The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress

Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ~35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48-61) large-scale proteolysis of the cytoskeleton during rupture ~48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release.     

The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

The malaria parasite exports numerous proteins into its host red blood cell (RBC). The trafficking of these exported effectors is complex. Proteins are first routed through the secretory system, into the parasitophorous vacuole (PV), a membranous compartment enclosing the parasite. Proteins are then translocated across the PV membrane in a process requiring ATP and unfolding. Once in the RBC compartment the exported proteins are then refolded and further trafficked to their final localizations. Chaperones are important in the unfolding and refolding processes. Recently, it was suggested that the parasite TRiC chaperonin complex is exported, and that it is involved in trafficking of exported effectors. Using a parasite‐specific antibody and epitope‐tagged transgenic parasites we could observe no export of Plasmodium TRiC into the RBC. We tested the importance of the parasite TRiC by creating a regulatable knockdown line of the TRiC‐θ subunit. Loss of the parasite TRiC‐θ led to a severe growth defect in asexual development, but did not alter protein export into the RBC. These observations indicate that the TRiC proteins play a critical role in parasite biology, though their function, within the parasite, appears unrelated to protein trafficking in the RBC compartment.     

Population dynamics of a pathogen: the conundrum of vivax malaria

Building a mathematical model of population dynamics of pathogens within their host involves considerations of factors similar to those in ecology, as pathogens can prey on cells in the host. But within the multicellular host, attacked cell types are integrated with other cellular systems, which in turn intervene in the infection. For example, immune responses attempt to sense and then eliminate or contain pathogens, and homeostatic mechanisms try to compensate for cell loss. This review focuses on modeling applied to malarias, diseases caused by single-cell eukaryote parasites that infect red blood cells, with special concern given to vivax malaria, a disease often thought to be benign (if sometimes incapacitating) because the parasite only attacks a small proportion of red blood cells, the very youngest ones. However, I will use mathematical modeling to argue that depletion of this pool of red blood cells can be disastrous to the host if growth of the parasite is not vigorously check by host immune responses. Also, modeling can elucidate aspects of new field observations that indicate that vivax malaria is more dangerous than previously thought.     

Protein and lipid trafficking induced in erythrocytes infected by malaria parasites

The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.     

The Homeostasis of Plasmodium falciparum-Infected Red Blood Cells

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15–32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before ~48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.     

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.     

Pfl I-I and Pf332: two giant proteins synthesized in erythrocytes infected with Plasmodium falciparum

Although the malaria parasite develops within erythrocytes, it has to modify the surrounding red blood cell membrane for its intracellular survival and maturation. These changes include the translocation of proteins across the parasite and the parasitophorous vacuole membranes to the host membrane. In this review, Denise Mattei, Katherine Hinterberg and Artur Scherf focus on two distinct giant parasite molecules of unprecedented size (approximately one MDa), called Pf332 and PflI-I, that are synthesized and exported into the cytoplasm of the host cell in the asexual and sexual blood stages of Plasmodium falciparum, respectively. The corresponding genes are located in genetically unstable subtelomeric chromosome regions.     

Protein export in malaria parasites: an update

Symptomatic malaria is caused by the infection of human red blood cells (RBCs) with Plasmodium parasites. The RBC is a peculiar environment for parasites to thrive in as they lack many of the normal cellular processes and resources present in other cells. Because of this, Plasmodium spp. have adapted to extensively remodel the host cell through the export of hundreds of proteins that have a range of functions, the best known of which are virulence‐associated. Many exported parasite proteins are themselves involved in generating a novel trafficking system in the RBC that further promotes export. In this review we provide an overview of the parasite synthesized export machinery as well as recent developments in how different classes of exported proteins are recognized by this machinery.     

Molecular and cellular biology of malaria

Thanks to the extensive use of recombinant DNA technology and immunological methods, much insight into cellular functions of the human malaria parasite Plasmodium falciparum has been gained since it was learnt ten years ago how to grow this organism in culture. The amino acid sequence of over a dozen surface proteins of the parasite and of several proteins the parasite excretes into its most important host cell, the erythrocyte, have been determined. Interestingly many of these proteins show blocks of repeated amino acids. Several proteins have been shown to be involved in specific aspects of the complex hostparasite interaction, such as penetration of host cells or increased stickiness of infected red blood cells in the blood vessels. Some of the proteins described here may be protective antigens and may become important in vaccine development.     

The twists and turns of Maurer's cleft trafficking in P. falciparum-infected erythrocytes

The malaria parasite, Plasmodium falciparum, invades the red blood cells (RBCs) of its human host and initiates a series of morphological rearrangements within the host cell cytoplasm. The mature RBC has no endogenous trafficking machinery; therefore, the parasite generates novel structures to mediate protein transport. These include compartments called the Maurer's clefts (MC), which play an important role in the trafficking of parasite proteins to the surface of the host cell. Recent electron tomography studies have revealed MC as convoluted flotillas of flattened discs that are tethered to the RBC membrane, prompting speculation that the MC could, in one respect, represent an extracellular equivalent of the Golgi apparatus. Visualization of both resident and cargo proteins has helped decipher the signals and routes for trafficking of parasite proteins to the MC and beyond.     

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite‐induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive ‘knob’ structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50–60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.     

Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes

Malaria parasites during intraerythrocytic development change the ultrastructure, biophysics, and the antigens of the host red blood cell membrane. Parasite-encoded proteins are associated with, inserted into, or secreted across the infected erythrocyte membrane. Since parasites of the genus Plasmodium are eukaryotic cells, it must be assumed that they possess essentially eukaryotic modes of vesicle-mediated transport and translocation of proteins and membranes. Numerous studies have demonstrated vesicular structures in the cytoplasm of malaria-infected red blood cells and an assortment of parasite proteins associated with the different vesicles, membranes, and membrane-defined compartments. Some parasite polypeptides remain trapped between the parasite and the parasitophorous vacuole membranes PVM, whereas others are associated with morphologically distinct membrane-limited vesicles and vacuoles. Some of these same parasite protein antigens also associate with the erythrocyte membrane or with parasite-induced ultrastructural modifications in the membrane of the parasitized red blood cells. This implies that intracellular transport occurs in malaria-infected erythrocytes, a capacity that uninfected red blood cells normally lose upon enucleation. The specific locations of parasite antigens within the infected cell also implys the existence of targeting signals in the translocated parasite polypeptides and perhaps transport-mediating proteins. The genes corresponding to some of these translocated proteins have been sequenced. Typical (and in some cases atypical) signal peptide sequences occur, as well as a number of sequences that may result in posttranslational modifications. How or if these features figure in to the translocation across, and targeting to a particular membrane compartment of the intraerythrocytic parasite remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes

Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.     

The status of zinc in malaria (Plasmodium falciparum) infected human red blood cells: stage dependent accumulation,compartmentation and effect of dipicolinate

The inhibitory effect of metal chelators on intraerythrocytic malarial parasites imply that trace metal have a vital role in the biology of these organisms. In the present work X-ray fluorometry was used to study the status of zinc and iron in human red blood cells infected with Plasmodium falciparum in culture conditions. It was found that while the iron level remains constant throughout the parasite cell cycle, that of zinc increases in parallel with parasite maturation to reach a 2.3-fold higher level than that of uninfected red blood cells. Compartment analysis of infected red blood cells indicated that most of this gain was associated with the parasite and some with the host-cell membrane. Analysis of the malarial pigment showed that the zinc/iron ratio was similar to that of red blood cells, implying the this compound, which results from the digestion of host-cell cytosol, sequesters the zinc of host metalloenzymes. Dipicolinic acid (DPA), like other chelators, was found to inhibit the intracellular development of the parasite with an ED₅₀ of 1 mM. DPA does not penetrate into normal red blood cells but readily permeates into infected cells, although it does not leach out their zinc. It is uncertain whether the inhibitory effect of DPA is exerted through alterations of host cell metabolism or by directly affecting that of the parasite. The putative receptors of zinc in the infected red blood cell are discussed.     

The Exported Protein PbCP1 Localises to Cleft-Like Structures in the Rodent Malaria Parasite Plasmodium berghei

Protein export into the host red blood cell is one of the key processes in the pathobiology of the malaria parasite Plasmodiumtrl falciparum, which extensively remodels the red blood cell to ensure its virulence and survival. In this study, we aimed to shed further light on the protein export mechanisms in the rodent malaria parasite P. berghei and provide further proof of the conserved nature of host cell remodeling in Plasmodium spp. Based on the presence of an export motif (R/KxLxE/Q/D) termed PEXEL (Plasmodium export element), we have generated transgenic P. berghei parasite lines expressing GFP chimera of putatively exported proteins and analysed one of the newly identified exported proteins in detail. This essential protein, termed PbCP1 (P. berghei Cleft-like Protein 1), harbours an atypical PEXEL motif (RxLxY) and is further characterised by two predicted transmembrane domains (2TMD) in the C-terminal end of the protein. We have functionally validated the unusual PEXEL motif in PbCP1 and analysed the role of the 2TMD region, which is required to recruit PbCP1 to discrete membranous structures in the red blood cell cytosol that have a convoluted, vesico-tubular morphology by electron microscopy. Importantly, this study reveals that rodent malaria species also induce modifications to their host red blood cell.     

Fate of Parasite and Host Organelle DNA during Cellular Transformation of Red Algae by Their Parasites

The transfer of a nucleus into a cytoplasm of a genetically foreign cell and its subsequent multiplication in the cytoplasm of this cell characterize most parasitic red algal species and their interactions with specific red algal hosts. Nuclei enter the host's cytoplasm upon cell fusion of parasite and host cell; here, they replicate, are spread to contiguous host cells, and ultimately are packaged into spores that reinfect other host thalli. In this study, we examined whether the proplastids and mitochondria that occur in these red algal adelphoparasites are acquired from their host or whether they are unique to the parasite and are brought into the host along with the parasite nucleus. To establish their origins and fates, plastid and mitochondrial restriction fragment length polymorphisms (RFLPs) of parasite cells were compared with those of their host plastid and mitochondrial DNA in three host and parasite pairs. For plastids, no RFLP differences were found between hosts and parasites, supporting an earlier conclusion, based on microscopic studies, that the proplastids of parasites are acquired from their hosts. For mitochondria, characteristic RFLP differences were detected between host and parasite for two of the pairs of species but not for the third. Evidence of the evolutionary difference between hosts and their parasites was shown by RFLP differences between nuclear ribosomal repeat regions.     

Blocking effect of a biotinylated protease inhibitor on the egress of Plasmodium falciparum merozoites from infected red blood cells

The malaria parasite Plasmodium falciparum invades human red blood cells. Before infecting new erythrocytes, the merozoites have to exit their host cell to get into the blood plasma. Knowledge about the mechanism of egress is scarce, but it is thought that proteases are basically involved in this step. We have introduced a biotinylated dibenzyl aziridine-2,3-dicarboxylate (bADA) as an irreversible cysteine protease inhibitor to study the mechanism of merozoite release and to identify the proteases involved. The compound acts on parasite proteins in the digestive vacuole and in the host cell cytosol, as judged by fluorescence microscopy. The inhibitor blocks rupture of the host cell membrane, leading to clustered merozoite structures, as evidenced by immunoelectron microscopy. Interestingly, bADA did not prevent rupture of the parasitophorous vacuole membrane (PVM) that surrounds the parasite during the period of intraerythrocytic maturation. The compound appears to be a valuable template for the development of inhibitors specific for individual plasmodial proteases, which would be useful tools to dissect the molecular mechanisms underlying the process of merozoite release and consequently to develop potent antimalarial drugs.     

Cyclic nucleotide signalling in malaria parasites

Cyclic nucleotides are so-called intracellular second messenger molecules used by all cells to transform environmental signals into an appropriate response. Interest in the cyclic nucleotides cAMP and cGMP in malaria parasites followed early observations that both molecules might be involved in distinct differentiation events within the sexual phase of the life cycle that is required for transmission of parasites to the mosquito vector. Completed genome sequences combined with biochemical and genetic studies have confirmed the presence of the main enzymatic components of cyclic nucleotide signalling in the parasite. Dissection of their functions is underway and is giving initial insights into some of the cellular processes, which are regulated by these signalling pathways. Malaria parasites occupy terminally differentiated red blood cells for a significant proportion of their life cycle, but although there is some evidence of potential roles for the residual host cell signalling machinery in parasite development, details are few. A major gap in our knowledge is the nature of the cell surface receptors, which might trigger cyclic nucleotide signalling in the parasite.     

A reticulocyte-binding protein complex of Plasmodium vivax merozoites.

Plasmodium vivax merozoites primarily invade reticulocytes. The basis of this restricted host cell preference has been debated. Here we introduce two novel P. vivax proteins that comigrate on reducing SDS-polyacrylamide gels, colocalize at the apical pole of merozoites, and adhere specifically to reticulocytes. The genes encoding these proteins, P. vivax reticulocyte-binding proteins 1 and 2 (PvRBP-1 and PvRBP-2), have been cloned and analyzed. Homologous genes are evident in the closely related simian malaria parasite, P. cynomolgi, which also prefers to invade reticulocytes, but are not evident in the genome of another related simian malaria parasite, P. knowlesi, which invades all red blood cell subpopulations. Native PvRBP-1 is likely a transmembrane-anchored disulfide-linked protein, and along with PvRBP-2 may function as an adhesive protein complex. We propose that the RBPs of P. vivax, and homologous proteins of P. cynomolgi, function to target the reticulocyte subpopulation of red blood cells for invasion.