Periodic Reporting for period 4 - ToxoPersist (Molecular Basis of Toxoplasma gondii Encystation and Persistence)
Okres sprawozdawczy: 2021-04-01 do 2022-09-30
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. 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. The specific aims are :
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 goal is to develop testable models as a basis for rational strategies aimed at breaching this barrier and overthrowing the current dogma that latent toxoplasmosis affecting 30% of the world’s human population is incurable.
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. The specific aims are :
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 goal is to develop testable models as a basis for rational strategies aimed at breaching this barrier and overthrowing the current dogma that latent toxoplasmosis affecting 30% of the world’s human population is incurable.
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).
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).
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 has identified ASP5 substrates and explored their contribution in the process of chronic infection and persistence.
Mmetabolic 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
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
Progress 4: We contributed to the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa.
The dynamics of tissue cysts development are poorly understood.
Progress 5: 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.
Progress 6: We could establish that the change in behaviour observed in chronic infected mice was correlating with neuroinflammation that persisted long after establishment of chronic infection.
Deviation to the project . TheASP3 closely related to ASP3 turned out to be implicated in invasion and egress (Dogga et et al, eLife 2017) and the orthologues in Plasmodium (Plasmepsins IX and X) are multistages antimalarial targets (Pino et al Science 2018, Mukherjee et al 2028 EMBO J ).
Progress 7 : These proteases entered in the drug discovery pipeline of two Pharma companies.
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 has identified ASP5 substrates and explored their contribution in the process of chronic infection and persistence.
Mmetabolic 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
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
Progress 4: We contributed to the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa.
The dynamics of tissue cysts development are poorly understood.
Progress 5: 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.
Progress 6: We could establish that the change in behaviour observed in chronic infected mice was correlating with neuroinflammation that persisted long after establishment of chronic infection.
Deviation to the project . TheASP3 closely related to ASP3 turned out to be implicated in invasion and egress (Dogga et et al, eLife 2017) and the orthologues in Plasmodium (Plasmepsins IX and X) are multistages antimalarial targets (Pino et al Science 2018, Mukherjee et al 2028 EMBO J ).
Progress 7 : These proteases entered in the drug discovery pipeline of two Pharma companies.