The moving junction, a key portal to host cell invasion by apicomplexan parasites

One defining feature of apicomplexan parasites is their special ability to actively invade host cells. Although rapid, invasion is a complicated process that requires coordinated activities of host cell attachment, protein secretion, and motility by the parasite. Central to this process is the establishment of a structure called moving junction (MJ), which forms a tight connection between invading parasite and host cell membranes through which the parasite passes to enter into the host. Although recognized microscopically for decades, molecular characterization of the MJ was only enabled by the recent discovery of components that make up this multi-protein complex. Exciting progress made during the past few years on both the structure and function of the components of the MJ is reviewed here.     

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.     

The kinomes of apicomplexan parasites

Protein phosphorylation plays a fundamental role in the biology of apicomplexan parasites. Many apicomplexan protein kinases are substantially different from their mammalian orthologues, and thus constitute a landscape of potential drug targets. Here, we integrate genomic, biochemical, genetic and evolutionary information to provide an integrated and up-to-date analysis of twelve apicomplexan kinomes. All kinome sequences are available through the Kinomer database.     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Genomics of apicomplexan parasites

The increasing prevalence of infections involving intracellular apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium (the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, respectively) represent a significant global healthcare burden. Despite their significance, few treatments are available; a situation that is likely to deteriorate with the emergence of new resistant strains of parasites. To lay the foundation for programs of drug discovery and vaccine development, genome sequences for many of these organisms have been generated, together with large-scale expression and proteomic datasets. Comparative analyses of these datasets are beginning to identify the molecular innovations supporting both conserved processes mediating fundamental roles in parasite survival and persistence, as well as lineage-specific adaptations associated with divergent life-cycle strategies. The challenge is how best to exploit these data to derive insights into parasite virulence and identify those genes representing the most amenable targets. In this review, we outline genomic datasets currently available for apicomplexans and discuss biological insights that have emerged as a consequence of their analysis. Of particular interest are systems-based resources, focusing on areas of metabolism and host invasion that are opening up opportunities for discovering new therapeutic targets.     

Distinct mechanisms govern proteolytic shedding of a key invasion protein in apicomplexan pathogens

Apical membrane antigen-1 (AMA1) is a conserved apicomplexan protein that plays an important but undefined role in host cell invasion. We have studied the fate of Plasmodium falciparum AMA1 (PfAMA1) during erythrocyte invasion by the malaria merozoite, and compared it with that of the Toxoplasma gondii orthologue, TgAMA1. Shedding of the PfAMA1 ectodomain goes essentially to completion during invasion, and occurs predominantly or exclusively via juxtamembrane cleavage at the previously identified sheddase cleavage site, Thr517. Only the resulting juxtamembrane stub of the ectodomain is efficiently carried into the host cell, and this remains distributed around the plasma membrane of the intracellular ring-stage parasite. Inhibition of normal shedding, however, results in proteolysis at an intramembrane, rhomboid-like cleavage site, and PfAMA1 is susceptible to cleavage by Drosophila rhomboid-1, showing that it can be a substrate for intramembrane cleavage but is not normally processed in this manner. In contrast, shedding of TgAMA1 from the surface of extracellular tachyzoites occurs exclusively via cleavage within the luminal half of its transmembrane domain by a rhomboid-like protease. Also unlike PfAMA1, complete TgAMA1 shedding does not accompany Toxoplasma invasion as the intact protein was readily detected on the surface of newly invaded tachyzoites. This work reveals unexpected differences in the manner in which Plasmodium and Toxoplasma shed AMA1 from the surface of invasive zoites, and demonstrates the presence at the malaria merozoite surface of a rhomboid-like protease.     

Aspartyl proteinase genes from apicomplexan parasites: evidence for evolution of the gene structure

Aspartyl proteinases are a widely distributed family of enzymes. All vertebrate aspartyl proteinases share a conserved nine-exon gene structure, but in other organisms the structure of aspartyl proteinase genes varies considerably. The exon-intron patterns generally reflect phylogeny based on amino acid sequences. However, close comparison of these gene structures reveals some striking features, such as the conservation of intron positions and intron phases between aspartyl proteinases from nematodes and apicomplexans. Here, we discuss the implications of gene structure for the possible evolution of the aspartyl proteinase family, with particular reference to the plasmepsins of Plasmodium falciparum and eimepsin from Eimeria tenella.     

The apicoplast: a review of the derived plastid of apicomplexan parasites

The apicoplast is a plastid organelle, homologous to chloroplasts of plants, that is found in apicomplexan parasites such as the causative agents of Malaria Plasmodium spp. It occurs throughout the Apicomplexa and is an ancient feature of this group acquired by the process of endosymbiosis. Like plant chloroplasts, apicoplasts are semi-autonomous with their own genome and expression machinery. In addition, apicoplasts import numerous proteins encoded by nuclear genes. These nuclear genes largely derive from the endosymbiont through a process of intracellular gene relocation. The exact role of a plastid in parasites is uncertain but early clues indicate synthesis of lipids, heme and isoprenoids as possibilities. The various metabolic processes of the apicoplast are potentially excellent targets for drug therapy.     

Rhoptry organelles of apicomplexan parasites

Rhoptries are the unifying structural feature of the intracellular, opicomplexon parasites and are implicated in having a central role in host cell invasion. Ultrastructural studies of zoites of different genera suggest that the participation of rhoptries in the invasion of the respective host cells is morphologically similar. However, biochemical analysis of their protein constituents reveals a considerable degree of diversity between different coccidion parasites. In this article Margaret Perkins asks whether there are common structural determinants of the rhoptry components of different genera and if the underlying mechanism of rhoptry function is similar in all opicomplexon parasites.     

LCCL proteins of apicomplexan parasites

The LCCL module is a conserved, autonomous protein-folding domain that has recently been found in several extracellular proteins of apicomplexan parasites including Plasmodium, Toxoplasma, Cryptosporidium and Theileria, identifying a new protein family in the Apicomplexa. The expression and structure of these modular proteins has fostered speculation about the roles of these novel molecules in immune evasion. Here, the current data and literature on the members of this protein family are reviewed, with a discussion on their possible roles in host-parasite interaction.     

Cytoskeleton of apicomplexan parasites.

The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elements in these parasites.     

Sex allocation and population structure in apicomplexan (protozoa) parasites

Establishing the selfing, rate of parasites is important for studies in clinical and epidemiological medicine as well as evolutionary biology Sex allocation theory offers a relatively cheap and easy way to estimate selfing rates in natural parasite populations. Local mate competition (LMC) theory predicts that the optimal sex ratio (r*; defined as proportion males) is related to the selfing rate (s) by the equation r* = (1-s)/2. In this paper, we generalize the application of sex allocation theory across parasitic protozoa in the phylum Apicomplexa. This cosmopolitan phylum consists entirely of parasites, and includes a number of species of medical and veterinary importance. We suggest that LMC theory should apply to eimeriorin intestinal parasites. As predicted, data from 13 eimeriorin species showed a female-biased sex ratio, with the sex ratios suggesting high levels of selfing (0.8-1.0). Importantly, our estimate of the selfing rate in one of these species, Toxoplasma gondii, is in agreement with previous genetic analyses. In contrast, we predict that LMC theory will not apply to the groups in which syzygy occurs (adeleorins, gregarines and piroplasms). Syzygy occurs when a single male gametocyte and a single female gametocyte pair together physically or in close proximity, just prior to fertilization. As predicted, data from four adeleorin species showed sex ratios not significantly different from 0.5.     

Mix and match modules: structure and function of microneme proteins in apicomplexan parasites

Microneme organelles are found in the apical complex of all apicomplexan parasites and play an important role in the invasion process. The recent identification of microneme proteins from different apicomplexan genera has revealed a striking conservation of structural domains, some of which show functional complementation across species. This supports the idea that the mechanism of host cell invasion across the phylum is conserved not only morphologically, but also functionally at the molecular level. Here, we review and summarize these recent findings.     

Signalling mechanisms involved in apical organelle discharge during host cell invasion by apicomplexan parasites

Malaria is caused by Plasmodium parasites, which belong to the phylum apicomplexa. The characteristic feature of apicomplexan parasites is the presence of apical organelles, referred to as micronemes and rhoptries, in the invasive stages of the parasite life cycle. Survival of these obligate intracellular parasites depends on successful invasion of host cells, which is mediated by specific molecular interactions between host receptors and parasite ligands that are commonly stored in these apical organelles. The timely release of these ligands from apical organelles to the parasite surface is crucial for receptor engagement and invasion. This article is a broad overview of the signalling mechanisms that control the regulated secretion of apical organelles during host cell invasion by apicomplexan parasites.     

Synthesis of chloroplast galactolipids in apicomplexan parasites

Monogalactosyldiacylglycerol and digalactosyldiacylglycerol are major chloroplast lipids of algae and land plants and are synthesized within the plastid envelope. Here we report that in Toxoplasma gondii and Plasmodium falciparum lysates, radiolabeled UDP-galactose is incorporated into monogalactosylcerebrosides, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol due to distinct enzymological activities. Furthermore, DGDG is immunologically detected in apicomplexans.     

Nutrient acquisition by intracellular apicomplexan parasites: staying in for dinner

The intracellular forms of the apicomplexan parasites Plasmodium, Toxoplasma and Eimeria reside within a parasitophorous vacuole. The nutrients required by these intracellular parasites to support their high rate of growth and replication originate from the host cell which, in turn, takes up such compounds from the extracellular milieu. Solutes moving from the external medium to the interior of the parasite, are confronted by a series of three membranes --the host cell membrane, the parasitophorous vacuole membrane and the parasite plasma membrane. Each constitutes a potential permeability barrier which must be either crossed or bypassed. It is the mechanisms by which this occurs that are the subject of this review.     

The paradox of the parasites: implications for biological invasion

The enemy-release hypothesis for biological invasions supposes that invasive species may be more successful in their introduced ranges than in their native ranges owing to the absence of coevolved natural enemies. Recent studies supporting this hypothesis have found that introduced plants and animals are less parasitized in their introduced ranges than in their native ranges. Expanding on this theory, I hypothesize that the role of enemy release may differ among the introduction, establishment and spread phases of an invasion. I present a simple model indicating that parasite release is unlikely to greatly affect the chance of establishment in populations with and without an immune subpopulation. The specific numerical relationship between the number of individuals introduced and the chance of establishment depends on a relationship between virulence, here conceptualized as the chance for the extinction of a lineage, and the fraction of the population infected at introduction. These results support the idea of a 'filter effect' in which different biological processes regulate the different phases of an invasion.     

Global protein expression analysis in apicomplexan parasites: current status

Members of the phylum Apicomplexa are important protozoan parasites that cause some of the most serious, and in some cases, deadly diseases in humans and animals. They include species from the genus Plasmodium, Toxoplasma, Eimeria, Neospora, Cryptosporidium, Babesia and Theileria. The medical, veterinary and economic impact of these pathogens on a global scale is enormous. Although chemo- and immuno-prophylactic strategies are available to control some of these parasites, they are inadequate. Currently, there is an urgent need to design new vaccines or chemotherapeutics for apicomplexan diseases. High-throughput global protein expression analyses using gel or non-gel based protein separation technologies coupled with mass spectrometry and bioinformatics provide a means to identify new drug and vaccine targets in these pathogens. Protein identification based proteomic projects in apicomplexan parasites is currently underway, with the most significant progress made in the malaria parasite, Plasmodium falciparum. More recently, preliminary two-dimensional gel electrophoresis maps of Toxoplasma gondii and Neospora caninum tachyzoites and Eimeria tenella sporozoites, have been produced, as well as for micronemes in E. tenella. In this review, the status of proteomics in the analysis of global protein expression in apicomplexan parasites will be compared and the challenges associated with these investigations discussed.