Metabolic interactions between adipocytes and trypanosomes, a new paradigm for trypanosomiases – AdipoTryp

We have recently established new growth conditions for bloodstream form (BSF) trypanosomes in which glucose was replaced by glycerol. This led us to show that BSF grown in glycerol are ~60-fold less sensitive to suramin (the first line drug used to treat stage 1 HAT patients infected with T. brucei rhodesiense), as compared to glucose conditions. This glycerol-linked suramin resistance provides us with a unique tool to study adipocyte-BSF metabolic interactions in vitro and in vivo. Indeed, the presence of suramin will push BSF trypanosomes to use the glycerol produced by adipocytes to feed their central carbon metabolism. We established the proof of concept for this original approach. Indeed, a BSF strain causing acute infections (monomorphic strain) in the mouse model was shown to reside for at least 3 weeks in the extravascular compartment of several tissues of infected mice treated with suramin, including in the skin and the white adipose tissues, while the parasitemia remained undetectable after parasite clearance by suramin. These experiments will be repeated with mutant cell lines (glycerol kinase and aquaglyceroporin among others). In order to address specific questions, in vivo experiments will be completed by in vitro experiments, performed on parental and mutant BSF cell lines. (i) Molecular dialog between trypanosomes and adipocytes. We will determine whether glycerol produced by adipocytes can sustain growth of BSF trypanosomes by developing BSF-adipocytes co-culture conditions. These metabolic interactions with be analysed by NMR spectrometry (exometabolome) and mass spectrometry (endometabolome). (ii) Sensing glycerol for tissues tropism. As glycerol is not evenly available in all tissues, we reasoned that the parasite tropism to the skin and adipose tissues may require sensing of chemo-attractants, such as glycerol. (iii) Differentiation of BSF in the tissues for transmission to the fly. Preliminary data showed that glycerol stimulates differentiation of dividing slender BSF into non-dividing stumpy BSF. We will use In vitro standard protocols for differentiation of slender BSF in stumpy BSF and then into procyclic trypanosomes (PCF). The role of glycerol metabolism in the development of PCF trypanosomes in the insect vector will be addressed in vitro and in vivo with different mutant cell lines. We will use a model already optimized in our network that mimics the parasite development upon inducible over-expression of RBP6. The production of epimastigote forms (salivary glands) and then of metacyclic forms (salivary glands) by the parental and mutant procyclic forms (midgut) over-expressing RBP6 will be quantified by stage-specific markers upon induction of RBP6 expression in the presence or the absence of glycerol. In vivo analyses of the development of trypanosomes in the insect vector will be done by monitoring the colonization of the midgut and salivary glands.

1- Metabolic interactions between adipocytes and trypanosomes in vivo - To study the in vivo aspects, we produced a triple knockout mutant KOaqp1/2/3 (aquaglyceroporin) which is incapable of transporting and therefore of metabolizing glycerol. In vivo in the mouse model, this mutant shows a significant delay in its ability to invade the tissues of the infected animal, in comparison with the KOaqp1, KOaqp2/3 and parental strains of the parasite, suggesting that glycerol has a role in the propagation of the parasite in vivo. Treatment of animals infected with the triple mutant with suramin, which is lethal to glucose-consuming parasites, is underway. We expect that the mutant will no longer be able to colonize the tissues, unlike the parental strain. - We also produced a KO mutant of the genes encoding glycerol kinase (GK) in BSF using a new approach based on CRISPR-Cas9 technology. In fact, the 11 copies of the GK gene were eliminated in a single transfection of the BSF with the guide, the cassette containing the resistance gene and the recombinant Cas9. This new approach will be submitted for publication by the end of the year. The KOgk and “rescues” lines (in production) will be used to infect mice, whether or not treated with suramin. - We also participated in the characterization of parasitic forms of adipose tissue (Trindade et al., Nat Microbiol, In revision). These forms multiply more slowly than BSF and exhibit the characteristics of persistent forms. 2- Metabolic interactions between adipocytes and trypanosomes in vitro - The in vitro aspect requires optimizing the culture of adipocytes so that they produce enough glycerol to feed the metabolism of trypanosomes in co-culture. We have observed that the addition of insulin and a lipolysis inhibitor makes it possible to multiply by 7 the production of glycerol in adipocytes, in concentrations compatible with the growth of the parasites. Preliminary data from co-cultures between adipocytes and BSF show that trypanosomes use the glycerol produced by adipocytes, even in the presence of glucose. These preliminary data suggest that adipocytes may influence the metabolism of trypanosomes, as the latter prefer glucose to glycerol, when cultured alone. - We recently showed that the procyclic forms (PCF) of trypanosomes prefer to metabolize glycerol to glucose using a new mode of regulation of the use of carbon sources called «metabolic contest« (Allemann, PLoS Biol, 2021). We believe that this is also the case for BSF in co-culture with adipocytes, a hypothesis that we are testing in vitro. 3- Glucogenesis from glycerol We have participated in the demonstration that PFK can function in vitro in the direction of gluconeogenesis (Fernandes, 2019). This work is consistent with our hypothesis that PFK is involved in the production of F6P from F1,6BP. To test this hypothesis, we generated an inducible RNAi line targeting PFK (RNAi-PFK) currently under analysis.

The data that we are going to accumulate will provide a better understanding of the development of the parasite in its mammalian host, in particular in adipose tissue and skin. We recently demonstrated for the first time that the patients tested had parasites in the skin, including those with negative serology (M. Camara et al., Clin Infect Dis, 2020). These results highlight that the skin is a potential reservoir for African trypanosomes, with implications for our understanding of the epidemiology of this disease in the context of its planned elimination. Beyond understanding the biology of the parasite in vivo, our work also aims to participate in the development of new therapeutic means against the tissue forms of the parasite.

MULTIPARTENAIRE
Allmann S., M. Wargnies, E. Cahoreau, M. Biran, N. Plazolles, P. Morand, E. Pineda, H. Kulyk-Babier, C. Asencio, O. Villafraz, L. Rivière, E. Tetaud, B. Rotureau, A. Mourier, J.-C. Portais, & F. Bringaud (2021) Glycerol suppresses glucose consumption in trypanosomes through metabolic contest. PLoS Biol. 19:e3001359. (article)

Villafraz O., M. Biran, N. Plazolles, E. Cahoreau, R. Ornitz Oliveira Souza, E. Tetaud, L. Rivière, A. Silber, M.P. Barrett, A. Zikova, M. Boshart, J.-C. Portais & F. Bringaud (2021) Procyclic Trypanosomes Recycle Glucose Catabolites and TCA Cycle Intermediates to Stimulate Growth in the Presence of Physiological Amounts of Proline. PLoS Pathog. 17:e1009204. (article)

Villafraz O., H. Baudouin, M. Mazet, H. Kulyk, J.-W. Dupuy, C. Botté, D. Inaoka, J.-C. Portais & F. Bringaud (2021) The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place. mBio 12:e0037521. (article)

MONOPARTENAIRE
Mochizuki K., D.K. Inaoka, M. Mazet, T. Shiba, K. Fukuda, H. Kurasawa, Y. Millerioux, M. Boshart, E.O. Balogun, S. Harada, K. Hirayama, F. Bringaud & K. Kita (2020) The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei. BBA Bioenergetics. 1861:148283. (article)

Michels P.A.M, O. Villafraz, E. Pineda, M.B. Alencar, A.J. Cáceres, A.M. Silber & F. Bringaud (2021) Carbohydrate metabolism in trypanosomatids is not quite so simple. Exp. Parasitol. 224:108102. (revue

Camara M, Soumah AM, Ilboudo H, Travaillé C, Clucas C, Cooper A, Kuispond Swar NR, Camara O, Sadissou I, Calvo Alvarez E, Crouzols A, Bart JM, Jamonneau V, Camara M, MacLeod A, Bucheton B, Rotureau B. (2021) Extravascular Dermal Trypanosomes in Suspected and Confirmed Cases of gambiense Human African Trypanosomiasis. Clin. Infect. Dis. Jul 1;73(1):12-20. (article)

Lagarde D, Jeanson Y, Portais JC, Galinier A, Ader I, Casteilla L, Carrière A. (2021) Lactate Fluxes and Plasticity of Adipose Tissues: A Redox Perspective. Front Physiol.12:689747. (revue)

Lagarde D, Jeanson Y, Barreau C, Moro C, Peyriga L, Cahoreau E, Guissard C, Arnaud E, Galinier A, Bouzier-Sore AK, Pellerin L, Chouchani ET, Pénicaud L, Ader I, Portais JC, Casteilla L, Carrière A. Lactate fluxes mediated by the monocarboxylate transporter-1 are key determinants of the metabolic activity of beige adipocytes. (2021) J Biol Chem. 296:100137. (article)

Trypanosoma brucei is a unicellular and extracellular parasite transmitted by the tsetse fly (Glossina genus) and causing Human African Trypanosomiasis (HAT) in Sub-Saharan Africa. If untreated, this neglected tropical disease has a case fatality rate close to 100%. There is no vaccine and the currently available drugs present significant side-effects, variable efficacies and/or do not cross the blood brain barrier. Other related parasite species are responsible for animal African trypanosomiases that remain a major constraint to productive livestock rearing in countries throughout Sub-Saharan Africa, causing major economic losses.
It has been considered for decades that trypanosomes propagate exclusively in the body fluids of their mammalian hosts and mostly in the blood. In a break with this dogma, three recent publications showed that most parasites actually reside in the extravascular compartment of mouse models, especially in the adipose tissues and the skin, from which transmission can occur. Within the skin, some parasites seem to tightly interact with adipocytes, the major constituent of fat, suggesting that the adipocytes-trypanosomes interactions might confer a selective advantage to the parasites. In resonance with this major discovery, we recently broke another strong dogma considering that T. brucei relied exclusively on the glucose provided by the mammalian host blood to feed its central carbon metabolism. Indeed, we established long-term conditions for growth of the parasite in glucose-free medium containing glycerol. Since adipocytes excrete large amounts of glycerol from lipolysis and glycolysis, we hypothesised that extravascular trypanosomes could take advantage of this glycerol production to (i) feed their central carbon metabolism and (ii) colonize the glycerol-rich tissues by chemotactism. Our preliminary data presented in details in the application support these two hypotheses.
The objectives of this project are (i) to evaluate the importance of the glycerol metabolism for parasites in vivo, including a potential role of the glycerol as a metabolic sensor to attract trypanosomes in specific tissues, (ii) to develop an in vitro assay to study the metabolic interactions between adipocytes and trypanosomes, (iii) to determine the probable alternative(s) to FBPase, the key gluconeogenic enzyme, as well as the role of the parasite gluconeogenesis in vivo in the mammalian host, (iv) to confirm the existence and determine the role of ß-oxidation of fatty acids previously proposed to be activated in tissue-dwelling parasites, and (v) to identify the most relevant metabolic step(s) targeted by suramin. All these questions will be tackled by leading experts in complementary fields forming a long-standing network.
This project will have major impacts in understanding the biology of tissue trypanosomes. It will also pave the way for the development of therapeutic approaches targeting extravascular parasites.