Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalisation

Abstract

Toxoplasma gondii is a common parasite of humans and animals, which is transmitted via oocysts in cat faeces or tissue cysts in contaminated meat. The robust oocyst and sporocyst walls protect the infective sporozoites from deleterious external attacks including disinfectants. Upon oocyst acquisition, these walls lose their integrity to let the sporozoites excyst and invade host cells following a process that remains poorly understood. Given the resistance of the oocyst wall to digestive enzymes and the ability of oocysts to cause parenteral infections, the present study investigated the possible contribution of macrophages in supporting sporozoite excystation following oocyst internalisation. By using single cell micromanipulations, real-time and time-point imaging techniques, we demonstrated that RAW macrophages could interact rapidly with oocysts and engulfed them by remodelling of their actin cytoskeleton. Internalised oocysts were associated to macrophage acidic compartments and showed evidences of wall disruption. Sporozoites were observed in macrophages containing oocyst remnants or in new macrophages, giving rise to dividing tachyzoites. All together, these results highlight an unexpected role of phagocytic cells in processing T. gondii oocysts, in line with non-classical routes of infection, and open new perspectives to identify chemical factors that lead to oocyst wall disruption under physiological conditions.

Dynamics of Toxoplasma gondii Oocyst Phagocytosis by Macrophages

“…We used the micropipette aspiration technique described by Freppel et al (2016). Briefly, a glass capillary (internal diameter 0.58 mm/outer diameter 1 mm) was heated using a micropipette puller (David Kopf Instruments, Model 700C) until melting, and separated in half to obtain two closed microneedles.…”

“…Macrophages were plated in triplicate at the density of 1.5.10 5 cells in 1 ml culture on 12 mm-glass coverslips (0.13-0.17 mm thick) precoated with 0.01% poly-L-lysine solution placed at the bottom of 24-well sterile plates as described previously (Freppel et al, 2016). After incubation for 24 h at 37 • C, cells were pre-treated for 30 min with 1 µM of the actin polymerization inhibitor latrunculin A (LatA, Sigma-Aldrich, Saint-Quentin-Fallavier, France) or 50 µM of the myosin-II inhibitor blebbistatin (Blebb, Sigma-Aldrich).…”

“…To investigate whether removal of the oocyst and sporocyst walls modifies the development of tachyzoites from sporozoites over time, macrophages were challenged either with oocysts, free sporocysts or sporozoites. Sporocysts and sporozoites were obtained following oocyst sonication and further incubation in an excystation medium as detailed by Freppel et al (2016). Macrophages (1.5.10 5 cells) were plated on coverslips as described above and challenged with oocysts (ratio 1:1), freshly released sporocysts or freshly excysted sporozoites in cell culture medium.…”

“…Interestingly, oocysts can cause parenteral infections, at least in laboratory mice, suggesting a possible excystation of sporozoites in absence of digestive factors (Dubey and Frenkel, 1973). From these observations, we developed in vitro oocyst-macrophage co-cultures to investigate whether phagocytic cells could mediate sporozoite excystation following oocyst phagocytosis (Freppel et al, 2016). Previous in vitro experiments showed that naïve RAW 264.7 macrophagic cells could ingest oocysts, open their walls in or near acidic compartments, and host the differentiation of the sporozoites into replicative tachyzoites.…”

rTgOWP1-f, a specific biomarker for Toxoplasma gondii oocysts

“…to evaluate the ability of antibodies anti-rHTgOWP1-f to detect oocysts previously treated with NaOCl, as prior described 18 . The immunofluorescence assays were done with oocysts in suspension and visualized without any fixation process.…”