INTRODUCTION

Toxoplasma gondii is an intracellular protozoan parasite which affects animals and humans causing encephalitis, chorioretinitis and death. The tachyzoite has structures that allow it to perform cell invasion. During the invasion, the tachyzoite adheres to the target cell membrane through proteins MIC that come from micronemes and then projects a highly 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 asociation between conoid extrusion and MIC secretion.

 
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

ACKNOWLEDGEMENTS

This work was supported by the project CONACYT # 165282 

Figures:

Figure 1. Conoid extrusion induced by incubation with ethanol. Tachyzoites were incubated ethanol and analyzed by scanning electron microscopy. Conoid projected (arrows) and the presence of secreted elements accumulate in the posterior end (arrow head) is observed. Scale = 2 μm

Figure 2. Temporal relationship between conoid extrusion and microneme secretion induced by ethanol. A. Time lapse of tachyzoites activated for conoid extrusion by exposure to ethanol showing the release of components from the posterior end. B. SEM micrograph of a tachyzoite exposed for 30 s to ethanol. Arrows indicate conoid position; arrowheads indicate secreted components adhered to the pellicle and posterior end. C. Immunodetection of MIC2 in tachyzoites activated for conoid extrusion after exposure to ethanol. Upper set of micrographs corresponds to phase contrast images of the respective fluorescent images shown in the lower set of micrographs.

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/induction-of-conoid-extrusion-is-asociated-to-mic-secretion-in-toxoplasma-gondii-tachyzoites/