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The role of host cell actin cytoskeleton in invasion by Apicomplexa parasites

Final Report Summary - API-INVASION (The role of host cell actin cytoskeleton in invasion by Apicomplexa parasites)

The phylum of Apicomplexa is one of the largest group of unicellular parasites as it contains about 5 000 members, the vast majority are polarised, harbouring an apical pole filled by a complex (Apicomplex) of secretory vesicules. In humans, the most pathogenic Apicomplexa are Plasmodium sp, the etiological agent of malaria which kills about 500 millions of persons each year and Toxoplasma gondii which is carried by about 30% of the world's human population and renders this population at high risk of severe diseases in case of deregulation of the immune system. After ingestion of a few Toxoplasma parasites and local infection of the small intestine, the population expands rapidly causing the acute phase of the infection. This phase is usually asymptomatic or of limited severity in immuno-competent individuals, which eventually control most of the parasitic load. However, some parasites can escape from the immune defence system and transform into quiescent cryptic stages in deep tissues such as heart, liver, brain and retina where they can last for life-long. These dormant parasites do not cause significant signs of pathology but if infected people become transiently or permanently immuno-compromised as during cancer or post-transplantation treatments or during human immunodeficiency virus (HIV) infection, Toxoplasma wakes up, massively multiplies leading to pronounced tissue inflammation and to life-threatening diseases such as encephalitis, chorioretinis, pneumonia. In addition, if Toxoplasma is first contracted during pregnancy, it traverses the placenta and infects the foetus leading to abortion or foetal malformations depending on the timing of infection.

Most if not all Apicomplexa parasites strictly depend on host cell nutrients to ensure growth or persistence. Gaining access to the host cell implies to have the capacity to invade host cells. The overall mode of entry appears well conserved amongst most Apicomplexa parasites and was mainly studied in Plasmodium and Toxoplasma. Although the mechanisms of cell invasion by these two parasites have been explored since several decades, numbers of black boxes remain to date and there is no satisfying picture on the molecular mechanisms supporting invasion. The current view is that parasites use their internal cytoskeleton driven motile capacities to approach and then penetrate the host cells. The penetration starts with an intimate contact between the apical pole of the parasite and host cell membrane which shapes a tight junctional platform, seen as a circumferential ring connecting the two cells and serving as an obligate entry door for the parasites. They indeed propel themselves into the host cell through the junction being tightly surrounded by a membrane derived from the invagination of host cell surface membrane. When the junction closes, the nascent vacuole pinches off to enclose the parasite intracellularly in a subcellular compartment called parasitophorous vacuole that acquires unique features to promote growth and survival.

A main part of the research programme aimed at revisiting how the junction forms and functions during invasion and three different orientations have been followed. Data from Isabelle Tardieux's lab, see Gonzalez et al. Cell Host Microbe (CHM) 2009, highlighted that, unlike stipulated by a well accepted previous dogma, assembly of cell host filaments consisted of the cytoskeleton protein actin contributes to invasion by providing a stable anchor to the junction onto which the parasite can propel itself. We further pursued first this investigation and focussed on molecules from the parasite origin that were expected to structure a functional junction. A parasite protein complex is known to be secreted from the apical vesicules called rhoptries, i.e. roptry neck proteins (RONs) and to contain a member, RON2, inserted into the host cell surface membrane while the other members are injected into the host cell. Since 2005, a prevailing dogma states that an additional parasite protein secreted from a second set of apical organelles and named apical major antigen one (AMA1) is an essential partner of RON2 without which a junction does not fold into the typical ring and does not function properly to promote invasion. Combining dynamic and static confocal microscope imaging, we were able to demonstrate that while the parasite population lacking more than 99% of the protein AMA1 is indeed significantly impaired in its invasive capacity, the individuals that enter the host cells build a ring-shaped and functional junction similarly to the parasites expressing normal levels of AMA1. In contrast, the mutants are unable to flatten onto the surface of the cell and rather stay more perpendicularly positioned toward the cell, suggesting that the defect in invasion results from an attachment and positioning step prior and during invasion. This study was conducted in collaboration with R. Ménard's lab working on the Plasmodium model and led to a publication in CHM (GIovaninni et al., 2011). More recently, we confirmed these results in collaboration with M. Meissner who engineered Toxoplasma parasites fully devoid of AMA1 molecules and using similar approaches we characterised these parasites and concluded that AMA1 is not an essential protein to form a functional junction for entry into host cells. These new set of data have been compiled with Plasmodium work and a manuscript has been recently submitted. Furthermore, we have gathered evidence that AMA1 could still have an important function in vivo using mice models and Toxoplasma parasites and this work will soon be submitted for publication.

In the course of this programme, we also participated to a study carried out in I. Tardieux's concerning a parasite protein contained in the rhoptry vesicules, named toxofilin and secreted into the host cell early during invasion. Applying new cell biology and Imaging techniques to the field of parasitology, we showed that toxofilin is targeted to the host cell actin cytoskeleton network underneath the host cell membrane where it accelerates actin filaments disassembly thereby creating the appropriate space for vacuole folding at the entry site. These data have been recently published in Delorme-Walker et al. JCS 2012. Collectively, these data strengthen that successful invasion by Toxoplasma might rely on a transient coupling of two remodelling events of the host cell actin cytoskeleton. It is tempting to speculate that toxofilin would act by generating actin molecules released from the actin filaments of the underlying meshwork and these actin molecules could then fuel the actin filaments assembly that stabilises the junction, but these fast coupling events now need to be verified.

The last part of this study extends the investigation on the host cell cortical actin meshwork property during invasion. This underlying dense and heterogenous layer of actin filaments is connected to the surface membrane by panoply of linker proteins and the level of interaction between membrane and this actin meshwork partly defines the membrane tension characterising the cells. Using pharmacological and genetical approaches to modify host cell membrane tension combined with real time recording of invasion, we pointed to the influence of membrane tension throughout the invasion process and in particular at the end of it, when the parasitophorous vacuole separates from the host cell membrane. Our data dissected how membrane fission proceeds and highlighted a peculiar twist of the membrane accompanying vacuole pinch off. This twist largely depends on the thickening and contractibility of the host cell sub-cortical actin meshwork. These data are currently confirmed by a series of assays and will be part of a coming manuscript in 2013.

In conclusion, considerable advances have been made during this research programme on how the tight junction, which is a hallmark of host cell invasion by Apicomplexa parasites, forms and functions. These data have led to revisit strong dogma and thus to open new avenues for futures studies on invasion. It is clear that both parasite and host cell-derived molecules have to be concomitantly analysed if we want to understand the mechanistic of host cell entry. On the host cell side, emphasis on membrane dynamics should also be considered as a priority. It seems therefore important to pursue this type of studies, with a possibility to identify targets for anti-invasion drug development but also beyond parasitology for improving knowledge on cell membrane dynamics and rigidity. These properties are currently widely investigated by biologists and biophysicists since they control fundamental processes such as cell motility and appear deregulated in many pathological situations in particular during cancer and metastasis. Toxoplasma certainly deserves to be analysed as a parasite to unveil the pathophysiological aspect of the parasite-host interaction but it should also be seen as a 'tool' for cell biologists in search for unravelling fundamental cell properties.