Invited ReviewCellular architecture of Plasmodium falciparum-infected erythrocytes
Graphical abstract
Introduction
Malaria remains one of the most devastating diseases of modern times, with one million deaths recorded annually (Snow et al., 2005). Of the human malaria species, Plasmodium falciparum is medically the most important and is responsible for most of the mortality and morbidity associated with this disease. The life cycle of P. falciparum is complex, involving invasive, trophic and replicative forms in two hosts – a human, and a female Anopheles mosquito.
Transmission between mosquitoes and humans involves a remarkable series of morphological transformations. The asexual stage begins with transmission of motile sporozoites into the host’s blood stream during mosquito feeding. The sporozoites enter hepatocytes where they multiply, then differentiate to generate many thousands of merozoites. Upon release the merozoites invade the host’s red blood cells (RBCs) to initiate the blood stage of the infection. The intraerythrocytic parasite develops through the ring, trophozoite and schizont stages, eventually bursting to release 16 daughter merozoites (Bannister et al., 2000a, Garcia et al., 2008). Each intraerythrocytic cycle takes approximately 2 days and rupture of infected RBCs induces periodic waves of fever in the patient (Miller et al., 2002). Symptoms range in severity from headaches, hypoglycaemia, anaemia and fevers to renal failure and cerebral malaria leading to death (Miller et al., 2002).
When it takes up residence in a human RBC, the asexual blood stage parasite exploits a highly specialized cellular niche. The mature RBC is effectively a sack of haemoglobin; it provides a ready source of protein building blocks, however, there are logistical difficulties in gaining access to some other nutrients, as well as problems in detoxification of haemoglobin breakdown products. The RBC is unable to process and present antigens and thereby provides protection from the host’s immune system, however, the intraerythrocytic parasite needs to avoid passage through the spleen where resident macrophages recognize and remove RBCs with compromised deformability or altered antigenicity. To do this, mature stage parasites alter the properties of the host RBC membrane to promote adhesion of the infected RBCs to the vascular endothelium.
The development of powerful new genomics tools and the advent of molecular transfection technologies have greatly improved our understanding of the malaria parasite. However, a full interpretation of the information from these studies requires similar advances in our knowledge of its ultrastructure, and for many years a range of electron microscopy techniques have been exploited to provided details of the cellular organization of the intraerythrocytic parasite (Bannister et al., 2000a, Garcia et al., 2008). As described below, new developments in electron microscopy are greatly enhancing our ability to image whole cells at high resolution.
Section snippets
Three-dimensional (3-D) electron microscopic reconstruction of whole P. falciparum-infected RBCs
Until recently, knowledge of the 3-D organization of the ultrastructural features of Plasmodium-infected RBCs was deduced from analyses of multiple randomly cut sections of different cells or by reconstructions of regions of single cells from serially cut sections. This approach has been used in studies of P. falciparum organelles, including the mitochondrion (Slomianny and Prensier, 1986), the digestive vacuole (Slomianny, 1990, Elliott et al., 2008), the apicoplast (Hopkins et al., 1999), the
Immuno-labelling of P. falciparum proteins for ultrastructural studies
Immuno-labelling of specific cellular components represents a major challenge in ultrastructural studies of P. falciparum. Protocols that enhance structural preservation invariably reduce antigenicity and this is exacerbated by the low abundance of many of the proteins of interest. Moreover, the epitopes of many P. falciparum antigens appear to be susceptible to disruption during preparation for electron microscopy.
Low concentrations of aldehydes are used in an effort to preserve parasite
Merozoites
The P. falciparum merozoite has an ellipsoidal shape with an apical prominence at one end. It is amongst the smallest of eukaryotic cells (∼1.2 μm in length) but has a full complement of the cellular components needed for invasion and growth (Bannister and Mitchell, 2009). As is the case for the invasive forms of all apicomplexan parasites, the merozoite is characterized by a set of specialized organelles located at its apical end. Ultrastructural studies and immuno-electron microscopy have been
Ring stage
During invasion the parasite encloses itself within a PV membrane that remains closely associated with the parasite plasma membrane during its intraerythrocytic development. Shortly after invasion material is released from the dense granules into the PV (Aikawa et al., 1990, de Souza, 2005). This initiates the export of a range of proteins to the RBC cytoplasm and the elaboration of different membranous structures in the RBC cytoplasm (Bannister et al., 2004, Lanzer et al., 2006, Tilley et al.,
Trophozoite stage – inside the parasite
By the trophozoite stage (∼20–38 h), the parasite is at its metabolically most active and is increasing in size (approximately 2–4 μm in diameter; Fig. 4A and D). Large numbers of free and ER-bound ribosomes are observed, consistent with an increase in the level of protein synthesis (Bannister et al., 2000a). A Golgi complex composed of clusters of tubular cisternae and vesicles is elaborated close to the nucleus (Bannister et al., 2000a). The mitochondrion (Fig. 4B) and apicoplast (Fig. 4C,
Trophozoite stage – host cell modifications
As the parasite matures it initiates modifications to its host RBC that both facilitate access to nutrients and cause adhesion of the infected RBC to the vascular endothelium (Haldar and Mohandas, 2007, Tilley et al., 2008). The properties of the RBC are modified by proteins that are secreted from the parasite, across the PV membrane, and trafficked to different sites in the RBC cytoplasm (Lanzer et al., 2006, Maier et al., 2009). An ATP-powered translocon transfers the exported proteins across
Schizont stage
The final stage of the intraerythrocytic life cycle (38–48 h post-invasion) generates merozoites that are capable of invading new RBCs. During this stage the parasite undergoes multiple rounds of mitotic DNA replication in order to produce 16–20 daughter merozoites (Bannister et al., 2000a). The designation of the schizont stage is based on the appearance of individual daughter cells in light micrographs of P. falciparum smears (Freeman and Holder, 1983). In fact, endomitotic nuclear division is
Conclusions
The first ultrastructural description of P. falciparum-infected RBCs was provided over 30 years ago (Langreth et al., 1978). Since then a range of electron microscopy techniques has been applied to the study of this organism and our view of the cellular organization of the intraerythrocytic parasite has been continuously informed by ultrastructural studies. Recently, improved methods for serial sectioning, electron tomography, 3-D reconstruction and rendering of tomographic data have been
Acknowledgments
The authors would like to acknowledge Ms. Samantha Deed, La Trobe University, for technical assistance with the sample preparation, Dr. Kenneth Goldie, Bio21 Institute, Melbourne, for technical advice and Prof. David Mastronarde, University of Colorado, for helpful discussion regarding the IMOD package. The authors acknowledge support from the Australian Research Council and the Australian National Health and Medical Research Council.
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