The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force

Abstract

BackgroundThe public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their related causative agents Toxoplasma and Plasmodium, respectively, to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their host cells has been highlighted as a priority research with the perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanism of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell-cell junction but this model lacks direct evidence and has been challenged by recent genetic studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyze characteristic features of the kinematics of penetration of more than 1,000 invasion events.ResultsThe majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into an invaginating host cell plasma membrane. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and, therefore, are unable to locally remodel the cortical actin cytoskeleton, the junction travels retrogradely with the host cell membrane along the parasite surface allowing the formation of a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However, about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RhOptry Neck (RON2) molecules are posteriorally capped before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics junction capping and entry failure are drastically reduced.ConclusionsThis kinematic analysis newly highlights that to invade cells parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0108-y) contains supplementary material, which is available to authorized users.

Genetic impairment of parasite myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces

“…In HeLa cells expressing the CAAX-mCherry (CAAX-mC) PM reporter, lox - MyoA (= MyoA + ) or Δ MyoB/C (= MyoA + ) tachyzoites entered the nascent PVs through static or capped TJs (Fig. 2a, blue trajectories; additional movie file shows these trajectories in real time (see Additional file 5)) as described for motor-competent tachyzoites [17]. In contrast, the apex of Δ MyoA tachyzoites appeared tightly enwrapped within a tight collar or a glove of host cell PM protrusions we will refer to as “PM ruffles” (Fig.…”

“…Human foreskin fibroblasts (HFFs), human epithelial cervical cancer cells (HeLa), human ARPE-19 retinal epithelial cells, and human U2OS osteosarcoma epithelial cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with GlutaMAX, 10 % heat-inactivated FCS, penicillin (100 U/mL), streptomycin (100 mg/mL), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). When specific fluorescent HeLa cell lines were used, they were grown in the presence of the appropriate antibiotics (puromycin or G418) used for selection: these lines stably express either the lipid- (non raft) CAAX or the lipid- (raft) MyrPalm PM targeting domain in fusion with mCherry and GFP, respectively [17]. Rat kangaroo kidney epithelial cells (PtK1) were cultured in Ham’s F12 medium (Sigma-Aldrich, Lyon, France) containing 25 mM HEPES, 10 % fetal bovine serum (FBS), and antibiotics.…”

“…The zoite then tracts itself into a PM invagination that arises below the TJ [14] and then evolves as a non-fusogenic parasitophorous vacuole (PV) that will support zoite growth and multiplication [15]. Our recent kinematic analysis has allowed tracking of the RON complex during its secretion and assembly into the PM and its establishment of a traction bridge with the host PM and its associated cortical actin lattice [16, 17]. In this scheme, the invasive force is thought to be provided by the single-headed unconventional myosin A (MyoA) of the apicomplexan-specific myosin class XIV [8, 18, 19].…”

Gliding motility powers invasion and egress in Apicomplexa

“…The content of the rhoptry neck contributes to the formation of the moving junction, which is visible as a ring-like structure by immunofluorescence (FIG. 5b) and forms a solid platform 112 with the host cell plasma membrane, through which the motile zoite glides (FIG. 5c,d).…”

“…The neck of the rhoptries is inserted into the apical polar rings (see the figure, part a), and the content of the rhoptry neck and bulb is successively secreted at the apical tip of the parasites. Invasion is correlated with the presence of rhoptry organelles, as several rhoptry neck proteins are involved in the moving junction, which is built by the parasite to support the traction forces generated during its penetration into the host cell 112,123 . Ookinetes are able to migrate through the epithelium of the mosquito midgut but do not invade.…”