Signalling and regulation of autophagy-related pathways during cellular differentiation of African trypanosomes.

For a long time, lysosomes have been seen as waste management organelles. Over the last two decades, however, this view has evolved. Lysosomes are now considered as central organelles for the maintenance of homeostasis (a state of steady internal, physical and chemical conditions maintained by the cell for proper functioning), cellular survival and differentiation. Autophagy (from greek "Auto" self and "phagos" eating) is an essential process by which cells target intracellular components, macromolecules, organelles or intracellular pathogens to the lysosome for degradation and recycling. Autophagy is essential during cell growth, differentiation, or in response to stressors such as nutrient limitation. Moreover, defects or loss of regulation of autophagy can contribute to cancer or to the progression of neurodegenerative diseases. In eukaryotes, protein kinases and kinase enzyme complexes create protein phosphorylations. These reversible regulatory modifications are essential to control autophagy and therefore promote cell survival and differentiation. Recent studies have identified an unconventional secretory pathway called lysosome exocytosis that is dependent on the autophagy machinery. Lysosome exocytosis is a fundamental mechanism for the release of proteins that lysosomes could not degrade, thus avoiding potential toxic effect for the cell. Additionally, it has been implicated in the breach of host cellular membrane to allow invasion by the worm C. elegans. Indeed, the worm releases its lysosome contents directly into the extracellular environment to perforate the membrane of the host cells, allowing their invasion by the worm within the infected host.

Studying how protein kinases regulate autophagy and lysosome exocytosis by phosphorylation events is of major importance to understand how insect-transmitted parasites invade and survive in human or animal hosts. These organisms persist in changing environments that demand finely-tuned and rapid adaptation for survival. A tractable model to explore the diversity of eukaryotic signalling networks are kinetoplastid parasites. Kinetoplastid parasites are of great clinical relevance affecting millions of people and animals with diseases such as Leishmaniasis, Chagas disease and Sleeping Sickness, leading to critical social-economic implications. Kinetoplastid are exquisitely sensitive to their environment and require autophagy to adapt drastic changes during host transitions.

I propose to use the kinetoplastid parasite Trypanosoma brucei (causative agent of African sleeping sickness and livestock 'nagana') to characterise signalling pathways that regulate autophagy and lysosome exocytosis during lifecycle differentiation. In this project, I will develop an unbiased approach that will combine the optimisation of a high-throughput live imaging system, a gene silencing screen targetting all protein kinases encoded in the genome and mass spectrometry. Results obtained will reveal the molecular composition and regulations by phosphorylation of autophagy and lysosome exocytosis in T. brucei. The function of these regulatory components will then be evaluated for their roles in differentiation during the parasite lifecycle.

The results of this study will not only provide a better understanding of how kinetoplastid parasites adapt to the different hosts encountered during their complex lifecycles and persist in vivo, but will lay the foundations for the use of T. brucei as model to study the regulation of, and crosstalk between, the autophagy and exocytosis pathways.

Autophagy and lysosome exocytosis are essential pathways that share molecular machinery and regulate the dynamic composition of lysosomes, central organelles for the homeostasis, cell survival and cell differentiation. Therefore, studying the regulation of these pathways is of a major importance to understand the complex mechanisms leading to the dynamic turnover of molecules in lysosomes. My project aims to decipher the regulation of autophagy and lysosome exocytosis and potential interplay during the Trypanosoma brucei differentiation. To understand how trypanosomes maintain homeostasis and adapt to different hosts is of paramount importance for animal and human health. These organisms are likely to have evolved alternative approaches since, for example, they lack the essential autophagosome initiation kinase complex ULK1.

By coupling a high-throughput live imaging system with a kinome-wide RNAi screen, I will identify all kinases implicated in the regulation of autophagy in T. brucei. I will then reveal the dynamic (phospho-)proteomes of autophagy and lysosome exocytosis using a proximity labelling system. Direct interactions of these components with the identified kinases and their interplays between both pathways will be investigated by CRISPr/Cas9 knock-outs, kinase assays and mass spectrometry. Thereafter, I will decipher their role during tissue invasion and the quorum-sensing dependent differentiation, in the mammal host, and differentiation in the insect-vector of T. brucei, using relevant in vitro and in vivo models.

By demonstrating how phosphorylation controls autophagy, exocytosis and differentiation, this project will provide a better understanding of how trypanosome parasites adapt to, and persist in the different hosts encountered during their complex lifecycles. Additionally, where core components are conserved, this project may prove informative for other eukaryotes and neurodegenerative diseases, for example.