Sitting in the driver's seat: Manipulation of mammalian cell Rab GTPase functions by apicomplexan parasites

Many intracellular microbial pathogens subvert, disrupt or otherwise modulate host membrane trafficking pathways to establish a successful infection. Among them, bacteria that are trapped in a phagosome during mammalian cell invasion, disengage the programmed degradation process by altering the identity of their replicative niche through the exclusion or recruitment of specific Rab GTPases to their vacuole. Many viruses co-opt essential cellular trafficking pathways to perform key steps in their lifecycles. Among protozoan parasites, Apicomplexa are obligate intracellular microbes that invade mammalian cells by creating a unique, nonfusogenic membrane-bound compartment that protects the parasites straightaway from lysosomal degradation. Recent compelling evidence demonstrates that apicomplexan parasites are master manipulators of mammalian Rab GTPase proteins, and benefit or antagonise Rab functions for development within host cells. This review covers the exploitation of mammalian Rab proteins and vesicles by Apicomplexa, focusing on Toxoplasma, Neospora, Plasmodium and Theileria parasites.     

Lipid metabolism in mucous-dwelling amitochondriate protozoa

Entamoeba, Giardia, and trichomonads are the prominent members of a group known as 'mucosal parasites'. While Entamoeba and Giardia trophozoites colonise the small intestine, trichomonads inhabit the genitourinary tracts of humans and animals. These protozoa lack mitochondria, well-developed Golgi complexes, and other organelles typical of higher eukaryotes. Nonetheless, they have developed unique metabolic pathways that allow them to survive and multiply in the small intestine and reproductive tracts by scavenging nutrients from the host. Various investigators have shown that these protozoa are unable to synthesise the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources. Therefore, questions of how they transport and utilise exogenous lipids for metabolic purposes are extremely important. There is evidence suggesting that these parasites can take up the lipids and cholesterol they need from lipoprotein particles present in the host and/or in the growth medium. Studies also support the idea that individual lipid and fatty acid molecules can be transported without the help of lipoproteins. Exogenous phospholipids have been shown to undergo fatty acid remodelling (by deacylation/reacylation reactions), which allows these protozoa to alter lipids, bypassing the synthesis of entirely new phospholipid molecules. In addition, many of these amitochondriates are, however, capable of elongating/desaturating long-chain fatty acids, and assembling novel glycophospholipid molecules. In this review, progress in various aspects of lipid research on these organisms is discussed. Attempts are also made to identify steps of lipid metabolic pathways that can be used to develop chemotherapeutic agents against these and other mucosal parasites.     

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.     

Biogenesis and maintenance of the apicoplast in model apicomplexan parasites

The apicoplast is a non-photosynthetic relict plastid of Apicomplexa that evolved from a secondary symbiotic system. During its evolution, most of the genes derived from its alga ancestor were lost. Only genes involved in several valuable metabolic pathways, such as the synthesis of isoprenoid precursors, heme, and fatty acids, have been transferred to the host genome and retained to help these parasites adapt to a complex life cycle and various living environments. The biological function of an apicoplast is essential for most apicomplexan parasites. Considering their potential as drug targets, the metabolic functions of this symbiotic organelle have been intensively investigated through computational and biological means. Moreover, we know that not only organellar metabolic functions are linked with other organelles, but also their biogenesis processes have developed and evolved to tailor their biological functions and proper inheritance. Several distinct features have been found in the biogenesis process of apicoplasts. For example, the apicoplast borrows a dynamin-related protein (DrpA) from its host to implement organelle division. The autophagy system has also been repurposed for linking the apicoplast and centrosome during replication and the division process. However, many vital questions remain to be answered about how these parasites maintain and properly inherit this symbiotic organelle. Here we review our current knowledge about its biogenesis process and discuss several critical questions remaining to be answered in this field.     

Directing traffic: Chaperone‐mediated protein transport in malaria parasites

The ability of eukaryotic parasites from the phylum Apicomplexa to cause devastating diseases is predicated upon their ability to maintain faithful and precise protein trafficking mechanisms. Their parasitic life cycle depends on the trafficking of effector proteins to the infected host cell, transport of proteins to several critical organelles required for survival, as well as transport of parasite and host proteins to the digestive organelles to generate the building blocks for parasite growth. Several recent studies have shed light on the molecular mechanisms parasites utilise to transform the infected host cells, transport proteins to essential metabolic organelles and for biogenesis of organelles required for continuation of their life cycle. Here, we review key pathways of protein transport originating and branching from the endoplasmic reticulum, focusing on the essential roles of chaperones in these processes. Further, we highlight key gaps in our knowledge that prevents us from building a holistic view of protein trafficking in these deadly human pathogens.     

Migration of Apicomplexa across biological barriers: the Toxoplasma and Plasmodium rides

The invasive stages of Apicomplexa parasites, called zoites, have been largely studied in in vitro systems, with a special emphasis on their unique gliding and host cell invasive capacities. In contrast, the means by which these parasites reach their destination in their hosts are still poorly understood. We summarize here our current understanding of the cellular basis of in vivo parasitism by two well-studied Apicomplexa zoites, the Toxoplasma tachyzoite and the Plasmodium sporozoite. Despite being close relatives, these two zoites use different strategies to reach their goal and establish infection.     

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.     

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.     

Invasion of red blood cells by malaria parasites

The malaria parasite is the most important member of the Apicomplexa, a large and highly successful phylum of intracellular parasites. Invasion of host cells allows apicomplexan parasites access to a rich source of nutrients in a niche that is largely protected from host defenses. All Apicomplexa adopt a common mode of host-cell entry, but individual species incorporate unique features and utilize a specific set of ligand-receptor interactions. These adhesins ultimately connect to a parasite actin-based motor, which provides the power for invasion. While some Apicomplexa can invade many different host cells, the disease-associated blood-stage form of the malaria parasite is restricted to erythrocytes.     

Versatility in the acquisition of energy and carbon sources by the Apicomplexa

Members of the phylum Apicomplexa are motile and rapidly dividing intracellular parasites, able to occupy a large spectrum of niches by infecting diverse hosts and invading various cell types. As obligate intracellular parasites, most apicomplexans only survive for a short period extracellularly, and, during this time, have a high energy demand to power gliding motility and invasion into new host cells. Similarly, these fast‐replicating intracellular parasites are critically dependent on host‐cell nutrients as energy and carbon sources, noticeably for the extensive membrane biogenesis imposed during growth and division. To access host‐cell metabolites, the apicomplexans Toxoplasma gondii and Plasmodium falciparum have evolved strategies that exquisitely reflect adaptation to their respective niches. In the present review, we summarize and compare some recent findings regarding the energetic metabolism and carbon sources used by these two genetically tractable apicomplexans during host‐cell invasion and intracellular growth and replication.     

Toxoplasma gondii Relies on Both Host and Parasite Isoprenoids and Can Be Rendered Sensitive to Atorvastatin

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.     

Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii

Host-cell invasion by apicomplexan parasites is extremely rapid and relies on a sequence of events that are tightly controlled in time and space. In most Apicomplexa, the gliding motility and host-cell invasion are tightly coupled to the release of microneme proteins at the apical tip of the parasites and their redistribution toward the posterior pole. This movement is dependent on an intact parasite actomyosin system. Micronemes are involved in the trafficking and storage of ligands (MICs) for host-cell receptors that are not only structurally related but also functionally conserved among the Apicomplexa. In Toxoplasma gondii, the repertoire of membrane-spanning microneme proteins includes adhesins such as TgMIC2 and escorters such as TgMIC6. The latter forms a complex with the soluble adhesins, TgMIC1 and TgMIC4 and assures their proper sorting to the mironemes. Escorters are also anticipated to bridge host-cell receptors to the parasite membrane during invasion. Most TgMICs are proteolytically cleaved either during their transport along the secretory pathway and/or after exocytosis. The biological significance of these processing events is largely unknown. One of these processing events targets a conserved motif close to the membrane-spanning domain causing the release of the processed form of the micronemes from the parasite surface. The cleavages occurring after release might contribute to the disassembly of the complexes and thus to fission between the parasitophorous vacuole and the host plasma membrane at the end of the invasion process. Gliding motility and host-cell penetration involve the redistribution of the micronemes toward the posterior pole of the parasites. This capping process involves actin polymerisation, myosin adenosine triphosphatase activation and the establishment of a connection between the MICs-receptor complexes and the actomyosin system of the parasite. The most carboxy-terminal end of the MICs cytoplasmic tails is implicated in this process, but the precise nature of the connection with the actomyosin system remains to be elucidated.     

Gliding motility and cell invasion by Apicomplexa: insights from the Plasmodium sporozoite

Apicomplexa constitute one of the largest phyla of protozoa. Most Apicomplexa, including those pathogenic to humans, are obligate intracellular parasites. Their extracellular forms, which are highly polarized and elongated cells, share two unique abilities: they glide on solid substrates without changing their shape and reach an intracellular compartment without active participation from the host cell. There is now ample ultrastructural evidence that these processes result from the backward movement of extracellular interactions along the anteroposterior axis of the parasite. Recent work in several Apicomplexa, including genetic studies in the Plasmodium sporozoite, has provided molecular support for this 'capping' model. It appears that the same machinery drives both gliding motility and host cell invasion. The cytoplasmic motor, a transmembrane bridge and surface ligands essential for cell invasion are conserved among the main apicomplexan pathogens.     

Climate variation influences host specificity in avian malaria parasites

Parasites with low host specificity (e.g. infecting a large diversity of host species) are of special interest in disease ecology, as they are likely more capable of circumventing ecological or evolutionary barriers to infect new hosts than are specialist parasites. Yet for many parasites, host specificity is not fixed and can vary in response to environmental conditions. Using data on host associations for avian malaria parasites (Apicomplexa: Haemosporida), we develop a hierarchical model that quantifies this environmental dependency by partitioning host specificity variation into region‐ and parasite‐level effects. Parasites were generally phylogenetic host specialists, infecting phylogenetically clustered subsets of available avian hosts. However, the magnitude of this specialisation varied biogeographically, with parasites exhibiting higher host specificity in regions with more pronounced rainfall seasonality and wetter dry seasons. Recognising the environmental dependency of parasite specialisation can provide useful leverage for improving predictions of infection risk in response to global climate change.     

LISP1 is important for the egress of Plasmodium berghei parasites from liver cells

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so-called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium , the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 ( li ver- s pecific p rotein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1-deficient liver-stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.     

Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences

Investigating the evolutionary relationships of the major groups of Apicomplexa remains an important area of study. Morphological features and host-parasite relationships continue to be important in the systematics of the adeleorinid coccidia (suborder Adeleorina), but the systematics of these parasites have not been well-supported or have been constrained by data that were lacking or difficult to interpret. Previous phylogenetic studies of the Adeleorina have been based on morphological and developmental characters of several well-described species or based on nuclear 18S ribosomal DNA (rDNA) sequences from taxa of limited taxonomic diversity. Twelve new 18S rDNA sequences from adeleorinid coccidia were combined with published sequences to study the molecular phylogeny of taxa within the Adeleorina and to investigate the evolutionary relationships of adeleorinid parasites within the Apicomplexa. Three phylogenetic methods supported strongly that the suborder Adeleorina formed a monophyletic clade within the Apicomplexa. Most widely recognized families within the Adeleorina were hypothesized to be monophyletic in all analyses, although the single Hemolivia species included in the analyses was the sister taxon to a Hepatozoon sp. within a larger clade that contained all other Hepatozoon spp. making the family Hepatozoidae paraphyletic. There was an apparent relationship between the various clades generated by the analyses and the definitive (invertebrate) host parasitized and, to lesser extent, the type of intermediate (vertebrate) host exploited by the adeleorinid parasites. We conclude that additional taxon sampling and use of other genetic markers apart from 18S rDNA will be required to better resolve relationships among these parasites.     

Mitosis in the human malaria parasite Plasmodium falciparum

Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes.     

Secretion of proteins into host cells by Apicomplexan parasites

The phylum Apicomplexa consists of a diverse group of obligate, intracellular parasites. The distinct evolutionary pressures on these protozoans as they have adapted to their respective niches have resulted in a variety of methods that they use to interact with and modify their hosts. One of these is the secretion and trafficking of parasite proteins into the host cell. We review this process for Theileria , Toxoplasma and Plasmodium . We also present what is known about the mechanisms by which parasite proteins are exported into the host cell, as well as information on their known and putative functions once they have reached their final destination.     

Individual and population effects of eugregarine, Gregarina niphandrodes (Eugregarinida: Gregarinidae), on Tenebrio molitor (Coleoptera: Tenebrionidae)

Gregarines are single-celled parasites in the phylum Apicomplexa that infect invertebrates. They are highly abundant on three levels: among a large diversity of invertebrates, in the proportion of population of organisms they infect, and within individually infected organisms. Because of their remarkable prevalence, we hypothesize that they play an important role in support of their hosts. However, studies done to date on the impact of gregarines on their host are conflicting. Therefore, we studied the impact of gregarines on their host using a model Gregarina niphandrodes infection in Tenebrio molitor. The impact of infection was measured by comparing beetles with no or low infection to those with artificially induced high infection. The numbers of individuals in each of the three easily visible developmental stages of the T. molitor (larva, pupa, and adult) were censused weekly. From these observations, fertilities and probabilities of survival with transition between stages were estimated. These estimated vital rates were used to construct a stage-classified projection matrix model. We also measured the longevity of individual beetles with low and high infection that were grown in isolation. The results indicate that there is no significant difference in the population dynamics of beetles with low and high infection. However, the longevity was significantly different between beetles with low infection than the deliberately highly infected group.