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Molecular Basis of Toxoplasma gondii Encystation and Persistence

Periodic Reporting for period 2 - ToxoPersist (Molecular Basis of Toxoplasma gondii Encystation and Persistence)

Reporting period: 2018-04-01 to 2019-09-30

Toxoplasma gondii is an ubiquitous obligate intracellular parasite that infects most warm blooded animals. The acute phase of infection initiates with the dissemination of the fast-replicating tachyzoites. At the onset of the immune response tachyzoites convert into slow-replicating bradyzoites that form cysts predominantly in the central nervous system and in striated and heart muscles. Encystation ensures life-long persistence and poses a significant threat of reactivation during immunosuppression and can lead to encephalitis and other severe clinical manifestations. Despite the central importance of encystation for pathogenesis and transmission, our insight into how T. gondii defies the innate and adaptive immune responses to take up permanent residence in the immunocompetent hosts is rudimentary. The encystation effectively sequesters bradyzoites from pharmacological intervention and currently makes it impossible to cure latent toxoplasmosis.
Understanding how host and parasite creates a microenvironment in the host brain that allows both controlled dissemination and long-term persistence while keeping inflammation at bay is key to develop successful therapeutic interventions. We propose to capitalize on the increased sensitivity of -omics approaches, the power of the CRISPR/Cas9 genome editing, the high-resolution microscopy, and on the ex-vivo tissue examination by MALDI imaging mass spectrometry and NanoSIMS technologies to address four objectives: i) – Identify the components of the niche into which the dormant parasite persists, including the Cyst Wall (CW), Parasitophorous Vacuole (PV) and PV Membrane (PVM) of the cyst. ii) Determine the parasite factors responsible for CW formation and maturation via targeted and unbiased approaches. iii) Define the metabolic functions of the bradyzoites to ensure encystation persistence. iv) Identify the host metabolic functions hijacked by the parasite to ensure survival and persistence.
The ToxoPersist project is articulated around four specific objectives:
1. Identification of the components of the Cyst Wall (CW), Parasitophorous Vacuole (PV) and PV Membrane (PVM) of the cyst
As planned we have generated all the recipient parasite lines to undertake reverse genetic approaches in cyst forming strains of Toxoplasma gondii. To identify bradyzoite specific parasite proteins targeted to the CV, PV, PVM and potentially exported in to the host cells, we capitalized on our recent parasites mutant ASP5-KO published in Hammoudi PM et al,. PLoS Pathog. 2015 Oct 16;11(10):e1005211 and on the TAILS approach that we established for T. gondii and described in Dogga et al, Elife. 2017 Sep 12;6. pii: e27480. We are currently tagging two dozen of candidates ASP5 substrates and disrupting the corresponding genes to assess their function in bradyzoites differentiated in vitro as well as in vivo (mouse model).

2. Determination of the parasite factors responsible for CW formation and maturation via targeted and unbiased approaches
In parallel and as planned, we have selected as series of bradyzoites specific genes based on stage specific RNAseq data (in collaboration with Dr. A Hehl in Zurich) and bioinformatics considerations (uniquely conserved in cysts forming Apicomplexans, trafficking determinants). The 15 first knockout mutants are currently under investigation in bradyzoites differentiated in vitro as well as in vivo (mouse model). In this context, we have unraveled the role of unconventional myosins in establishing cell-cell communication between intravacuolar parasites. This communication is interrupted upon bradyzoite differentiation and cyst maturation to all parasite growth in a slow and unsynchronized manner Frénal K et al, Nat Commun. 2017 Jun 8;8:15710.

3. Identification of the metabolic functions of the bradyzoites to ensure encystation persistence
To understand the metabolic capabilities of life-cycle stages in T. gondii we have combined computational and experimental approaches (-omics data) to build well-curated stage-specific metabolic networks, ToxoNet2 and ToxoNet3 capable of accurately predicting novel and experimentally observed phenotypes. Upon integrating the CRISPR-based genome-wide screen for genes contributing to parasite fitness, we have acquired important insights into the physiological conditions of encystation. Three key metabolic pathways are currently under comparative investigation between acute and chronic stages of infection: Pentothenate, Coenzyme A and Heme biosynthesis, and sphingolipids biosynthesis and salvage pathways.

4. Identification of the subverted host metabolic functions to ensure cyst formation and persistence
This objective has not started yet although the methodological approaches including MALDI imaging and whole brain imaging of T. gondii infected mice are currently under development. During this process we have validated the application of Serial sections Transmission Electron microscopy (ssTEM) and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and 3D reconstruction to T. gondii. Hamoundi et al, Mol. Micro. 2018, In press.
The aspartyl protease ASP5 is implicated in the maturation of proteins involved in the establishment of parasitism by Toxoplasma gondii.
The ASP5 substrates are either sent into the parasitophorous vacuole or crossing the parasitoporous vacuole membrane and exported into the host cells to subvert host cellular functions.
Progress 1: We have explored the technology based on terminal amine isotopic labeling of substrates (TAILS) N-terminomic analyses and successfully identified ASP5 substrates involved on host parasite interplay during encystation.
We are currently investigating their contribution in the process of chronic infection and persistence.

Virtually nothing is known about the metabolic capabilities that ensure the very slow growth rate of encysted parasites and about the extent of nutrients exchanges between the parasite and the host cells during persistence.
Progress 2: We have identified three metabolic pathways (pantothenate/CoA, Heme and Sphingolipids) that are distinctly modulated between the acute and chronic phase of infection, as a result of a differential accessibility to host cell nutrients.
Progress 3: We have constructed the first central carbon metabolic networks of the acute and chronic stage of T. gondii

The dynamics of tissue cysts development are poorly understood.
Progress 4: To investigate the replicative potential of bradyzoites and the cyst burden during the course of chronic infection we have applied new technologies to establish the cartography of the cysts numbers and size in the whole brain of infected mice using the Serial Two Photon Tomography imaging technology.
Comparison of Toxoplasma gondii tachyzoites in a parasitophous vacuole and bradyzoites in a cyst