Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

Sébastien Besteiro,Maryse Lebrun,Joël Poncet,Adeline Michelin,Jean-François Dubremetz,Vern B Carruthers

doi:10.1371/journal.ppat.1000309

Sébastien Besteiro, Maryse Lebrun + Show 4 more

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https://doi.org/10.1371/journal.ppat.1000309

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Abstract

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

Highlights

Invasion by Apicomplexa is an essential step of the pathologies associated with these protozoan parasites that include Plasmodium spp., the causative agents of malaria, as well as Toxoplasma gondii, responsible for human and animal toxoplasmosis
A unique feature of apicomplexan parasites is the formation of an intimate contact between the apex of the parasite and the host cell membrane called the moving junction that moves along the parasite during invasion
Our study demonstrates that all the RON proteins are translocated into the host cell, where RON4/5/8 remain associated with the cytoplasmic face of the host cell plasma membrane

Summary

Introduction

Invasion by Apicomplexa is an essential step of the pathologies associated with these protozoan parasites that include Plasmodium spp., the causative agents of malaria, as well as Toxoplasma gondii, responsible for human and animal toxoplasmosis. Proteins located on the surface of the parasite probably mediate the initial interaction with the target cell. This is followed by an intimate contact between the apical tip of the parasite and the host cell membrane, called the moving junction (MJ) [2]. This singular structure, likely linked to the subpellicular cytoskeleton motor of the parasite, might serve as a support to propel the parasite into the parasitophorous vacuole (PV) that forms inside the host cell. The major reason for this was its transient nature, since host cell invasion is a very rapid process (a few seconds), and isolating the structure was not possible

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