**7. Merozoite egress from the red cell: A split second event likely depending on very early changes to the red blood cell membrane**

The release of infectious merozoites from the host cell requires the opening of the parasitophorous and red cell membranes. Dvorak and collaborators have first observed that the swelling of the infected erythrocyte precedes the egress of *Plasmodium falciparum* merozoites by a few minutes (Dvorak *et al.*, 1975). In addition, the use of amphiphiles, osmotic stress and protease inhibitors strongly suggested that merozoite release is pressure driven (Glushakova *et al.*, 2009; Glushakova *et al.*, 2005). Shortly before merozoite egress, the intracellular parasites seem to move more freely while the red cell membrane is still intact

Human Erythrocyte Remodelling by *Plasmodium falciparum* 123

As described before, parasite-induced changes at the red blood cell membrane affecting its stability occur as early as parasite entry and very early intra-erythrocytic growth. Moreover, phosphorylation of host peripheral proteins increases upon parasite growth and might modulate the bio-physical properties of the red cell membrane throughout the parasite development ( Pantaleo *et al.,* 2010). One might thus consider that on one hand the parasite weakens its host cell membrane and on the other hand it stabilizes it by exporting proteins to the red cell sub-membrane skeleton and recruiting host proteins to or from the sub-

However, the ability to curl and buckle has also been proposed to be an intrinsic property of the erythrocyte membrane when the cell is exposed to certain osmotic stress (Lew, 2011) although with marked kinetic differences as compared to the infected erythrocyte. Whether the parasite explores a property of its host cell and at what extent the changes of the red cell membrane and sub-membrane skeleton induced by the parasite are essential for efficient

As described in this chapter, Apicomplexan parasites widely transform the parasitophorous vacuole in which they grow and multiply and which constitutes the interface between the parasite and its extracellular environment. Changes of its closed environment, the red blood cell cytoplasm and plasma membrane, induced by the life-threatening human malaria parasite *Plasmodium falciparum* have been extensively studied because these changes are crucial for the parasite development and some, referred to as knobs, are specific for this species and central to the pathogenesis of severe malaria. In the last decade, the set up of *P. falciparum* genetic engineering and the spectacular advances of imaging technologies, have considerably highlighted our knowledge of the red cell remodelling by the parasite, the

Upon intra-erythrocytic parasite growth, new permeation pathways in the red cell membrane and extensions of the parasitophorous vacuole membrane in the host cell cytosol, named the tubovesicular network, participate in the import of nutrients from the extracellular milieu. Other membrane structures transposed by the parasite in the cytoplasm of its host cell, referred to as Maurer's clefts, and proposed to generate from the parasitophorous vacuole membrane, are central to the transport of parasite proteins to the red blood cell. They tightly interact with the host cell membrane even upon merozoite release. This interaction together with exported parasite proteins interacting with the host cell sub-membrane skeleton might prevent the premature rupture of the red cell membrane and consequent release of immature merozoites. Maintaining the integrity of the red cell membrane upon its growth is likely crucial for the parasite because it has weakened its host cell membrane by altering the cohesion between the plasma membrane and sub-membrane skeleton *via* the phosphorylation and the recruitment of host cell membrane and skeletal proteins. On the other hand, one can consider that the parasite has prepared its host cell membrane not only for entry but also for egress because reversing the parasite-induced modifications, for example by the activation of phosphatases, would highly facilitate the

membrane skeleton.

**8. Concluding remarks** 

merozoite release need further investigations.

rupture of the red cell plasma membrane.

processes involved and their importance for the parasite survival.

(Abkarian *et al.*, 2011), comforting previous studies providing evidence that, when the merozoites are close to egress, the PVM enlarges and ruptures before the erythrocyte membrane (Wickham *et al.*, 2003). What drives a sudden increase in the osmotic pressure? A premature release of immature merozoites has been recently described which results from the inhibition of RNA degradation and is preceded by swelling of the infected erythrocyte (Balu *et al.*, 2011). In addition, parasite proteases specifically active just prior to merozoite release could also participate in the increased osmolarity (Koussis *et al.*, 2009). Noteworthy, proteases of both parasite and host origin have likely numerous roles in merozoite egress and might also participate in both the rupture of the PVM and the subsequent opening of the erythrocyte membrane (Arastu-Kapur *et al.*, 2008; Chandramohanadas *et al.*, 2009; Yeoh *et al.*, 2007).

Indeed, although first considered as an explosive event, merozoite egress from the red blood cell has been shown recently to occur through the opening and stabilization of an osmotic pore in the host cell membrane allowing the release of a limited number of merozoites (Abkarian *et al.*, 2011). The pore opening is followed by the curling and buckling of the erythrocyte membrane, and this results in the wide-angular dispersion of the remaining merozoites. These events happen when a critical radius of the osmotic pore is reached. Abkarian *et al* 2011 hypothesized that this instability is biologically relevant as it disperses the merozoites and contributes to separate them efficiently from the infected cell membrane. Indeed, abortive egress events have been observed with a stop of curling and no buckling, resulting in the merozoites remaining stuck together inside the open erythrocyte and thus unable to further invade new red blood cells (Abkarian *et al.*, 2011). Noteworthy, these data have been obtained with infected erythrocytes in suspension and it is important to determine whether merozoites release proceeds through similar steps *in vivo*, when red cells with mature parasites are sequestered in the microvasculature, adhering to endothelial cells. Observations of infected erythrocytes adhered to a glass substrate shed some light on this process: over 5 merozoites were sequentially released through a pore of similar radius (1 µm) and with higher velocity as compared to non adhering cells, before curling occurred. The membrane was then projected backwards, thereby releasing merozoites but without actually pushing them forward. In brief, while similar steps are involved, the resulting dispersion of the merozoites looks different. These results suggest that adhesion maintains a membrane tension high enough to produce the overpressure driving more merozoites out of the host cell. Considering that *P. falciparum* infected erythrocytes are also able to adhere to non-infected erythrocytes, the merozoites would be released appropriately to re-invade *in vivo* efficiently.

The curling and buckling of the infected erythrocyte membrane can originate from an additional elastic energy due to an asymmetry between the membrane leaflets (Abkarian *et al.*, 2011). A nice illustration of this effect is the curling of a gift ribbon after one slides it between the thumb and a scissor blade, thus creating an excess area of the outer leaflet (Klales *et al.*, 2007). In *P. falciparum*- infected erythrocytes, this asymmetry between the two membrane leaflets could originate from a lipid excess in the inner leaflet caused by a lipid release of parasite origin, a modification of the mechanical properties of the red cell membrane through changes of the cytoskeleton/membrane interactions [reviewed in (An & Mohandas, 2010)] and/or interactions of the erythrocyte membrane with the Maurer's clefts.

As described before, parasite-induced changes at the red blood cell membrane affecting its stability occur as early as parasite entry and very early intra-erythrocytic growth. Moreover, phosphorylation of host peripheral proteins increases upon parasite growth and might modulate the bio-physical properties of the red cell membrane throughout the parasite development ( Pantaleo *et al.,* 2010). One might thus consider that on one hand the parasite weakens its host cell membrane and on the other hand it stabilizes it by exporting proteins to the red cell sub-membrane skeleton and recruiting host proteins to or from the submembrane skeleton.

However, the ability to curl and buckle has also been proposed to be an intrinsic property of the erythrocyte membrane when the cell is exposed to certain osmotic stress (Lew, 2011) although with marked kinetic differences as compared to the infected erythrocyte. Whether the parasite explores a property of its host cell and at what extent the changes of the red cell membrane and sub-membrane skeleton induced by the parasite are essential for efficient merozoite release need further investigations.
