**8. Concluding remarks**

122 Malaria Parasites

(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

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* 

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

*et al.*, 2007).

*vivo* efficiently.

clefts.

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 processes involved and their importance for the parasite survival.

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 rupture of the red cell plasma membrane.

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The merozoite release, following the engulfment of the infected erythrocyte, relies on an unique site of opening allowing the egress of the first one or two merozoites; the release of the remaining merozoites results from the curling and eversion of the red blood cell membrane. Importantly, the same sequence of events has been observed whether the infected erythrocytes were in suspension or adhering to the substrate (which is the usual status of *P. falciparum* infected erythrocytes because of cytoadherence to the micro-vessel endothelium and to non-infected erythrocytes). The physical parameters of curling and eversion of the red cell membrane emphasized once more the importance of parasiteinduced changes to the host cell membrane.

Red blood cell remodelling by the malaria parasite necessitates both efficient export of parasite proteins to the host cell and extensive membrane synthesis. These processes, together with the parasite enzymatic activities, such as proteases, protein kinases and phosphatases, which are crucial for the intra-cellular survival of the parasite and evasion from splenic clearance and host immune response, deserve precise characterization because they are Achilles heels that could be targeted by specific drugs or antibodies.

#### **9. Acknowledgements**

We are very grateful to Maryse Lebrun for critical reading of the manuscript and apologize to researchers whose work has not been directly cited in this review because of limiting space. This work was supported by the University Montpellier 2 and the CNRS. XY Yam was supported by the MalParTraining FP6 Marie Curie Action under contract No. MEST-CT-2005-020492. A Mbengue is supported by a PhD fellowship from the French Ministère de l'Education Nationale, de la Recherche et de la Technologie.

#### **10. References**


The merozoite release, following the engulfment of the infected erythrocyte, relies on an unique site of opening allowing the egress of the first one or two merozoites; the release of the remaining merozoites results from the curling and eversion of the red blood cell membrane. Importantly, the same sequence of events has been observed whether the infected erythrocytes were in suspension or adhering to the substrate (which is the usual status of *P. falciparum* infected erythrocytes because of cytoadherence to the micro-vessel endothelium and to non-infected erythrocytes). The physical parameters of curling and eversion of the red cell membrane emphasized once more the importance of parasite-

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**1. Introduction** 

policies in 21th century.

continents.

**2. Studies on avian malaria parasites** 

**8** 

*Spain* 

Alfonso Marzal

*University of Extremadura* 

**Recent Advances in Studies** 

In 1902, Sir Ronald Ross was awarded the Nobel Prize in Medicine for his discovery of the mosquito transmission of malaria. This finding was achieved working with avian malaria and its vector, giving him a control over his experimental subjects difficult to attain with human models. Since then, malaria parasites of birds have played an essential role as a model in human malaria studies. Important advances in medical parasitology such of the study of the life cycle, development of chemotherapy, and cultivation *in vitro* have initially been developed using bird haemosporidian models. Significant anti-malarial compounds such as plasmochin, primaquine and atebrin were evaluated in bird model. In the same way bird parasites were used for drug testing and for further malaria-associated experiments. Nowadays, research on bird malaria is at the very peak since scientists have realized the benefits of using studies on avian malaria to answer ecological, behavioural and evolutionary questions. This review will highlight the importance of studies on avian malaria, showing the results of some recent investigations on this topic and describing new applications of avian malaria researches that could be useful for conservation and health

Haemosporidians (Sporozoa: Haemosporida) are one of the most well known groups of parasitic protists. They include the agents of malaria, one of the most lethal human diseases. But the systematic and ecological diversity of malaria parasites is much larger. Systematic parasitologists have erected more than 500 described species belonging to 15 genera within the order Haemosporidia (Phylum Apicomplexa) that infect squamate reptiles, turtles, birds, and mammals, and use at least seven families of dipteran vectors (Levine, 1988; Martinsen et al., 2008). These parasites are distributed in every terrestrial habitat on all the warm

Bird haemosporidians are the largest group of haemosporidians by number of species. Avian malaria and related haemosporidians are widespread, abundant and diverse and are easily sampled without disrupting the host populations. In addition, experimental studies on bird malaria usually present bigger samples sizes than primate or human studies, achieving a high degree of precision and confidence in the outcome of the study. More than

**on Avian Malaria Parasites** 

