Eukaryotic Intracellular Parasites: Immunity and Chemoresistance
Coordinator : N. Blanchard
T cell immunity during Toxoplasma gondii and Plasmodium infections
Malaria pathophysiology and chemoresistance
Our research addresses how immunity develops and is modulated by two apicomplexan parasites, Toxoplasma gondii and Plasmodium, which respectively cause toxoplasmosis and malaria. We also work on the epidemiology and mechanisms of drug resistance of malaria parasites.
Malaria affects 200 million people worldwide and kills almost half a million people every year. The biggest toll is on children of sub-Saharan Africa who suffer from deadly complications such as cerebral malaria.
Toxoplasmosis poses serious risk for fetuses if contracted during pregnancy. T. gondii infection may lead to chorioretinitis or encephalitis in immunocompromised individuals. Even in immunocompetent people, latent toxoplasmosis may have underestimated consequences on behavior and neuropsychiatric disorders.
These two apicomplexan parasites are unicellular eukaryotic microorganisms residing in a vacuole inside infected cells. The relatively simple asexual cycle and genetic amenability of T. gondii has made it a valuable model parasite for the community interested in apicomplexan parasitic diseases.
AXIS 1: Study antigen presentation and CD8 T cell function during toxoplasmosis
CD8 T cells are key players in protective immunity against T. gondii. Because T. gondii dwells within a vacuole segregated from the host cytosol, parasite-derived antigens need to reach beyond the vacuole membrane in order to access the MHC I pathway. This poses major cell biology challenges which are still poorly understood. Moreover the modalities of CD8 T cell recognition of the parasite in the brain, where the parasite resides during chronic stage, remain completely mysterious.
The life cycle of T. gondii Mouse hippocampal neurons (MAP2 staining in white) infected with cysts (yellow structures) of T. gondii (red fluorescence).
Using genetically modified T. gondii and antigen-specific tools (e.g. T cell hybridomas), we have identified critical parameters that control processing of T. gondii antigens in infected cells. Building up on our discovery of a strongly immunogenic protein of T. gondii (GRA6), we demonstrated that the location of the epitope at the C-terminus of the source antigen critically determines processing, immunodominance and protection by GRA6-specific CD8 T cells (Feliu et al, PLoS Path 2013). We found that although ER recruitment via Sec22b is involved in MHC I presentation of luminal antigens (Cebrian et al, Cell 2011), it is not required for GRA6 presentation. For GRA6, topology of membrane insertion determines processing efficiency (Buaillon*, Guerrero* et al, Eur J Immunol 2017).
The host-T. gondii interface comprises the vacuole membrane and an IntraVacuolar Network of tubular membranes (IVN) of mysterious function (Santi-Rocca & Blanchard, Curr Op Microbiology 2017). Using parasite mutants with perturbed GRA6 transport or disrupted IVN, we showed that membrane binding regulates access of T. gondii membrane-bound Ag to the MHC I pathway and that association to the IVN limits presentation and curtails GRA6-specific CD8 responses. Our data indicate that IVN membrane deformations play a role in immune modulation (Lopez et al, Cell Reports 2015).
Recently, we have investigated the mechanisms underlying parasite detection by CD8 T cells infiltrating the brain. We have shown that neurons are able to present parasite antigens to CD8 T lymphocytes and that this mechanism is essential for the control of the parasite in the brain (Salvioni et al, Cell Reports 2019)
Given that there is no pharmacological treatment available against the persistent intracerebral forms of Toxoplasma gondii (cysts), CD8 T cells represent interesting cellular targets to try and eliminate the parasite.
On this basis, our projects aim to:
1- Determine the immune evasion mechanisms of cysts with respect to CD8 T cell recognition
2- Analyze how CD8 T cell / neuronal interactions contribute to T. gondii induced behavioral changes (collaboration with E. Suberbielle, Dunia team)
3- Decipher how intra-cerebral memory CD8 T cells are generated in the context of latent toxoplasmosis (collaboration with F. Masson, Liblau / Saoudi team)
‘Mosaic’ of vacuoles containing the Toxoplasma gondii parasite in human fibroblasts
Mouse macrophage invaded by 2 T. gondii parasites
PI: N. Blanchard
AXIS 2: Decipher how the interplay between dendritic cells and CD4 T cells control protective immunity during blood stage malaria
Cerebral Malaria is a severe complication of malaria, characterized by vascular leakage of brain capillaries, microhemorrhages, edema and coma/death if not treated. Cerebral Malaria is associated with sequestration of parasitized Red Blood Cells (pRBC) and leukocytes in microcapillaries.
In experimental rodent models, CD8 T cells are the major effectors of endothelial damage but CD4 Th1 cells also underlie disease development. Yet the parasite antigens presented by MHC class II on dendritic cells (DC) and the roles played by conventional DC in Th polarization are poorly defined. This is particularly relevant since CD4 T cells differentiate into Th subsets, including the B cell-helping T Follicular Helper (TFH), which ultimately determine the balance between antibody-mediated parasite control and immunopathology.
Using a mouse model of Cerebral Malaria induced by Plasmodium berghei ANKA (Pb ANKA), we have employed proteomics to identify the first MHC II immunopeptidome in malaria (Draheim et al., EMBO Mol Med 2017). We validated the peptides in vivo in infected mice, as well as in long-term protected mice vaccinated with an attenuated parasite. We found that XCR1+ CD8a+ cDC1 are superior to other cells for MHC II presentation of Plasmodium-derived antigens and we showed that cDC1 promote the differentiation of Th1 cells, which play a pathogenic role in cerebral malaria.
We are now setting out to map the immunopeptidome of P. falciparum in humans.
In a separate project, we are keen to understand why different lines of rodent-adapted Plasmodium elicit different disease outcomes, a total mystery up to now.
Mouse red blood cells parasitized with Plasmodium berghei parasites.
PI: N. Blanchard
AXIS 3: Monitor the spreading of drug-resistant malaria parasites and analyze the molecular mechanisms of resistance
Regular monitoring of the levels of anti-malarial resistance of P. falciparum is an essential policy to adapt therapy and improve malaria control. In collaboration with Institute for Research and Development, we have assessed the impact of anti-malarial treatments on the emergence and spread of resistant parasites in Cameroon (Ménard et al, Malar J 2012).
Artemisinin-derivatives (ART) are the last still effective anti-malarial class but emergence of resistant P. falciparum raises dramatic public health concerns. In collaboration with F. Benoit-Vical (LCC CNRS, Toulouse), we have demonstrated that F32-ART parasites, a highly ART-resistant, in vitro-selected strain, survive toxic effects of ART through temporary growth arrest, as has been recently reported in malaria isolates from patients in Asia (Witkowski et al, AAC 2010 and AAC 2013). By sequencing the whole genome of F32-ART and its parental line in collaboration with the Pasteur Institutes of Paris and Cambodia, we found mutations which were also present in clinical ART-resistant malaria isolates from South-East Asia. These findings allowed the identification of the first gene strongly associated with ART-resistance (Ariey et al, Nature 2014).
Based on this work, we are addressing 2 specific aims:
1- Monitor the emergence and spread of P. falciparum chemoresistance in Africa and South-East Asia
2- Characterize the mechanisms underlying quiescence of ART-resistant P. falciparum and infer potential strategies to revert this quiescent state
A quiescent Plasmodium falciparum parasite in a human erythrocyte following artemisinin treatment
OCEAC, Organisation de Coordination et de Coopération pour la lutte contre les grandes Endémies en Afrique Centrale (Yaoundé, Cameroun).
Screening children for malaria infection in the Mfou district (Cameroun)
PI: A. Berry
Robust Control of a Brain-Persisting Parasite through MHC I Presentation by Infected Neurons Journal Article
Cell Rep, 27 (11), pp. 3254-3268 e8, 2019, (Jun 11;27(11):3254-3268.e8. doi: 10.1016/j.celrep.2019.05.051.).
Performance evaluation of different strategies based on microscopy techniques, rapid diagnostic test and molecular loop-mediated isothermal amplification assay for the diagnosis of imported malaria Journal Article
Clin Microbiol Infect, 2019, (May 31. pii: S1198-743X(19)30225-3. doi: 10.1016/j.cmi.2019.05.010.).
Toxoplasma and Dendritic Cells: An Intimate Relationship That Deserves Further Scrutiny Journal Article
Trends Parasitol, 2019, (Sep 3. pii: S1471-4922(19)30210-7. doi: 10.1016/j.pt.2019.08.001.).
Real-time PCR for diagnosis of imported schistosomiasis Journal Article
PLoS Negl Trop Dis, 13 (9), pp. e0007711, 2019.
Multiple Phenotypic and Genotypic Artemisinin Sensitivity Evaluation of Malian Plasmodium falciparum Isolates Journal Article
Am J Trop Med Hyg, 2018, (Feb 12. doi: 10.4269/ajtmh.17-0798.).
Protease-Activated Receptor 2 contributes to Toxoplasma gondii-mediated gut inflammation Journal Article
Parasite Immunol, 2017, (Sep 7. doi: 10.1111/pim.12489.).
MHC I presentation of Toxoplasma gondii immunodominant antigen does not require Sec22b and is regulated by antigen orientation at the vacuole membrane Journal Article
Eur J Immunol, 47 (7), pp. 1160-1170, 2017, (Jul;47(7):1160-1170. doi: 10.1002/eji.201646859. Epub 2017 Jun 8.).
Profiling MHC II immunopeptidome of blood-stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells Journal Article
EMBO Mol Med, 2017, (Sep 21. pii: e201708123. doi: 10.15252/emmm.201708123.).
Young Sprague Dawley rats infected by Plasmodium berghei: A relevant experimental model to study cerebral malaria Journal Article
PLoS One, 12 (7), pp. e0181300, 2017, (Jul 24;12(7):e0181300. doi: 10.1371/journal.pone.0181300. eCollection 2017.).
Editorial overview: Host-microbe interactions: parasites Journal Article
Curr Opin Microbiol, 40 , pp. viii-xi, 2017, (Dec;40:viii-xi. doi: 10.1016/j.mib.2017.11.028.).
Membrane trafficking and remodeling at the host-parasite interface Journal Article
Curr Opin Microbiol, 40 , pp. 145-151, 2017, (Dec;40:145-151. doi: 10.1016/j.mib.2017.11.013. Epub 2017 Nov 22.).
Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells Journal Article
EMBO Rep, 2016, (Oct 10. pii: e201642358.).
Continuous Effector CD8(+) T Cell Production in a Controlled Persistent Infection Is Sustained by a Proliferative Intermediate Population Journal Article
Immunity, 45 (1), pp. 159-71, 2016, (Jul 19;45(1):159-71. doi: 10.1016/j.immuni.2016.06.013. Epub 2016 Jul 12.).
Erythroferrone contributes to hepcidin repression in a mouse model of malarial anemia Journal Article
Haematologica, 2016, (Sep 22. pii: haematol.2016.150227.).
A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms Journal Article
N Engl J Med, 374 (25), pp. 2453-64, 2016, (Jun 23;374(25):2453-64. doi: 10.1056/NEJMoa1513137.).
Insight into k13-propeller gene polymorphism and ex vivo DHA-response profiles from Cameroonian isolates Journal Article
Malar J, 15 (1), pp. 572, 2016, (Nov 26;15(1):572.).
Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute-phase Toxoplasma gondii burden Journal Article
Immunology, 2016, (Jul 5. doi: 10.1111/imm.12643.).
An epidemiologically successful Escherichia coli sequence type modulates Plasmodium falciparum infection in the mosquito midgut Journal Article
Infect Genet Evol, 43 , pp. 22-30, 2016, (Sep;43:22-30. doi: 10.1016/j.meegid.2016.05.002. Epub 2016 May 3.).
Persistence of Toxoplasma gondii in the central nervous system: a fine tuned balance between the parasite, the brain and the immune system Journal Article
Parasite Immunol, 2015, (Jan 9. doi: 10.1111/pim.12173.).
Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in pregnant women in Yaounde, Cameroon: emergence of highly resistant pfdhfr/pfdhps alleles Journal Article
J Antimicrob Chemother, 70 (9), pp. 2566-71, 2015, (Sep;70(9):2566-71. doi: 10.1093/jac/dkv160. Epub 2015 Jun 16.).
Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence Journal Article
Nat Commun, 6 , pp. 6174, 2015, (Jan 28;6:6174. doi: 10.1038/ncomms7174.).
Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen Journal Article
Cell Rep, 2015, (Nov 23. pii: S2211-1247(15)01288-7. doi: 10.1016/j.celrep.2015.11.001.).
Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure Journal Article
Emerg Infect Dis, 21 (10), pp. 1733-41, 2015, (Oct;21(10):1733-41. doi: 10.3201/eid2110.150682.).
A molecular marker of artemisinin-resistant Plasmodium falciparum malaria Journal Article
Nature, 2013, (Dec 18. doi: 10.1038/nature12876.).
Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite Journal Article
PLoS pathogens, 9 (6), pp. e1003449, 2013, (Jun;9(6):e1003449. doi: 10.1371/journal.ppat.1003449. Epub 2013 Jun 20.).
Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia Journal Article
Antimicrob Agents Chemother, 57 (2), pp. 914-23, 2013, (Feb;57(2):914-23. doi: 10.1128/AAC.01868-12. Epub 2012 Dec 3.).
Molecular monitoring of plasmodium falciparum drug susceptibility at the time of the introduction of artemisinin-based combination therapy in Yaounde, Cameroon: implications for the future Journal Article
Malaria journal, 11 , pp. 113, 2012, (Apr 12;11:113.).
Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells Journal Article
Cell, 147 (6), pp. 1355-68, 2011, (Dec 9;147(6):1355-68.).
Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum Journal Article
Nat Immunol, 9 (8), pp. 937-44, 2008, (NLM In-Process Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't DEP - 20080629).
Impact on the society
Our field work in Africa will provide valuable information on the spread of Plasmodium isolates that are resistant to artemisinin derivatives as well as to other drugs effective for treating malaria.
These data should allow us to swiftly develop new control strategies in order to limit the burden of these resistant clones. A better understanding of the biology of resistant parasites is also essential to devise new pharmacological strategies.
Our fundamental work on immunity to Toxoplasma gondii and Plasmodium has the potential to uncover novel pathways and/or molecules by which intracellular parasites manipulate presentation of their own antigens, modulate immune responses and, more generally, subvert their host.
Our studies could help identify or down-select new vaccine targets. They may also suggest approaches to use CD8 T cells in order to eradicate T. gondii cysts, against which there is currently no effective pharmacological treatment.
Alliance-Laure Otam (M2 student, 2019)
Emilie Guemas (M2 student, 2019)
Anna Salvioni (PhD student 2016-2019)
Jérémy Aboagye (M2 student, 2019)
Erine Prévost (M2 student, 2019)
Amel Aida (M1 student, 2019)
Sébastien Margueres (L3 student, 2019)
Naïs Rolland Clavel (L3 student, 2019)
Thomas Peyret (L3 student, 2019)
Nian-Zhang Zhang (Visiting Scholar from Lanzhou Veterinary Research Institute, China, 2017-2019)
Thomas Galaup (M1 student, 2018)
Alexis Audibert (L3 student, 2018)
Vincent Cantaloube-Ferrieu (M1 student, 2018)
Julien Santi-Rocca (post-doc 2015-2017)
Myriam Wlodarczyk (post-doc 2012-2017)
Marion Draheim (PhD Student, 2014-2016)
Camille Motbal (M1 student, 2016)
Guillaume Jacob (BTS student, 2016)
Virginie Vasseur (Engineer, 2010-2016)
Marine Le Bouar (M2R student, 2015-2016)
Francesca de Giorgi (Unipharma Graduate Program / Erasmus student, 2016)
Célia Rollin (M2R student 2015-2016)
Célia Buaillon (PhD student 2012-2016)
Jodie Lopez (Graduate student 2012-2015)
Nestor Guerrero (post-doc, 2013-2015)
Giulia Fornabaio (European Unipharma Graduate Program student, 2015)
Noémie Gaudré (Medical student, Master 2R student, 2013-2014)
Valeria Bellini (European Unipharma Graduate Program student, 2012-2013)
Virginie Feliu (Engineer)
Mathilde François (Medical student, Master Student, 2012-2013)
Rose-Anne Lavergne (Intern, Internal Medicine, 2011-2013)
Albano Lima-Perez (Master 2R student, 2012-2013)
Sophie Blanié (post-doc, 2010-2012)
Benjamin Berteloite (Master 1 student, 2011)
Coraline Chéneau (Master 1 student, 2010)
E. Suberbielle, Dunia lab, CPTP
F. Masson, Liblau/Saoudi team, CPTP
A. Saoudi, CPTP
F. Benoit-Vical, LCC CNRS UPR 8241, Toulouse
S. Marion, CIIL, Pasteur Lille
I. Morlais & J.-J. Lupez Rubio, MIVEGEC IRD UMR 5290, Montpellier
J.-M. Saliou, P3M proteomics platform, CIIL, Institut Pasteur, Lille
O. Silvie, CIMI-UPMC INSERM U1135, Paris
P. Awono-Ambene, Yaoundé, Cameroon
I. Cebrian & L. Mayorga, IHEM-CONICET, University of Cuyo, Mendoza, Argentina
D. Ménard, Pasteur Institute, Cambodia
E. Robey, University of California, Berkeley, USA