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Exploring the hidden life of African trypanosomes: parasite fat tropism and implications for disease

Periodic Reporting for period 1 - FatTryp (Exploring the hidden life of African trypanosomes: parasite fat tropism and implications for disease)

Reporting period: 2018-08-01 to 2020-01-31

We have recently discovered that the adipose tissue (fat) is a major reservoir for the extracellular protozoan Trypanosoma brucei and that, within this environment, parasites become phenotypically different from those in the blood. Our study exposed novel biology of the T. brucei life cycle, yet it remains unknown how parasites adapt to the fat and how parasite fat tropism affects disease. Our first aim is to determine the molecular and cellular mechanisms underlying T. brucei fat tropism. We will perform a genetic screen in mice to identify key parasite genes required for establishing and maintaining chronic infection in the fat. Together with the information of the transcriptome and proteome, we will identify the mechanistic steps underlying parasite tissue-adaptation. Our second aim is to identify the consequences of T. brucei fat tropism for the host and the importance for disease. We will first investigate if parasites can egress from the fat. We will also determine if parasites induce lipid breakdown in the host, leading to loss of fat mass. Finally, we will measure the impact of fat tropism in general traits of disease, including host survival and transmission potential.

This project represents a completely novel research avenue built on recent work from my laboratory. By uncovering fundamental aspects of the biology of T. brucei, we will also improve the understanding of clinically relevant features of African trypanosomiasis, including relapses and weight loss. In addition, since parasite fat tropism has also been observed in malaria and Chagas’ disease, our findings will help elucidate disease mechanisms relevant to other infectious diseases.
Aim 1. Determine the molecular and cellular mechanisms behind parasite fat tropism

We performed a proteome analysis of monomorphic T. brucei parasites isolated from blood and gonadal fat, revealing 166 protein groups differentially expressed. The biological process ontology showed that the most significant alteration in ATFs is the down-regulation of proteins involved in translation. We established a fluorescence-based method to measure the rate of protein synthesis, which confirmed that protein synthesis is 24% lower in ATFs than BSFs. We are currently investigating the mechanisms that regulate these changes in gene expression.

To identify HOF genes in T. brucei, we have initiated the RNAi screen (in collaboration with Catarina Gadelha, Nottingham). A 24,000-clone surfeome specific library has been produced and allowed relative fitness quantification at very high accuracy both in vitro and in vivo, demonstrating that 1/4 surfeome genes are required for normal parasite growth. While the large-scale screen is being established, with the help of our collaborators Terry Smith and Fred Bringaud, we generated mutant parasite lines to test the role of candidate HOF genes (TFEa, ASCT, NUBM, NUKM). None of them resulted in a reduced load of parasites in adipose tissue and thus they were discarded.

Aim 2. Determine the consequences of parasite fat tropism for host and disease

We showed that lipolysis in activated in white adipose tissue early during infection independently of disease-induced hypophagia. This process is dependent of adipose triglyceride lipase (ATGL). ATGL deficiency leads to shorter host survival, suggesting adipose tissue may act as a parasite sponge, promoting host survival. Activation of Lipolysis results in the release of Glycerol by adipocytes. We found that Glycerol has a major impact in the life cycle of T. brucei, triggering differentiation from slender to stumpy forms.

To have a broader picture of the changes undergone by host tissue during T. brucei infection, we compared the transcriptome of adipocytes and liver at multiple days of infection. In both tissues, he observed a very robust upregulation of genes involved with immune response. We are currently focusing our attention in the downregulated genes, whose functions are more diverse.

We established intravital microscopy and CUBIC protocol to analyse transparent organs by Light Sheet fluorescence microscopy. We succeeded to track and quantify T. brucei behaviour in the arterial and venous vasculature, and characterize parasite behaviour in the extravascular space of multiple organs. We found significant differences between brown and white adipose tissues. Unexpectedly, we identified the pancreas as an additional large reservoir of T. brucei parasites.
All results described in section above are novel and go beyond the state of the art. Till the end of the project, we expect to show:
1. The metabolic adaptations of parasites to adipose tissue and consequences of this adaptation to parasite virulence.
2. The impact of T. brucei infection on host lipid metabolism and implications for cachexia.
3. The mechanism by which parasites cross from blood into tissues.
Graphical abstract