form Trypanosoma brucei brucei

The biosynthesis of N-linked glycan precursors in the endoplasmic reticulum is important for many eukaryotes. In particular, the synthesis of Man5GlcNAc2-PP-dolichol (M5-DLO) at the cytoplasmic face of the endoplasmic reticulum is essential for maintaining cellular functions. In Trypanosoma brucei, the unicellular organism that causes African trypanosomiasis, homologs of the mannosyltransferases ALG2 and ALG11, which are involved in the biosynthesis of M5-DLO, are found, but the effects of their deletion on cells remain unknown. In this study, we generated conditional gene knockout strains of TbALG2 and TbALG11 in the bloodstream form T. brucei. Decreased N-linked glycosylation and cell death were observed in both strains under non-permissive conditions, with TbALG2 having a greater effect than TbALG11. Transcriptomic analysis of cells losing expression of TbALG11 showed decrease in mRNAs for enzymes involved in glucose metabolism and increase in mRNAs for procyclins and variant surface glycoproteins. These results indicate that the M5-DLO biosynthetic pathway is essential for the proliferation of the bloodstream form T. brucei. They also suggest that the failure of this pathway induces the transcriptomic change.

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

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