African trypanosomes are transmitted by the bite of the tsetse fly and cause the debilitating, and often fatal, neglected tropical disease sleeping sickness, or Human African Trypanosomiasis (HAT). Trypanosoma brucei gambiense, the parasites responsible for 98% of human cases, first reside in the patient blood and skin for months to years before invading the central nervous system, where they cause the neurological symptoms of the disease. HAT is approaching elimination, with the number of cases reported in 2017 dropping to approximately 1,442 from only a dozen African countries. In this context, HAT was included in the WHO roadmap on neglected tropical diseases, with 2020 set as target date for elimination as a public health problem. A secondary goal of zero transmission by 2030 has also been set. These targets have, in part, been encouraged by the success of surveillance efforts that rely on detecting extracellular trypanosomes in human blood. Nevertheless, the reduction in case numbers brings about other challenges. For example, the sensitivity of any diagnostic test diminishes as the disease burden drops, and this is being seen with the serological tests available for HAT. In this context, new highly sensitive and specific diagnostic tools will be required to accurately monitor the occurrence of new cases and the possible emergence of drug-resistant trypanosomes during the elimination phase. Most diagnostic tests currently under development are based on optimization of existing methods that may not combine all the requirements to stand up to the harsh constraints imposed by the elimination phase requirements, especially in terms of sensitivity and specificity. We propose that adapting the recently developed Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) technology that combines a CRISPR-Cas system and lateral flow test to trypanosomes will provide the sensitivity and specificity required for a diagnostic test in the elimination and post elimination phases. The SHERLOCK system relies on the collateral effect of Cas13a promiscuous RNA cleavage activity upon target recognition. Combining the collateral effect with pre-amplification of RNAs resulted in rapid RNA detection with attomolar (10^-18 moles/l) sensitivity and single-base mismatch specificity, in a diagnostic setting. This technology has been used to detect specific strains of Zika and Dengue viruses and distinguish pathogenic bacteria in a mixed sample. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications. A lateral flow test for a simple and rapid readout can be easily implemented after a reaction that does not exceed two hours from the sampling step. The first step of this project is to identify promising RNA targets in silico by data mining and multiple alignements of all available transcriptomic data on bloodstream African trypanosomes.

Centrosomes are the main microtubule organizing center of eukaryotic cells with critical roles in cell division, polarity, signaling and structure. In most cells, one or both centrioles act as basal body (BB), nucleating microtubules to form cilia or flagella, sensory and motile organelles of vital importance for a wide range of biological functions. Notorious deadly diseases such as cancer, microcephaly and ciliopathies correlate with dysregulation in the number and/or structure of the centrosome/BB. Defects in centriolar proteins also impact cell division and flagellar function of parasitic protists. Notably, T. gondii can assemble flagella during its sexual cycle within the cat’s enteroepithelial tissue, a largely unattainable life stage in vitro. The state of the art of the field points at the centrosome and basal body of apicomplexan and trypanosomatid parasites as potentially rich sources of novel therapeutic targets to fight parasitic diseases. However, their molecular composition and the regulation of their biogenesis remain ill-described. Albeit a number of structural components appear to be conserved between parasitic protozoa and their vertebrate hosts, the absence of conserved homologs of regulatory components, suggests that their biogenesis is likely controlled by divergent triggers of unknown targets. Within the framework of a funded ACIP grant (076-2017), this team pursued the characterization of the centrosome composition in T. gondii, and explored the localization of newly identified principles in T. brucei. This proposal focuses on deciphering the role of the newly identified proteins in the biology of the centrosome in Toxoplasma gondii, as a model for the phylum apicomplexa, and to analyze the role of these conserved proteins in basal body biogenesis and function in Trypanosoma brucei. Based on our preliminary identification of novel centrosomal/basal body components and the powerful tools available in our model organisms, we now propose: 1. To analyze the phylogenetic distribution and functional domains of 20 novel proteins of T. gondii through bioinformatic approaches. 2. To assess the localization of these 20 proteins, and the function and cell cycle dynamics of those localized to the centrosome, in T. gondii. 3. To characterize the function of a protein complex linking the centrosome to nascent daughter cells in T. gondii. 4. To characterize the role of 3 novel T. brucei proteins homologs in basal body biology.

African trypanosomes are flagellated protist parasites transmitted to mammals by the infectious bite of the tsetse fly. They are responsible for sleeping sickness in humans and nagana in cattle. Trypanosomes first proliferate freely in the blood, and then, about six hours after being inoculated, leave the bloodstream to invade various organs, including the skin, which is an anatomic reservoir for the parasites. Few details are known on the metabolic aspects of the different parasite stages in the blood and in the skin, as well as the immune response of the mammalian host against the different stages of development of dermal parasites. Two strains of trypanosomes (“AnTat1.1E” and “Lister 427”) with different genetic profiles were studied here: one having only one developmental stage named "SL" or slender in reference to their tapered shape in mice, the second having two developmental stages "SL", and "ST" for stumpy in reference to their stocky shape in mice. This will allow us to distinguish the transcriptomic signature of parasites at the ST stage. Mice were sampled 5 days or 4 weeks after experimental infection in order to evaluate how the parasite transcriptome evolves over the course of an infection. In all these conditions, blood and skin samples were taken in order to compare the transcriptomes of blood and dermal parasites on the one hand, and transcriptomes of murine blood and dermal tissues on the other. We will especially scrutinize the metabolic pathways of the two parasite strains in the blood and the skin, as well as the immune response of the host in each compartment, two crucial elements determining the development of the infection.