The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.DOI: http://dx.doi.org/10.7554/eLife.17716.001

Abstract Background The diagnosis of gambiense Human African Trypanosomiasis (gHAT) typically involves two steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who we hypothesize are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. Methods To test this hypothesis, we conducted a prospective observational cohort study in the gHAT focus of Forecariah, Republic of Guinea. Of the 5,417 subjects serologically screened for gHAT, 66 were enrolled into our study and underwent a dermatological examination. At enrolment, 11 seronegative, 8 unconfirmed seropositive and 18 confirmed seropositive individuals had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immuno-histological methods. Results In seropositive individuals, dermatological symptoms were significantly more frequent, relative to seronegative controls. T.b. gambiense parasites were present in the blood of all confirmed cases (n=18) but not in unconfirmed seropositive individuals (n=8). However, T. brucei parasites were detected in the extravascular dermis of all unconfirmed seropositive individuals and all confirmed cases. Skin biopsies of all treated cases and most seropositive untreated individuals progressively became negative for trypanosomes 6 and 20 months later. Conclusions Our results highlight the skin as a potential reservoir for African trypanosomes, with implications for our understanding of this disease’s epidemiology in the context of its planned elimination and underlining the skin as a novel target for gHAT diagnostics.

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.

Highlights • New triple reporter combining a red luciferase and a tdTomato fused by a Ty1-tag • Tested in two different stages of both T. brucei and L. major parasites • Tested in vitro and in vivo in tsetse flies and murine models • Useful for multimodal imaging at different scales, including intravital monitoring

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway.

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

The African trypanosome flagellum is essential in multiple aspects of the parasite development. In the mammalian infective form of this protist, FLAgellar Member 8 (FLAM8) is a large protein distributed along the entire flagellum that is suspected to be involved in host-parasite interactions. Analyses of knockdown and knockout trypanosomes demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and slender to stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes are able to establish and maintain the infection in the blood circulation, and to differentiate into transmissible stumpy forms. However, bioluminescence imaging revealed that FLAM8-null parasites exhibit an impaired dissemination in the extravascular compartment, especially in the skin, that is partially restored by the addition of a single rescue copy of FLAM8. To our knowledge, FLAM8 is the first example of a flagellar protein that modulates T. brucei parasite distribution in the host tissues, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches.Author summaryAfrican trypanosomes are blood and tissue-dwelling protists responsible for African trypanosomiases, a group of neglected tropical diseases in sub-Saharan Africa, including sleeping sickness in humans and nagana in cattle. Although underestimated for long, the importance of extravascular parasite niches has been recently re-discovered both in animal models and in infected individuals. The trypanosome flagellum is an essential organelle for the parasites, yet its contribution to host-parasite interactions, parasite tropism and spatiotemporal dissemination in the mammalian host remain scarce. To this end, we investigated the role of a flagellar protein termed FLAgellar Member 8 (FLAM8), present only at the distal tip of the flagellum in insect parasites, but differentially localized to the entire flagellum length in trypanosomes found in the vertebrate host. Our findings revealed that FLAM8 is dispensable in vitro for survival, growth, motility and differentiation of T. brucei. However, interestingly, FLAM8 modulated the extravascular dissemination of trypanosomes in the mammalian host, especially in the skin. FLAM8 is the first example of a flagellar protein that modulates T. brucei parasite distribution in extravascular host tissues.