A Nanobody/Monoclonal Antibody "hybrid" sandwich technology offers an improved immunoassay strategy for detection of African trypanosome infections.

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly, laborious, time-consuming, complex, and require skilled personnel. Hence, utilisation of most diagnostics for AAT is impracticable in rural areas, where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection, without relying on expensive equipment, would facilitate AAT detection. In turn, early management, would reduce disease incidence and severity. Today, several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context, we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T. congolense infections, which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here, originated from a past study. Briefly, the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T. congolense glycosomal fructose-1,6-bisphosphate aldolase (TcoALD) was discovered as the cognate Ag of Nb474H. In this study, splenocytes were harvested from a mouse immunized with recombinant TcoALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on TcoALD retrieved a lone binder, designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native TcoALD released during infection by T. congolense parasites. Hitherto, development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) "hybrid" sandwich technology offers prospects for exploration, using the unique specificity of Nb as a key determinant in Ag capturing, while using the versatility of monoclonal Ab to adapt to various detection conditions.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study.

BACKGROUND: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. METHODS: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. RESULTS: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1-99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5-88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7-84.2%); DCN HAT RDT 78.2% (95% CI: 75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n = 399) and 93.0-100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. CONCLUSIONS: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. TRIAL REGISTRATION: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

Animal models of neglected parasitic diseases: In vivo multimodal imaging of experimental trypanosomatid infections.

African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (µCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.

Reproductive, productive and financial consequences of chronic Trypanosoma vivax infection in a dairy cattle herd in a region without a cyclic vector.

This study evaluated the reproductive, productive and financial consequences of chronic Trypanosoma vivax infection in a dairy cattle herd located in a region without the cyclic vector during two years. Animals were categorized as either positive (chronically infected) or negative for T. vivax antibodies using a commercial rapid test. Additionally, serum samples from cows were analyzed for the presence of anti-Neospora caninum antibodies. Pregnancy diagnoses were performed through rectal palpation and ultrasonography after 30, 60 and every 21 days until the 144th day of pregnancy. If an abortion occurred in the final trimester, serology and cPCR were performed on calves for T. vivax and N. caninum. The breeding period, calving interval and pregnancy losses were recorded. The milk production of each animal during the 305 days of lactation was measured, and the annual financial impact of milk production was calculated using a revenue minus feed cost (RMFC) indicator. Out of 177 cows, 71.75 % were chronically infected, and 13.50 % were T. vivax-negative. No correlation (p = 0.8854) of co-infection between T. vivax and N. caninum was observed. Negative cows required fewer (p≤0.05) artificial inseminations than chronically infected ones. T. vivax was not significantly associated (p = 0.7893) with pregnancy loss up to 81 days of pregnancy. Cows chronically infected by T. vivax had 4-fold greater chance (p = 0.0280) of experiencing pregnancy loss between 82 and 144 days of gestation. Eighteen cows aborted, two were positive for T. vivax antibodies, and one for N. caninum antibodies. The calves were negative for T. vivax and N. caninum. Chronically infected cows and negative cows for T. vivax that experienced pregnancy loss (82-144 days of pregnancy) had a longer (p≤0.05) breeding period to become pregnant, and consequently a longer calving interval compared to cows that maintained pregnancy. The difference (p≤0.05) in milk production was evident when pregnancy loss occurred between 82 and 144 days of gestation in cows chronically infected by T. vivax. The RMFC indicated a negative impact of 38.2 % on the farm's annual milk revenue due to the presence of chronically infected cows.

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

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

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

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.

Improved serodiagnosis of Trypanosoma vivax infections in cattle reveals high infection rates in the livestock regions of Argentina.

Bovine trypanosomosis, caused by Trypanosoma vivax, currently affects cattle and has a significant economic impact in sub-Saharan Africa and South America. The development of new diagnostic antigens is essential to improve and refine existing methods. Our study evaluated the efficacy of two recombinant antigens in detecting specific antibodies in cattle. These antigens are derivatives of an invariant surface glycoprotein (ISG) from T. vivax. A fraction of a previously described antigen (TvY486_0045500), designated TvISGAf, from an African strain was evaluated, and a new ISG antigen from an American isolate, TvISGAm, was identified. The two antigens were expressed as fusion proteins in Escherichia coli: TvISGAf was fused to the MBP-His-tag, and TvISGAm was obtained as a His-tag fused protein. An ELISA evaluation was conducted using these antigens on 149 positive and 63 negative bovine samples. The diagnostic performance was enhanced by the use of a combination of both antigens (referred to as TvISG-based ELISA), achieving a sensitivity of 89.6% and specificity of 93.8%. Following the validation of the TvISG-based ELISA, the seroprevalence of T. vivax infection in 892 field samples from cattle in the central region of Argentina was determined. The mean seroprevalence of T. vivax was 53%, with variation ranging from 21% to 69% among the six departments studied. These results support the use of the TvISG ELISA as a valuable serological tool for the detection and monitoring of T. vivax infection in cattle. Furthermore, we report for the first time the seroprevalence of T. vivax in Argentina, which highlights the widespread endemic nature of the disease in the region. In order to effectively manage the increasing spread of T. vivax in the vast livestock production areas of South America, it is essential to implement consistent surveillance programs and to adopt preventive strategies.

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

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.

Adipose and skin distribution of African trypanosomes in natural animal infections.

BACKGROUND: Animal African trypanosomiasis, which is caused by different species of African trypanosomes, is a deadly disease in livestock. Although African trypanosomes are often described as blood-borne parasites, there have been recent reappraisals of the ability of these parasites to reside in a wide range of tissues. However, the majority of those studies were conducted on non-natural hosts infected with only one species of trypanosome, and it is unclear whether a similar phenomenon occurs during natural animal infections, where multiple species of these parasites may be present. METHODS: The infective trypanosome species in the blood and other tissues (adipose and skin) of a natural host (cows, goats and sheep) were determined using a polymerase chain reaction-based diagnostic. RESULTS: The animals were found to harbour multiple species of trypanosomes. Different patterns of distribution were observed within the host tissues; for instance, in some animals, the blood was positive for the DNA of one species of trypanosome and the skin and adipose were positive for the DNA of another species. Moreover, the rate of detection of trypanosome DNA was highest for skin adipose and lowest for the blood. CONCLUSIONS: The findings reported here emphasise the complexity of trypanosome infections in a natural setting, and may indicate different tissue tropisms between the different parasite species. The results also highlight the need to include adipose and skin tissues in future diagnostic and treatment strategies.

Toll-like receptor 9 signaling is associated with immune responses to Trypanosoma brucei infection.

Trypanosoma brucei, a causative agent of human and animal trypanosomiasis, regularly switches its major surface antigen to avoid elimination by the immune system. Toll-like receptor 9 (TLR9) is a key modulator for resistance to host-infective trypanosomes; however, the underlying molecular mechanism remains indistinct. Thus, we first approached the issue using Tlr9-mutant mice that render them non-responsive to TLR9 agonists. After infection, T cells in the spleens of Tlr9-mutant mice were analyzed by flow cytometry and a reduction in CD8+, CD4+ T, and NKT cells was observed in Tlr9-mutant mice compared to WT mice. We further found that the responses of inflammatory cytokines in the sera were reduced in Tlr9-mutant mice after T. brucei infection. The underlying molecular mechanism was that T. b. brucei DNA activated TLR9, which consequently upregulated the expression of p38 and ERK/MAPK, resulting in host resistance to trypanosome infection. In conclusion, these findings provide novel insights into the TLR9-mediated host responses to trypanosome infection.

Trypanosoma vivax in and outside cattle blood: Parasitological, molecular, and serological detection, reservoir tissues, histopathological lesions, and vertical transmission evaluation.

This study reports assessment of the sensitivity of diagnostic techniques to detect T. vivax in experimentally infected cattle. Additionally, it describes T. vivax extravascular parasitism during the acute and chronic phases of trypanosomosis and congenital transmission. The T. vivax diagnosis was compared using blood samples collected from the jugular, coccygeal and ear tip veins. For this study, 13 males and two females were infected with ≈ 1 × 106 viable T. vivax trypomastigotes (D0). One animal was kept as a negative control during the entire study. The 13 infected males were euthanized between 14 and 749 days post-infection (DPI). After confirming the cyclicity of both females (9 months of age), they were naturally mated with a bull. One female was euthanized at 840 DPI, and the other at 924 DPI. The two calves, one from each female, were euthanized at six months of age (924 DPI), and the negative control at 924 DPI. During this period, T. vivax in blood was assessed using direct methods (Woo test, cPCR, microscopic examination of fresh wet blood films and parasite quantification - Brener method), and serological methods (IFAT, ELISA, and IA). Tissue samples were collected from the liver, spleen, brain, cerebellum, heart, testicles, epididymis, kidneys, eyeballs, pre-scapular lymph nodes, ear tips, mammary glands, uterus, and ovaries. The protozoan DNA was examined using LAMP. There was no difference in the detection of T. vivax using the Woo test and Brener method among the jugular, coccygeal, and ear tip veins. The sensitivity of the detection methods varied depending on the disease phase. Direct methods (Woo test, Brener method, and cPCR) demonstrated higher sensitivity during the acute phase, while serological methods (IFAT, ELISA, and IA) were more sensitive during the chronic phase. Anti-T. vivax antibodies were detected up to 924 DPI. Tissue evaluation using LAMP demonstrated the presence of T. vivax DNA and associated histopathological changes up to 840 or 924 DPI. Only in mammary glands and ovaries was no DNA detected. The most frequently observed histopathological alteration was lymphohistioplasmocytic inflammatory infiltrate. No transplacental transmission of T. vivax was observed.

Comparison of Three Widely Employed Extraction Methods for Metabolomic Analysis of Trypanosoma brucei.

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites' physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). © 2024 Wiley Periodicals LLC. Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei.

Synthesis and biological evaluation of orally active anti-Trypanosoma agents.

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

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

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

The Transmission of Animal African Trypanosomiasis in Two Districts in the Forest Zone of Ghana.

Animal African trypanosomiasis, also known as nagana, is caused by Trypanosoma species, which cause significant clinical diseases and lead to losses in animal production. We carried out a cross-sectional survey to investigate the composition of vectors and parasite diversity in two districts in the eastern region of Ghana where pigs and cattle were exposed to tsetse bites. We performed cytochrome c oxidase subunit 1 polymerase chain reaction (PCR) to identify tsetse species and internal transcribed spacer 1 PCR to identify Trypanosoma species. Also, we investigated the source of tsetse blood meal based on mitochondrial cytochrome b gene sequence analysis. A total of 229 tsetse, 65 pigs, and 20 cattle were investigated for trypanosomes. An overall vector density of 4.3 tsetse/trap/day was observed. A trypanosome prevalence of 58.9% (95% CI = 52.5-65.1%), 46.2% (95% CI = 34.6-58.1%), and 0.0% (95% CI = 0.0-16.1%) in tsetse, pigs, and cattle, respectively, was detected. Trypanosoma congolense was predominant, with a prevalence of 33.3% (95% CI = 73.3-86.5%) in tsetse. There was evidence of multiple infections in tsetse and pigs. Approximately 39% of the tsetse were positive for multiple infections of T. congolense and Trypanosoma simiae. Parasite prevalence in pigs across the communities was high, with significant differences associated between locations (χ2 = 28.06, 95% CI = 0.05-0.81, P = 0.0009). Tsetse blood meal analysis revealed feeding on domestic Sus scrofa domesticus (pigs) and Phacochoerus africanus (warthogs). Infective tsetse may transmit trypanosomes to livestock and humans in the communities studied.

Depolymerization of SUMO chains induces slender to stumpy differentiation in T. brucei bloodstream parasites.

Trypanosoma brucei are protozoan parasites that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, a quorum sensing-like mechanism coordinates its differentiation from a slender replicative form into a quiescent stumpy form, limiting growth and activating metabolic pathways that are beneficial to the parasite in the insect host. The post-translational modification of proteins with the Small Ubiquitin-like MOdifier (SUMO) enables dynamic regulation of cellular metabolism. SUMO can be conjugated to its targets as a monomer but can also form oligomeric chains. Here, we have investigated the role of SUMO chains in T. brucei by abolishing the ability of SUMO to polymerize. We have found that parasites able to conjugate only SUMO monomers are primed for differentiation. This was demonstrated for monomorphic lines that are normally unable to produce stumpy forms in response to quorum sensing signaling in mice, and also for pleomorphic cell lines in which stumpy cells were observed at unusually low parasitemia levels. SUMO chain mutants showed a stumpy compatible transcriptional profile and better competence to differentiate into procyclics. Our study indicates that SUMO depolymerization may represent a coordinated signal triggered during stumpy activation program.

Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

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-ß-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-ß-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.