PH Puech

Collaboration A. Dumêtre (URMITE)

Resistance to physical and chemical degradation is essential for the perpetuation of the life cycle of environmental microbial pathogens. In the intracellular parasite Toxoplasma gondii, this function is served by the oocyst, the only stage of the parasite structurally equipped to survive in harsh environments. Over the last few years, oocyst-related infections in humans and other warm-blooded animals worldwide have been increasingly reported as more prevalent and severe than previously thought. Infections lead to possible deleterious ocular and neurological complications, and even death. The Toxoplasma oocyst wall is the key structure that helps the parasite to survive to different environmental conditions and disinfectants and allow successful infection of the host. The bilayered oocyst wall acts as a primary barrier to physical and chemical external attacks as long as its complex polymeric organization is perfectly maintained. Upon ingestion with water or food, the Toxoplasma oocyst travels rapidly in the host digestive tract down to the small intestine. These walls have then to open or be opened in the host digestive tract in order to release the infective sporozoites. These processes have been recognized to likely involve physical (mechanical) stimuli (of unknown nature) in conjunction with the action of digestive enzymes and biliary salts.

In this context, addressing the structure and chemistry of the Toxoplasma oocyst wall, in terms of mechanics and adhesion, is a crucial prerequisite to better understand the oocyst dynamics inside the host. Using AFM, EM and fluorescence microscopy, we have characterized the structure, mechanics and adhesion of oocysts and the effect of common disinfectants and thermal treatment on this compartiment (Dumêtre et al. PNAS 2013). We complemented this study by designing microfabricated 10 µm-sized PDMS wells to mechanically trap the oocysts in order to be able to press on them with higher forces, in order to document the breakout forces. Aside, we performed quantifications of sporocyst mechanics and found that its wall possesses a hardness comparable to the one of the oocyst wall, which is supported by our EM imaging of the structures that shows that they are quite similar in structure and thickness.

Interestingly, the T. gondii oocysts are also infective following parenteral inoculation (e.g. through dermal injection in mice) suggesting that a contact between the host digestive microenvironment and the oocyst is not the one and only route for opening its walls. Finally, how the oocyst and sporocyst walls are processed far from the intestine, and by which mechanism(s), is currently unknown. Our hypothesis is that wall opening could be linked to the processing of the oocyst by professional phagocytic cells such as macrophages. We started to investigate using micropipette manipulations, optical and fluorescence microscopy the dynamics of the interaction of macrophages and oocysts, and observed that, indeed, macrophages are able to recognize non opsonized oocysts and to ingest them, ultimately rupturing the oocyst wall layer (Freppel et al. Sci. Rep. 2016)

We are currently complementing our study by (a) studying the mechanics of sporocyst walls, treated or not with disifectants, (b) evaluating the effect of disinfectants and thermal treatments on the process of phagocytosis of oocysts by macrophages, (c) studying the effects of gastric-like conditions and (c) interfering with different elements of the cytoskeleton that are at play in the engulfment processes. We are extending this type of study to the interaction of other pathogens with macrophages, such as yeasts, fungi and other coccidian such as Cryptosporidium spp.

 

See also : https://toxophysics.wordpress.com/

 

Parasite-host cell interaction via production of parasite-derived extracellular Vesicles

CL Forestier

Extracellular vesicles (EVs) have been isolated from nearly all cell type ranging from prokaryote to unicellular eukaryote and multicellular eukaryotic systems. Secretion of such vesicular carriers by living cells has emerged as a specific and universal mechanism for intercellular communication across all domains of life. EVs allow the transfer of information between producing and target recipient cells via the packaging and subsequent delivery of their molecular cargo. The exosomes (30-100 nm) are specifically secreted by eukaryotic cells, they originate from the endocytic pathway and are released upon exocytosis of multivesicular bodies. The ectosomes (0.1- 1 mm) are ubiquitous vesicles formed through the direct budding of the plasma membrane and shed into the extracellular space.

Recent insights into the biology of parasitic eukaryotes-derived EVs show that these vesicular vehicle constitute potent element for host-pathogen communication and host manipulation, thus playing a critical role in parasite infectivity and pathogenicity (Szempruch, Nature reviews microbiology, 2016). Leishmania promastigotes parasites release EV first defined as exosomes in 2010 during parasite culture in vitro, within host cell following infection (Silverman JM et al 2010, Hassani 2014; Silverman and Reiner 2011a) and in the sand fly midgut (Diniz Atayde et al 2015). Our goal is to investigate the impact of Leishmania secreted vesicular devices on i) parasite host cell mechanics and functions and ii) the immunoregulation of parasite-specific T cells.