An update on the rapid advances in malaria parasite cell biology

Abstract

Recent years have seen rapid advances in our understanding of malaria parasite cell biology. Some of this progress has been the result of developments in genetic techniques, advances in imaging technology, and new molecular tools. Herein, three aspects of parasite cell biology will be the topic of our focus: (i) plastid metabolism, (ii) sporozoite biology, and (iii) protein transport to and from the host erythrocyte. In each case, recent work has led to a deeper understanding of parasite biology, often at the expense of previously accepted paradigms. These studies also highlight the impediments, technical and otherwise, which will have to be overcome for continued rapid progress in these fields.

Research advances in malaria

Malaria cell biology has entered an exciting phase of discovery made possible by new genetic and molecular tools, as well as advances in imaging technology. In this review, we focus on three areas which have been the subject of exciting new developments over the past year. The first part of this review describes recent insights into apicoplast metabolic pathways, and the roles that these pathways play in liver and blood stage parasites. These findings change how we think about these pathways as drug targets and introduce the exciting possibility that parasites with defective apicoplast metabolism could be developed as live parasite vaccines. The second part of this review focuses on how proteins are imported into malaria parasites and the export machinery necessary to traffic proteins across the parasitophorous vacuole membrane to destinations in the host cell. Lastly, recent work illuminates the development of malaria sporozoites, including the exciting finding that sporozoites can develop in the skin. These topics were highlighted at the recent Research Advances in Malaria meeting (RAM 2009), and additional information can be found at the conference website^*.

Apicoplast metabolism: drug target or vaccine target?

The apicoplast has long been considered as an exciting source of drug targets. This organelle is thought to have arisen through secondary endosymbiosis of an algal cell which had previously incorporated a cyanobacterium [1–3]. Due to its prokaryotic origin, the apicoplast contains a range of metabolic pathways that differ significantly from those of the host and have the potential to be excellent drug targets for treating blood stage malaria. To unlock this potential, it is important to understand which metabolic pathways function in the apicoplast, and what their roles are during blood stage parasite growth. Recently, significant advances have been made on both fronts. Two parasite transporters, iTPT (inner-membrane Triose-Phosphate Transporter) and oTPT (outer-membrane Triose-Phosphate Transporter), have been characterized and may act in concert to traffic products of glycolysis into the apicoplast [4]. In particular, phosphorylated three carbon compounds such as DHAP (dihydroxyacetone phosphate) and PEP (phosphoenolpyruvate) could provide the apicoplast with three commodities: carbon, reducing equivalents and ATP (Figure 1). The need for these commodities is best illustrated by examining two of the most important apicoplast metabolic pathways: (i) the type II fatty acid synthesis (FAS-II) and (ii) the isoprenoid biosynthesis pathway DOXP, named after one of its intermediates (1-deoxy-D-xyulose-5-phosphate). The FAS-II pathway requires pyruvate, ATP, NADH and NADPH - all of which could be obtained from imported PEP and DHAP (Figure 1). Similarly, these two metabolites could be the source of pyruvate, NADPH, ATP and glyceraldehyde-3-phosphate used by the DOXP pathway.

Figure 1.

DOXP and FAS-II pathway precursors derived from glycolysis products. Glycolysis in the parasite cytosol produces two products (DHAP and PEP) which may be imported into the apicoplast. The triose-phosphate transporters (TPT) may be responsible for transport across the outermost (oTPT) and innermost (iTPT) membranes. Enzymes are colored in blue while key metabolites are colored in red or green if they are predicted to be required for the FAS or DOXP pathways, respectively. Both the FAS pathway and the DOXP pathway consume NADPH, which could be produced by GAPDH, or could be generated from NADH by a transhydrogenase such as the hypothetical enzyme encoded by PF14_0508. Enzyme abbreviations: TPI (triosephosphate isomerase), GAPDH (glyceraldehyde phosphate dehydrogenase), PyrK (pyruvate kinase), PDH (pyruvate dehydrogenase), ACCase (Acetyl-CoA Carboxylase), LipA (lipoate synthase), and LipB (lipoyl [octanoyl] transferase). Metabolite abbreviations: DHAP (dihydroxyacetone phosphate), GA3P (glyceraldehyde 3-phosphate), 1,3-BPG (1,3-bisphosphoglycerate), PEP (phosphoenolpyruvate), Ac-CoA (acetyl-Coenzyme A), Mal-CoA (malonyl-Coenzyme A). The cofactor lipoate is necessary for PDH activity and can be synthesized through the action of LipB and LipA.

The parasite enzymes comprising the DOXP and FAS-II pathways have been pursued as drug targets. Pharmacological evidence suggests that both pathways are essential for the growth of blood-stage parasites. The compound fosmidomycin inhibits the DXR enzyme (DOXP reductoisomerase) of the DOXP pathway and has been used clinically to treat malaria in humans [5–6]. Amplification of the dxr gene in Plasmodium falciparum confers fosmidomycin resistance in vitro, suggesting that the DOXP pathway is essential for asexual blood stage growth [7]. Similarly, the biocide triclosan is a potent inhibitor of the FAS-II enzyme ENR (Enoyl-ACP Reductase), and has antimalarial activity both in vitro (P. falciparum) and in vivo (P. berghei) [8]. Over the past seven years, structure-based drug discovery efforts zeroed in on ENR [9–14], generating a growing collection of potent inhibitors; however, the correlation between ENR inhibition and antimalarial activity proved to be poor. This observation led to attempts to genetically validate ENR as a drug target [15]. Ultimately, Fidock and coworkers were able to generate ENR knockout strains of P. falciparum and P. berghei, demonstrating that ENR is not essential for blood stage growth in vitro or in vivo [15]. In an independent study, P. yoelii strains were generated with two other FAS-II enzymes deleted, HAD (β-Hydroxyacyl-ACP Dehydratase) and KASII (β-Ketoacyl-ACP Synthase II) [16]. Since these studies targeted different FAS-II genes in different Plasmodium species, the overall conclusion is that FAS-II is not required for blood-stage parasite development.

This conclusion raises interesting questions concerning the roles of the related metabolic pathways shown in Figure 1. If the FAS-II pathway is dispensable in the blood stages, then the ACCase (Acetyl-Coenzyme A Carboxylase) and four proteins comprising the PDH (Pyruvate Dehydrogenase) could be dispensable as well. Recently, the E1α and the E3 subunits of the PDH were deleted in P. yoelii resulting in parasite lines with liver stage, but not blood stage, growth defects [17]. These results suggest that synthesis of the PDH cofactor lipoate is not required for blood-stage growth. PDH is exclusively located in the apicoplast [18] and is the only known lipoylated protein in this compartment [19]. Thus, the lipoylation pathway enzymes LipA and LipB should be dispensable for blood stage growth. Interestingly, LipB has been disrupted [20], but efforts to disrupt LipA have failed [21]. It is possible that LipA is required for lipoylation of the PDH E2 subunit, and that the lipoylated E2 subunit may have a role independent of traditional PDH activity. Even though PDH activity is not required for blood stage parasite growth, the pyruvate kinase (PyrK in Figure 1) may be. This enzyme generates ATP and pyruvate that would help to drive the DOXP pathway. Thus, if either the FAS-II or DOXP pathways would be required for asexual blood stage growth, pyruvate kinase and the triose-phosphate transporters iTPT and oTPT should be needed to provide key metabolites.

Based on the results of genetic studies, it is reasonable to conclude that FAS-II pathway enzymes are not appropriate drug targets for blood stage malaria. Nevertheless, the situation may be more complicated. A recent study examined the in vivo expression profiles of P. falciparum parasites collected from 43 patients in Senegal [22]. A population of parasites was identified in which the genes encoding metabolic enzymes, including those of the FAS-II pathway, were upregulated. This result is still under debate [23–24], but may reveal the importance of the FAS-II pathway in vivo under conditions that have not been observed using in vitro parasite culture. This scenario is not hard to imagine when one considers the differences between in vivo and in vitro growth conditions. Differences may involve exposure to certain host factors, but they may also be a simple matter of exposure to nutrients. The HEPES buffered Roswell Park Memorial Institute (RPMI) medium used in almost all P. falciparum culture systems exposes the parasites to radically different concentrations of nutrients compared to what they would likely encounter in vivo. Recently, a profile of the distribution of metabolites found in cultured P. falciparum highlighted some of these differences. One of the most abundant intracellular metabolites, found at a concentration of 12 mM, was the buffer HEPES [25]. This certainly would not be the case in vivo!

Although the FAS-II pathway and related apicoplast metabolism might make poor drug targets, they open an unexpected avenue for vaccine development. An exciting outcome of recent work on the FAS-II enzymes is that hadΔ and kasIIΔ P. yoelii parasites are unable to complete liver stage development. These parasite strains appear to progress normally through early developmental stages but manifest abnormal morphological phenotypes after 40 hours [16]. Similar results were observed with enrΔ P. berghei, except that breakthrough blood stage infections were occasionally observed, albeit with significantly delayed patency [15]. These observations make FAS-II, and perhaps related genes, exciting targets for the development of genetically attenuated parasites for use in live parasite vaccines. The developmental blockade late in liver stage infection could provide an extended window of host exposure to parasite antigens. This approach may prove superior to other approaches which lead to arrest at an early stage of hepatocyte infection, such as the use of UIS (Up-regulated in Infective Sporozoites) deletion strains, or the use of irradiated sporozoites [26–27]. Paradoxically, the study of key metabolic pathways in malaria parasites may ultimately lead to a new vaccine, rather than a drug.

The parasite protein trade: import and export

During asexual development in erythrocytes, malaria parasites export a surprisingly large number of proteins. Most exported proteins require a conserved motif, termed the Plasmodium export element (PEXEL) for targeting beyond the parasitophorous vacuole membrane to destinations in the host cell [28–29]. A comparison of the ‘secretomes’ predicted using different criteria identified eight candidate proteins with a high likelihood of being exported [30]. In subsequent experiments, only five of these proteins were found to be exported, indicating that there may be additional determinants of protein export that are not currently recognized by in silico prediction programs. Overall, several hundred proteins (2–5% of the genome) are thought to be exported. Although many of these proteins have unknown functions, those that have been characterized seem to have roles in virulence, such as altering erythrocyte cell adhesion and rigidity [31]. Membrane rigidity was recently quantified with optically-trapped, infected red blood cells under flow conditions. The method was used to determine the effect of conditioned culture medium on membrane rigidity. Exported parasite molecules alter host membrane rigidity of infected as well as uninfected erythrocytes, possibly by interacting with the underlying spectrin network [32].

Exported parasite proteins must cross not only the parasite plasma membrane, but also the parasitophorous vacuole (PV) membrane in order to reach destinations in the erythrocyte. A translocon of exported proteins (PTEX) located in the vacuole membrane was identified using a combination of bioinformatic and proteomic approaches [33]. The translocon is composed of at least five proteins, including a pore-forming protein (EXP2) homologous to hemolysin E. Interestingly, a human HSP70 was found to be associated with the PTEX complex, presumably recruited by the complex to assist in the passage or refolding of exported proteins [33]. Consistent with this hypothesis, recent evidence demonstrates that proteins must be unfolded to be efficiently trafficked across the PV membrane. Proteins tagged with murine dihydrofolate reductase (DHFR) were no longer competent for export in the presence of the small molecule inhibitor WR99210, which stabilizes DHFR and traps the tagged proteins in the lumen of the PV [34].

Exported proteins are instrumental in organizing membrane structures in the erythrocyte cytosol. For example, normal formation of the tubovesicular network depends on the proper expression of the Erythrocyte Vesicle Protein 1 [35]. Similarly, the deletion of proteins exported to Maurer’s clefts can disrupt the morphology of the clefts [36] and their function [37–39]. In particular, deletion of the skeleton-binding protein (SBP1) [37–38] or the membrane-associated histidine-rich protein 1 (MAHRP1) [39] prevents the export of erythrocyte membrane protein-1 (PfEMP1), highlighting the role that Maurer’s clefts play in protein trafficking to the erythrocyte membrane. Using immunoelectron tomography, Maurer’s clefts were shown to be physically isolated compartments, but are sometimes connected via electron dense tethers about 30 nm in diameter, to either the erythrocyte membrane [40] or the parasitophorous vacuole membrane [41]. It is unclear, however, how these tethers help to traffic proteins such as PfEMP1 to the host membrane and it is possible that these tethers have some other function unrelated to protein trafficking.

Ultrastructure studies have also provided insight into the mechanism of protein import into parasite digestive vacuoles. A long-standing question concerns the biogenesis of digestive vacuoles. Recently, it was proposed that ring stage parasites form a cup shape which closes to pinch off erythrocyte cytosol, forming a large vacuole between 0.5 and 1.6 femtoliters in volume [42]. This phagotrophic process, named ‘the big gulp’ is consistent with the observation of vacuoles of 0.5 femtoliters at 15 hours post invasion [43], around the time at which hemozoin is first observed [44]. Phagotrophic ingestion of hemoglobin was first suggested by Rudzinska and Trager over 50 years ago using the avian malaria parasite, P. lophurae [45], but was not supported by Aikawa’s early electron microscopy studies on the primate malaria parasite, P. knowlesi [46]. The window will always be short that captures closure of the cup shaped ring from the instant the vacuole is a pseudo vacuole to the new food vacuole. Further evidence of ‘the big gulp’ would be to demonstrate a concentration of vacuolar ATPases on the plasma membrane of the cup shaped rings ready to begin acidification. The conflicting models of digestive vacuole biogenesis will hopefully be reconciled before another 50 years pass.

A less controversial method of hemoglobin import is mediated by endocytic vesicles which originate from a structure called the cytostome. Cytostomes (originally referred to as micropyle) were first described by Garnham as structures responsible for exporting parasite factors into host cells [47]. Aikawa, however, correctly assigned cytostomes a role in parasite feeding [48], and subsequent electron-microscopy studies provided more detail into this process [49–50]. Both the parasite plasma membrane and the parasitophorous vacuole membrane are drawn through the cytostome to form a double membrane-bound invagination which subsequently fuses with the digestive vacuole, either through direct contact [51], or after vesicle-mediated trafficking [42]. In both cases, the delivery of hemoglobin to the digestive vacuole is actin-dependent [42,51–52] and is most consistent with fluid-phase endocytosis, a process typically regulated by the endocytic marker Rab5. Analysis of the Rab GTPases found in malaria parasites identified three Rab5 family proteins [53], one of which has been implicated in hemoglobin uptake. Rab5A was found to be associated with hemoglobin-containing vesicles in parasites expressing a constitutively active Rab5A mutant [42]. It will be interesting to track the presence of Rab5A shortly after merozoites invade host red blood cells. Cytostomes have been observed on the surface of merozoites [54], raising the possibility that invasive parasites are also capable of bulk fluid phase endocytosis.

Surprising twists in the journey of sporozoites in the mammalian host

Before the pathogenic symptoms of blood stage malaria appear, the clinically silent sporozoites invade the body and develop in the liver. Plasmodium sporozoites make a remarkable journey from the skin, where they are deposited by an infectious mosquito, to the liver via the blood [55]. For a long time, the traditional assumption was that sporozoites reached the liver quickly following the insect bite; however, recent quantitative imaging studies on Plasmodium transmission from mosquito to mammals revealed that once in the skin, sporozoites display robust motility following what appears to be a random path within the dermis, rather than being directly targeted to blood vessels [56–57]. These observations led to the hypothesis that there is perhaps a skin stage of malaria infection, and raise many exciting questions about the dermis as a site of infection. What is the proportion of inoculated sporozoites that will remain permanently in the dermis, and will they have a fate different from those that reach the liver? Recently, Robert Ménard and coworkers used intravital microscopy to show that at least 50% of P. berghei sporozoites are retained in the skin. Surprisingly, sporozoites can develop inside cells associated with the hair follicle, the epidermis, and the dermis, and can ultimately generate merozoites (R. Ménard et al., unpublished). Although the sporozoites that remain in the skin multiply more slowly than the parasites growing in the liver, the significant number of these parasites in the skin makes this organ a secondary site for sporozoite development in mammals. Perhaps more importantly, these data show that many parasite antigens are left in the skin, originating from both sporozoites and developing parasites, which will be secondarily delivered to the draining lymph node [56–57] where adaptive immune response is initiated [58].

All malaria zoites move by gliding motility, a form of locomotion that is powered by a parasite subpellicular motor which translocates proteins posteriorly, resulting in the forward movement of the parasite. Several studies document the role played by parasite effectors of sporozoite motility and invasion [59]. The best characterized proteins are CSP, the major surface protein of the sporozoite, and two micronemal proteins, TRAP (Thrombospondin Related Antigenic Protein) and AMA-1 (Apical Membrane Antigen 1). CSP is responsible for the initial recognition of hepatocytes, followed by the specific attachment of the parasite to the unique heparan sulfate proteoglycans of these cells [60]. TRAP and AMA-1 are type I transmembrane proteins with adhesive motifs in their extracellular domains which allow the sporozoite to bind to hepatic receptors. It has been hypothesized that proteolytic cleavage of parasite surface proteins disengages adhesive interactions between the zoite and the extracellular substrate, enabling forward movement [61]. So far, the only parasite protease that has been shown to play a role in sporozoite infection of hepatocytes is the rhomboid protease 1 [62]. It is not clear, however, how rhomboid proteases promote gliding motility and hepatocyte penetration, and what their substrates are. During invasion, both TRAP and AMA-1 are shed from the parasite surface via proteolytic cleavage by a parasite serine protease [61]. Experiments using a heterologous expression system show that TRAP can be cleaved by rhomboid protease 4, while AMA-1 is efficiently cleaved by rhomboid protease 1 [63]. Thus, both rhomboid proteases could have a role in sporozoite invasion of hepatocytes analogous to the role proposed for rhomboid protease 4 in merozoites invasion of erythrocytes [64].

An intricate cycling between mosquito vectors and vertebrate hosts is necessary for the lifecycle of malaria parasites [65]. Following hepatocyte invasion, the sporozoite develops into an intracellular form which rapidly generates thousands of infectious merozoites. The fine structure of the hepatic forms of P. berghei demonstrated that parasite secretory organelles are lost during the development of hepatic trophozoites [66]. Until now, details of the cellular events and the mechanism of organelle elimination during sporozoite development have remained unexplored. Ultrastructural studies by Isabelle Coppens and coworkers detail the remodeling processes that occur in the liver stage developmental cascade. Sporozoite metamorphosis is associated with a spectacular change in morphology, and a major remodeling of the cellular architecture, including the elimination of organelles no longer required for the liver stage. During this process parasites discharge some organelles implicated in invasion into the parasitophorous vacuole, and also trigger an autophagic process to selectively sequester cytosolic organelles, such as micronemes.

The parasitophorous vacuole plays a central role in the development of liver schizonts. Only a few proteins have been identified on the parasitophorous vacuole membrane of liver forms, such as Exp-1 (Exported Protein-1), UIS3 (Up-regulated in Infective Sporozoites gene 3) and UIS4 (Up-regulated in Infective Sporozoites gene 4) [67]. The location of some of these proteins at the host-parasite interface suggests that they may interact with host cytosolic factors essential for parasite development. In this context, it has been shown that UIS3 specifically interacts with the host fatty acid binding protein, the main carrier of fatty acids for delivery to cytoplasmic compartments [68]. A collection of mutant parasites were engineered in order to pursue the functional characterization of parasitophorous vacuole membrane proteins. Sporozoites deficient in the protein P52, located in secretory organelles, cannot form a proper vacuolar membrane and do not persist in the liver [26]. Parasites lacking either UIS3 or UIS4 were able to form a typical vacuole but exhibited a growth arrest 14 h post-invasion [26]. This raises the interesting possibility that some of these parasite proteins may have a role in funneling nutrients to the fast growing liver forms. In addition to the FAS-II knock out parasites described above, many of these parasite lines are also being evaluated for their potential as genetically attenuated live parasite vaccines [27].

Concluding remarks and future directions

Many exciting advances in parasite cell biology, including some not highlighted in this review were presented at the RAM 2009 meeting. We can look forward to future progress in these fields as current impediments are overcome. A key question involves the potential differences between parasite metabolism in vivo and in vitro. Could a P. falciparum enzyme be essential for blood stage proliferation in vivo even though it can be deleted in vitro? Another challenge will be to use proteomics to identify the proteins which are exported by the parasite, as well as those responsible for endocytosis and subsequent endosomal trafficking. more complete catalog of trafficked proteins will facilitate unraveling the mechanisms underlying these processes. The recent demonstration that sporozoites can proliferate in the dermis as well as the liver raises a host of questions: How do sporozoites know they have contacted their target cells to switch from a migratory to an invasive phenotype? What are the host surface receptors that interact with invading sporozoites? What is the signal that triggers sporozoite development into liver forms? New approaches to genetically manipulate sporozoites [69], and to enhance the production of preerythrocytic forms will be instrumental in answering these questions.