LS-7-P-5904 Induction of calcium-dependent events relating to cell invasion in Toxoplasma gondii tachyzoites.

INTRODUCTION

Toxoplasma gondii is an intracellular protozoan parasite which affects humans causing encephalitis, chorioretinitis and death. The tachyzoite has structures that allow it to perform cell invasion. During the invasion, tachyzoite adheres to the target cell membrane through proteins from micronemes and then projects a dynamic structure called conoid [1,2]. Subsequently the parasite enters the cell and is housed in a parasitophorous vacuole proliferating by endodyogeny. After several cycles of replication, the parasites leave the infected cell by mechanisms such as conoid extrusion and secretion, which enables the parasite to invade neighboring cells. This replication cycle results in cell destruction and is responsible for the major clinical manifestations of toxoplasmosis. Ethanol is a well-characterized inductor calcium-dependent events in different cell models. In this work we used ethanol and we analyze the morphological changes that occur in isolated T. gondii tachyzoites.

METHODS

Cell culture. The cell model used for our invasion, proliferation and egress assays was HEP-2 cells (human epithelial laryngeal carcinoma cells) (Hep-2,ATCCCCL-23).

Parasites and conoid extrusion. The RH strain of T. gondii was maintained in BALB/c mice, purified and incubated with 0.5M ethanol, fixed, and processed for electron microscopy and immunofluorescence.

Eletronic microscopy. The samples were fixed, dehydrated and infiltrated in resin or processed for scanning electron microscopy. Thin sections obtained were contrasted with uranyl acetate and examined in a transmission electron microscope (Jeol 2000 EX).
Immunodetection. MIC2 protein detection was performed without permeating in extracellular tachyzoites. The samples were fixed and incubated with specific primary antibodies and analyzed using confocal microscopy.

RESULTS

The exposure of tachyzoites to ethanol induces reversible conoid extrusion, clearly observed the projection of the apical part of tachyzoites (Fig. 1). We also observed the presence of vesicle components accumulating in the back of tachyzoites, associated with some changes in the plasma membrane. We could detected that these vesicular components presenting on its surface was always associated with the extruded conoid(Fig. 1). For immunofluorescence, we showed that the secreted products during extrusion conoid micronemes come from. These results suggest a similar mechanism of secretion and induction for extrusion.

REFERENCES

[1] Mondragón y Frixione . J. Euk. Microbiol. 43 (1996) 120-127.

[2] González-Del Carmen, et al. Cell Microbiol 11(2009):967-82