1. Identification of the components of the Cyst Wall (CW)
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 fully characterized a dozen of candidates ASP5 substrates and disrupted the corresponding genes to assess their function in bradyzoites differentiated in vitro as well as in vivo (mouse model) (Hammoudi et al, Mol Microbiol. 2022). In this work we established by two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.
We developed a way assess the function of genes in the badyzoite stage and demonstrated that GRA60 represents a novel parasite effector conferring resistance to IRGs in type I parasites (Nyonda Cell Micro 2021). A signaling linked factor was shown not only is essential during acute infection but also plays a pivotal role during natural oral infection with tissue cysts' dissemination and persistence (Ye S et al, mBio 2022)
2. Determination of the parasite factors responsible for CW formation and maturation
We have revealed 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).
3. Identification of the metabolic functions of the bradyzoites to ensure encystation persistence
We have generated a computational metabolic model uncovering T. gondii’s metabolic needs and capabilities (Koehn et al, Curr Opin Biotechnol. 2021). Next it was curated by interrogating experimentally the parasite’s needs and capabilities both in vitro, and in vivo and by applying targeted metabolomics (Krishnan et al, Cell Host Microbe, 2020). Importantly, a reconstruction with experimental validation was also conducted for the rodent malaria parasites (Stanway et al, Cell, 2019).
We uncovered versatility between biosynthesis and salvage of vitamins and cofactors (Krishnan et al, J Biol Chem 2020 and Kloehn et al, BMC Biol 2020).
We unraveled the balance between uptake and salvage of i) sphingolipid biosynthesis (Nyonda et al, Cell Rep 2022) ii) vitamin B5 (pantothenate, Pan) (Lunghi et al, Nat Comm, 2022) ;(de Vrie et al, PloS Pathogens 2021). We identified of essential transporters (Kloehn et al, Curr Opi Microbiol, 2021); (Kloehn et al, Metabolites 2021).
4. Identification of the subverted host metabolic functions to ensure cyst formation and persistence
This objective was technologically very challenging and methodological approaches including MALDI imaging . Instead Serial sections Transmission Electron microscopy (ssTEM) and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) were applied Hamoundi et al, Mol. Micro. 2018. The whole brain imaging of T. gondii infected mice was successful and was applised to an important study addressing the molecular basis of the manipulation of mouse predator fear by T. gondii that correlated with the level of neuroinflammation (Boillat et al, Cell Report, 2020).
