Tsetse flies are the sole vectors of Trypanosoma brucei parasites that cause sleeping sickness. Our knowledge on the early interface between the infective metacyclic forms and the mammalian host skin is currently highly limited. Glossina morsitans flies infected with fluorescently tagged T. brucei parasites were used in this study to initiate natural infections in mice. Metacyclic trypanosomes were found to be highly infectious through the intradermal route in sharp contrast with blood stream form trypanosomes. Parasite emigration from the dermal inoculation site resulted in detectable parasite levels in the draining lymph nodes within 18 hours and in the peripheral blood within 42 h. A subset of parasites remained and actively proliferated in the dermis. By initiating mixed infections with differentially labeled parasites, dermal parasites were unequivocally shown to arise from the initial inoculum and not from a re-invasion from the blood circulation. Scanning electron microscopy demonstrated intricate interactions of these skin-residing parasites with adipocytes in the connective tissue, entanglement by reticular fibers of the periadipocytic baskets and embedment between collagen bundles. Experimental transmission experiments combined with molecular parasite detection in blood fed flies provided evidence that dermal trypanosomes can be acquired from the inoculation site immediately after the initial transmission. High resolution thermographic imaging also revealed that intradermal parasite expansion induces elevated skin surface temperatures. Collectively, the dermis represents a delivery site of the highly infective metacyclic trypanosomes from which the host is systemically colonized and where a proliferative subpopulation remains that is physically constrained by intricate interactions with adipocytes and collagen fibrous structures.

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein–encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.

Background Tsetse flies ( Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans ( G. morsitans morsitans , G. pallidipes , G. austeni ), Palpalis ( G. palpalis , G. fuscipes ), and Fusca ( G. brevipalpis ) which represent different habitats, host preferences, and vectorial capacity. Results Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. Conclusions Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies. Electronic supplementary material The online version of this article (10.1186/s13059-019-1768-2) contains supplementary material, which is available to authorized users.

Chagas disease, the leading cause of cardiac-related mortality in endemic Latin American countries.Trypanosoma cruzi, the disease-causing pathogen, is unable to synthesize purines de novo, necessitating salvage of pre-formed host purines. Therefore, purine and purine nucleoside analogs might constitute an attractive source to identify antitrypanosomal hits. In this study, structural elements of two purine nucleoside analogs, i.e. cordycepin and a recently discovered 7-substituted 7-deazaadenosine led to the identification of novel nucleoside analogs with potent in vitro activity. The structure-activity relationship of substituents at C7 was investigated, ultimately leading to the selection of compound 5 having a C7 para-chlorophenyl group for in vivo evaluation. This derivative showed complete suppression of T. cruzi Y-strain blood parasitemia when orally administered twice daily for 5 days at 25 mg/kg and was able to protect infected mice from parasite-induced mortality. However, sterile cure by immunosuppression could not be demonstrated. improved efficacy and safety profiles and preferentially with a new mode-of-action should be pursued. 4 Recently, several reports on new lead molecules have been published, marking a renewed hope in finding drugs to treat Chagas disease. [6][7][8][9][10] Trypanosoma cruzi, like their related T. brucei counterparts, are purine auxotrophs, i.e. they rely on the salvage of pre-formed purine analogs from the host as they are unable to synthesize the purine ring themselves. [11][12][13][14] Hence, focused purine (nucleoside) libraries are a promising source for discovering new antitrypanosomal agents. Natural antibiotics such as tubercidin, 15 Formycin A 16 and Formycin B, [17][18][19] cordycepin, [19][20][21][22][23] stylomycin aminonucleoside (also known as puromycin aminonucleoside) [24][25] as well as allopurinol [26][27] and certain other inosine analogs 20,[28][29] have been found to possess activity against T. cruzi (Figure 1). Allopurinol, although not a nucleoside analog sensu stricto (its active metabolite is generated by phosphoribosylation in the parasite 26 ), has been evaluated in clinical trials. 30 Figure 1: Examples of purine (nucleoside) analogs active against T. cruzi.Our group recently revisited the natural nucleoside antibiotic tubercidin and a series of 7-substituted analogs [in the body of the text, purine numbering will be used for nucleoside analogs; however, in the experimental section, IUPAC nomenclature and numbering of the pyrrolo[2,3-d]pyrimidine system will be applied] in search of novel hits active against T. brucei spp. parasites. 31 This study indicated that certain phenyl-substituted analogs (e.g. 2) also showed promising in vitro activity against intracellular T. cruzi amastigotes, which motivated us to explore related 7-substituted 7-deazapurine moieties with the carbohydrate group of cordycepin, i.e. a 3'-deoxy-D-ribofuranose for their activity against T. cruzi.Related sugar-modified 7-deazapurine nucleosides 32-34 have previously been assayed ...

African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3’-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3’-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3’-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.

Tsetse flies are the notorious transmitters of African trypanosomiasis, a disease caused by the Trypanosoma parasite that affects humans and livestock on the African continent. Metacyclic infection rates in natural tsetse populations with Trypanosoma brucei, including the two human-pathogenic subspecies, are very low, even in epidemic situations. Therefore, the infected fly/host contact frequency is a key determinant of the transmission dynamics. As an obligate blood feeder, tsetse flies rely on their complex salivary potion to inhibit host haemostatic reactions ensuring an efficient feeding. The results of this experimental study suggest that the parasite might promote its transmission through manipulation of the tsetse feeding behavior by modifying the saliva composition. Indeed, salivary gland Trypanosoma brucei-infected flies display a significantly prolonged feeding time, thereby enhancing the likelihood of infecting multiple hosts during the process of a single blood meal cycle. Comparison of the two major anti-haemostatic activities i.e. anti-platelet aggregation and anti-coagulation activity in these flies versus non-infected tsetse flies demonstrates a significant suppression of these activities as a result of the trypanosome-infection status. This effect was mainly related to the parasite-induced reduction in salivary gland gene transcription, resulting in a strong decrease in protein content and related biological activities. Additionally, the anti-thrombin activity and inhibition of thrombin-induced coagulation was even more severely hampered as a result of the trypanosome infection. Indeed, while naive tsetse saliva strongly inhibited human thrombin activity and thrombin-induced blood coagulation, saliva from T. brucei-infected flies showed a significantly enhanced thrombinase activity resulting in a far less potent anti-coagulation activity. These data clearly provide evidence for a trypanosome-mediated modification of the tsetse salivary composition that results in a drastically reduced anti-haemostatic potential and a hampered feeding performance which could lead to an increase of the vector/host contact and parasite transmission in field conditions.

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