Coccidia

A. Dumètre, PH. Puech

Collaboration: J. Husson (LadHyX), S. La Carbona (Actalia St Lô), I. Villena (Reims), A. Silvestre (INRAE Tours), JM. Repérant (ANSES Ploufragan), A. Kosta (IMM, Marseille), M. Pawlowic (Dundee Univ)

Resistance to physical and chemical degradation is essential for the perpetuation of the life cycle of environmental microbial pathogens. In the coccidian parasites Cryptosporidium, Eimeria and Toxoplasma, this function is served by the oocyst form. The oocyst wall is the key structure that helps the parasites to survive different environmental conditions and disinfectants and allows successful infection of the host (Freppel et al., 2019). It acts as a primary barrier to physical and chemical external attacks as long as its complex polymeric organization is perfectly maintained. Then, following ingestion with water or food, oocysts travel rapidly in the host digestive tract down to the small intestine where the oocyst wall has to open to release the infective forms (sporozoites) it contains. Depending on the coccidian species, this process has been recognized to likely involve physical (mechanical) stimuli (of unknown nature) and/or the action of digestive factors from the host (enzymes and biliary salts) or the parasite (sporozoite proteases). However, in certain coccidian species such as Toxoplasma, parenteral inoculation of oocysts can lead to the same burden of infection as the digestive route, suggesting that the digestive microenvironment is dispensable for a successful infection (Freppel et al., 2016), and that host cells such as phagocytes may contribute to initiating infection.

In this context, addressing the structure and chemistry of the coccidian oocyst wall, in terms of mechanics and adhesion, is an essential prerequisite to better understand the parasite dynamics outside and then inside the host. Using atomic force microscopy, advanced micropipette aspiration techniques, electron microscopy, fluorescence imaging, and automated AI-based data analysis, we aim to quantify the structure, mechanics and adhesion of the oocyst wall exposed to chemical and physical factors the parasite can encounter outside and inside the host. As a complementary approach, we are interested in the contribution of phagocytic cells such as macrophages and neutrophils in processing the oocyst wall and hosting parasite development by studying, using micropipettes  and optical tweezer techniques, the dynamics of oocyst internalization by phagocytes and the ability of parasites to overcome degradation to replicate and/or use these cells as Trojan horses (Freppel et al., 2016; Ndao et al., 2020).

Funding: ANR BreakingTheWall (2022-2027, coord. A. Dumètre); ANR STRIP (2018-2022; coord. I. Villena, Reims)

Related publications: (LAI members’ names are underlined)

Dynamics of Toxoplasma gondii oocyst phagocytosis by macrophages. Omar Ndao, Pierre-Henri Puech, Camille Bérard, Laurent Limozin, Sameh Rabhi, Nadine Azas, Jitender P. Dubey, Aurélien Dumètre. Frontiers in Cellular and Infection Microbiology 2020 https://doi.org/10.3389/fcimb.2020.00207

Structure, composition, and roles of the Toxoplasma gondii oocyst and sporocyst walls. Wesley Freppel, David J.P. Ferguson, Karen Shapiro, Jitender P. Dubey, Pierre-Henri Puech, Aurélien Dumètre. The Cell Surface, 2019 https://doi.org/10.1016/j.tcsw.2018.100016

Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalization. Wesley Freppel, Pierre-Henri Puech, David J. P. Ferguson, Nadine Azas, Jitender P. Dubey, Aurélien Dumètre. Scientific Reports 2016 https://doi.org/10.1038/srep33654

Mechanics of the Toxoplasma gondii oocyst wall. Aurélien Dumètre, Jitender P. Dubey, David J. P. Ferguson, Pierre Bongrand, Nadine Azas, Pierre-Henri Puech. PNAS 2013  https://doi.org/10.1073/pnas.1308425110

Leishmania

M. Casanova

Collaboration: S. Pomel (BioCIS, Univ. Paris-Saclay), Y. Sterkers (MIVEGEC, Univ. Montpellier)

Leishmania are protozoan parasites responsible for leishmaniases, which are neglected tropical diseases from the World Health Organization priority research program. Present in 88 countries on 5 continents, these vector-born diseases are responsible for around 30,000 deaths a year, with an annual incidence of 700,000 to 1 million new cases.

Leishmaniases are transmitted by the bite of a female sandfly. More precisely, inside the insect digestive tract, Leishmania cells are in an elongated flagellated form. Once they have been injected into the mammalian host, Leishmania cells are in a round form with a very reduced flagellum called the amastigote form, inside phagocytic cells, notably macrophages.

For the moment, no human vaccine exists, as well as no prophylaxis, vector control is difficult, and there is no satisfactory treatment for these diseases, due to the cost, side-effects and mode of administration of current drugs, but also to the development of drug resistance.

In this context, I am developing two research areas, in collaboration with the ‘Centre National de Référence’ on leishmaniases in Montpellier.

Consequences of global warming on Leishmania cells

As neglected tropical diseases, leishmaniases are rife in poor tropical countries. However, these diseases are also a problem in other countries, like in Europe and North America. This is not only due to human and pet movements. Indeed, in westernized countries, leishmaniases are a major problem in immunocompromimised people, whereas the number of such people is increasing. Furthermore, the increaseing ofin the number of Leishmania strains resistant to the usual drugs is becoming a problem. Last, the temperature increase due to global warming is inducing a significant increase in the number of leishmaniases. More precisely, the temperature increase would induce an increase in the survival, and the mobility of sandflies, as well as an increase in their population density and the number of sandfly bites. However, these current studies focus on the consequences of global warming on sandflies, whereas quite no study concerns the pathogen itself. Yet, the promastigote forms are subjected to the same temperature variations as the insect. Thus, I am interested in the consequences of temperature increase on Leishmania parasites: notably, are the growth, the cell cycle, the motility, or the virulence of the parasite affected? I am studying these features on different Leishmania species and with different temperature conditions (increase during a few hours, or during several days). For this, I am using the facilities in the LAI: cell culture platform, cytometry, microscopy, biophysics tools to study cell motility, … This research area is based on a ‘one health’ approach.

Study of Leishmania apoptosis

Leishmania parasites have a very original organization in comparison to higher eukaryotes. For example, despite a classic pattern of apoptosis, key mammalian apoptotic proteins are not present in Leishmania, such as caspases, cell death receptors and anti-apoptotic molecules. Even if some features have been described on this very original apoptosis, a lot of things have to be done to better understand it. This would allow a better understanding of the organization of living beings and of the eukaryote evolution. This would also allow the identification of new therapeutic tools based on Leishmania apoptosis, thus new, specific and so non-cytotoxic treatments. For this, I am notably trying to identify proteins and molecules involved in Leishmania apoptosis, to describe the different apoptotic pathways in Leishmania, and to encapsulate key proteins or DNA to induce Leishmania apoptosis.

Related publications:

Basmaciyan L, Casanova M. Cell death in Leishmania. Parasite. 2019;26:71. doi: 10.1051/parasite/2019071. Epub 2019 Dec 11. PMID: 31825305; PMCID: PMC6905399.

Basmaciyan L, Azas N, Casanova M. Different apoptosis pathways in Leishmania parasites. Cell Death Discov. 2018 Aug 20;4:27. doi: 10.1038/s41420-018-0092-z. Erratum in: Cell Death Discov. 2019 Jul 10;5:116. doi: 10.1038/s41420-019-0186-2. PMID: 30155277; PMCID: PMC6102309.

Basmaciyan L, Berry L, Gros J, Azas N, Casanova M. Temporal analysis of the autophagic and apoptotic phenotypes in Leishmania parasites. Microb Cell. 2018 Aug 1;5(9):404-417. doi: 10.15698/mic2018.09.646. PMID: 30280103; PMCID: PMC6167523.

Casanova M, Gonzalez IJ, Sprissler C, Zalila H, Dacher M, Basmaciyan L, Späth GF, Azas N, Fasel N. Implication of different domains of the Leishmania major metacaspase in cell death and autophagy. Cell Death Dis. 2015 Oct 22;6(10):e1933. doi: 10.1038/cddis.2015.288. PMID: 26492367; PMCID: PMC4632311.