Biology subjects

Rotureau, B.

Publications and source records attributed to Rotureau, B..

11 recordsLinked to original sources

bioRxiv2025

Glycerol metabolism triggers trypanosome differentiation into transmissible forms in mammalian tissue-like conditions

In the mammalian bloodstream, Trypanosoma brucei, the parasite responsible for sleeping sickness, proliferates as slender forms before undergoing quorum-sensing (QS)-mediated differentiation into cell cycle-arrested stumpy forms (stumpy-QS), a transition that regulates parasitaemia and primes parasites for tsetse fly transmission. Beyond the bloodstream, T. brucei also occupies extravascular, adipose-rich tissues such as the skin, a potential reservoir for transmission. Here, we identify an alternative slender-to-stumpy differentiation pathway driven by glycerol, a host metabolite abundant in adipose-rich tissues, that could resolve the long-standing paradox of successful parasite transmission from human hosts during chronic infection despite low parasitaemia. We show that high (10 mM) and non-physiological glycerol concentrations under low glucose conditions (0.5 mM) induce differentiation, generating distinct stumpy-Glyc forms that resemble stumpy-QS parasites but have an extended lifespan. Under conditions mimicking dermal tissue interstitial fluids (4 mM glucose, 0.25 mM glycerol), we show that glycerol promotes the emergence of proliferative intermediate forms that retain transmission potential and can differentiate into fly host-specific procyclic forms in vitro and within tsetse flies. These findings open the door for reevaluation of the model of T. brucei transmission and supports a dominant role for adipocyte-derived glycerol in the skin in sustaining parasite transmission.

microbiology↗

bioRxiv2025

A SHERLOCK toolbox for the eco-epidemiological surveillance of animal African trypanosomosis reveals a similar parasite diversity in domestic pigs in two ancient sleeping sickness foci in Western Africa.

Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan- trypanosomatid, Trypanozoon, T. vivax, T. congolense, T. theileri, T. simiae and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Cote dIvoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7 % [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense, a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.

microbiology↗

bioRxiv2023

Stumpy forms are the predominant transmissible forms of Trypanosoma brucei.

Schuster et al. demonstrated that bloodstream slender forms of African trypanosomes are readily transmissible to young tsetse flies where they can complete their complex life cycle (1). In their experimental conditions, a single slender parasite was sufficient for productive infection. Here, we compared the infectivity of slender and stumpy bloodstream forms in adult flies with a mature immune system, and without using any chemical compounds that would alter the insect immune response and/or promote the infection. After ingestion of slender forms, infected flies were observed only in one out of 24 batches of non-immunocompetent teneral flies and with a high number of parasites. In contrast, infected flies were detected in 75% (18/24) of the batches infected with stumpy parasites, and as few as 10 stumpy parasites produced mature infections in immune adult flies. We discuss that, although Schuster et al. have demonstrated the intrinsic capacity of slender form trypanosomes to infect young and naive tsetse flies, highlighting the remarkable plasticity and adaptability of these protists, this phenomenon is unlikely to significantly contribute to the epidemiology of African trypanosomiases. According to both experimental and field observations, stumpy forms appear to be the most adapted forms for African trypanosome transmission from the mammalian host to the tsetse fly vector in natural conditions.

microbiology↗

bioRxiv2023

FLAgellum Member 8 modulates extravasation and 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. Author SummaryTrypanosoma brucei parasites cause neglected tropical diseases termed human and animal African trypanosomiases. Transmitted by the bite of an infected tsetse fly, upon deposition in the skin of a mammalian host, these parasites occupy both the vasculature and extravascular tissues. Currently, the biology of tissue-resident parasites is not well understood, and the parasite factors that mediate extravascular colonization are not known. Using quantitative in vivo bioluminescence imaging and ex vivo gene expression quantification in host infected tissues and blood, we reveal that the flagellar parasite protein FLAM8 modulates the extravascular dissemination of trypanosomes in the mammalian host. FLAM8 is known to act as a docking platform for signaling complexes in the flagellum, but we observe that it does not influence parasite differentiation into transmissible stages. However, we show that the absence of FLAM8 results in the loss of a key component of the flagellar adenylate cyclase signaling complexes, and reduces parasite migration through endothelial cell monolayers, suggesting that FLAM8 is important for parasite exchanges between the intravascular and the extravascular compartments. This work identifies a key trypanosome flagellar component involved in host-parasite interactions, including the modulation of parasite tropism and extravascular dissemination.

microbiology↗

bioRxiv2021

Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei within the tsetse fly vector

The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasites mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.

molecular biology↗

bioRxiv2021

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands.

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.

molecular biology↗

bioRxiv2021

FLAgellum Member 8 modulates extravascular trypanosome distribution in the mammalian host

The African trypanosome flagellum is an essential organelle in multiple aspects of the parasites development. Here, we investigated the role of a flagellar protein termed FLAgellar Member 8 (FLAM8) that is specifically distributed along the entire flagellum in trypanosomes stages of the vertebrate host. 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 insect transmissible forms. However, quantitative bioluminescence imaging revealed that FLAM8-null parasites exhibit an impaired dissemination in the extravascular compartment, that is partially restored by the addition of a single rescue copy of FLAM8. Interestingly, among all dissected organs scrutinized individually, only the skin of mice infected with FLAM8-deprived parasites showed a significant reduction in extravascular trypanosome population as compared to mice infected with parental controls. 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, especially in the skin. Take awayO_LIFLAM8 is dispensable in vitro for survival, growth, motility and differentiation of T. brucei. C_LIO_LIFLAM8 depletion does not affect parasitemia and bloodstream form differentiation in vivo. C_LIO_LIFLAM8 modulates the extravascular dissemination of trypanosomes in the mammalian host, especially in the skin. C_LI

microbiology↗

bioRxiv2020

Intraflagellar transport during the assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies

Multicellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. This is the case of Trypanosoma brucei that assembles flagella of 3 to 30 {micro}m during its development in the tsetse fly. It provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3-D electron microscopy and live cell imaging revealed that IFT was present in all life cycle stages. IFT proteins are concentrated at the base, IFT trains are located along doublets 3-4 & 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed. Summary statementThis work investigated the assembly of flagella of different length during the development of Trypanosoma brucei in the tsetse fly, revealing a direct correlation between the amount of intraflagellar transport proteins and flagellum length.

cell biology↗

bioRxiv2019

\"Metabolic contest\", a new way to control carbon source preference

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, allowing to redirect cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism, named \"metabolic contest\", for regulating the use of carbon sources without nutrient sensing and signaling. In contrast to most microorganisms, trypanosomes show a glycerol-to-glucose preference that depends on the combination of three conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-like organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways being both ATP-dependent (glycerol kinase and hexokinase, respectively) and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being ~80-fold higher than the hexokinase activity.

microbiology↗

bioRxiv2019

Timing and original features of flagellum assembly in trypanosomes during development in the tsetse fly

Trypanosoma brucei exhibits a complex life cycle alternating between tsetse flies and mammalian hosts. When parasites infect the fly, cells differentiate to adapt to life in various tissues, which is accompanied by drastic morphological and biochemical modifications especially in the proventriculus. This key step represents a bottleneck for salivary gland infection. Here we monitored flagellum assembly in trypanosomes during differentiation from the trypomastigote to the epimastigote stage, i.e. when the nucleus migrates to the posterior end of the cell. Three-dimensional electron microscopy (Focused Ion Bean Scanning Electron Microscopy, FIB-SEM) and immunofluorescence assays provided structural and molecular evidence that the new flagellum is assembled while the nucleus migrates towards the posterior region of the body. Two major differences with well known procyclic cells are reported. First, growth of the new flagellum begins when the associated basal body is found in a posterior position relative to the mature one. Second, the new flagellum acquires its own flagellar pocket before rotating on the left side of the anterior-posterior axis. FIB-SEM revealed the presence of a structure connecting the new and mature flagellum and serial sectioning confirmed morphological similarities with the flagella connector of procyclic cells. We discuss potential function of the flagella connector in trypanosomes from the proventriculus. These findings show that T. brucei finely modulates its cytoskeletal components to generate highly variable morphologies.\n\nAuthor SummaryTrypanosoma brucei is a flagellated parasitic protist that causes human African trypanosomiasis, or sleeping sickness and that is transmitted by the bite of tsetse flies. The complex life cycle of T. brucei inside the tsetse digestive tract requires adaptation to specific organs and follow a strictly defined order. It is marked by morphological modifications in cell shape and size, as well organelle positioning. In the proventriculus of tsetse flies, T. brucei undergoes a unique asymmetric division leading to two very different daughter cells: one with a short and one with a long flagellum. This organelle is crucial for the trypanosome life cycle as it is involved in motility, adhesion and morphogenesis. Here we investigated flagellum assembly using molecular and 3D Electron Microscopy approaches revealing that flagellum construction in proventricular trypanosomes is concomitant with parasite differentiation. During flagellum growth, the new flagellum is connected to the mature one and rotates around the mature one after its emergence at the cell surface. The sequence of events is different from what is observed in the well-studied procyclic stage in culture revealing different processes governing morphological development. These results highlight the importance to study pathogen development in their natural environment.

microbiology↗

bioRxiv2018

Control of flagellum length by a grow-and-lock model

Several models have been proposed to explain how eukaryotic cells control the length of their cilia and flagella. Here, we investigated this process in the protist Trypanosoma brucei, an excellent system for cells with stable cilia like photoreceptors or spermatozoa. We show that the total amount of intraflagellar transport material (IFT, the machinery responsible for flagellum construction) increases during flagellum elongation, consistent with constant delivery of precursors and the previously reported linear growth. Reducing the IFT frequency by RNAi knockdown of the IFT kinesin motors slows down the elongation rate and results in the assembly of shorter flagella. These keep on elongating after cell division but fail to reach the normal length. This failure is neither due to an absence of precursors nor to a morphogenetic control by the cell body. We propose that the flagellum is locked after cell division, preventing further elongation or shortening. This is supported by the fact that subsequent increase in the IFT rate does not lead to further elongation. The distal tip FLAM8 protein was identified as a marker for the locking event. It is initiated prior cell division, leading to an arrest of elongation in the daughter cell. Here, we propose a new model termed grow-and-lock where the flagellum elongates until a locking event takes place in a timely defined manner hence fixing length. Alteration in the growth rate and/or in the timing of the locking event would lead to the formation of flagella of different lengths.

cell biology↗