Toxoplasma gondii and sex: essential or optional extra?

The evolutionary and biological significance of a female-biased sex ratio within apicomplexan parasites has been the subject of much discussion. It is proposed that the sex allocation theory, as applied to inbreeding populations, can explain the sex ratios observed for this diverse group of parasites. This is based on a mathematical model, which assumes that the majority of microgametes will succeed in fertilizing macrogametes. Is this a realistic assumption? It is possible, for different reasons, that the theory may not to be applicable to either malaria parasites or Toxoplasma gondii.     

Host cell preference and variable transmission strategies in malaria parasites

Malaria and other haemosporin parasites must undergo a round of sexual reproduction in their insect vector in order to produce stages that can be transmitted to vertebrate hosts. Consequently, it is crucial that parasites produce the sex ratio (proportion of male sexual stages) that will maximize the number of fertilization and thus, transmission to new vertebrate hosts. There is some evidence to show that, consistent with evolutionary theory, the sex ratios of malaria parasites are negatively correlated to their inbreeding rate. However, recent theory has shown that when fertilization success is compromised, parasites should respond by increasing their investment in sexual stages or by producing a less female biased ration than predicted by their inbreeding rate alone. Here, we show that two species of rodent malaria, Plasmodium chabaudi and Plasmodium vinckei petteri, adopt different strategies in response to host anaemia, a factor though to compromise transmission success: P. chabaudi increases investment in sexual stages, whereas P. vinckei produces a less female biased sex ratio. We suggest that these different transmission strategies may be due to marked differences in host cell preference.     

Fertility insurance and the sex ratios of malaria and related hemospororin blood parasites

The sex ratio (z*; proportion of gametocytes that are male) of malaria and related hemospororin blood parasites has been predicted to be related to the inbreeding rate (f) by the simple equation z* = (1 - f)/2. Although there is some empirical support for this prediction, there are several cases where the sex ratio is less female biased or more variable than expected. Here, we present a theoretical model that may be able to explain some of these discrepancies. We show that if low gametocyte densities lead to a danger that female gametes may not encounter any male gametes, then natural selection favors a less female-biased sex ratio as a form of 'fertility insurance' to ensure that female gametes are mated. This model can be applied to a number of situations. In particular, (1) empirical data suggest that the number of gametocyes per blood meal can be low enough to favor fertility insurance in some Plasmodium infections in humans and (2) our model predicts facultative shifting toward less-biased sex ratios in response to immune pressure that reduces gametocyte or gamete survival or mobility, consistent with some recent experimental data from Plasmodium species of birds and mice.     

Male gametocyte fecundity and sex ratio of a malaria parasite, Plasmodium mexicanum

Evolutionary theory predicts that the sex ratio of Plasmodium gametocytes will be determined by the number of gametes produced per male gametocyte (male fecundity), parasite clonal diversity and any factor that reduces male gametes' ability to find and combine with female gametes. Despite the importance of male gametocyte fecundity for sex ratio theory as applied to malaria parasites, few data are available on gamete production by male gametocytes. In this study, exflagellating gametes, a measure of male fecundity, were counted for 866 gametocytes from 26 natural infections of the lizard malaria parasite, Plasmodium mexicanum. The maximum male fecundity observed was 8, but most gametocytes produced 2-3 gametes, a value consistent with the typical sex ratio observed for P. mexicanum. Male gametocytes in infections with higher gametocytaemia had lower fecundity. Male fecundity was not correlated with gametocyte size, but differed among infections, suggesting genetic variation for fecundity. Fecundity and sex ratio were correlated (more female gametocytes with higher fecundity) as predicted by theory. Results agree with evolutionary theory, but also suggest a possible tradeoff between production time and fecundity, which could explain the low fecundity of this species, the variation among infections, and the correlation with gametocytaemia.     

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.     

MARveling at parasite invasion

Micronemal proteins (MICs) are key mediators of cytoadherence and invasion for Toxoplasma gondii. Emerging evidence indicates that carbohydrate binding facilitates Toxoplasma entry into host cells. The recently solved Toxoplasma MIC1s (TgMIC1s) structure reveals the presence of novel specialized domains that can discriminate between glycan residues. Comparison with Plasmodium erythrocyte-binding antigen 175 reveals that terminal sialic acid residues might represent a shared but tailored invasion pathway among apicomplexan parasites.     

Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling

Some apicomplexan parasites have evolved distinct protein kinase families to modulate host cell structure and function. Toxoplasma gondii rhoptry protein kinases and pseudokinases are involved in virulence and modulation of host cell signalling. The proteome of Plasmodium falciparum contains a family of putative kinases called FIKKs, some of which are exported to the host red blood cell and might play a role in erythrocyte remodelling. In this review we will discuss kinases known to be critical for host cell invasion, intracellular growth and egress, focusing on (i) calcium-dependent protein kinases and (ii) the secreted kinases that are unique to Toxoplasma (rhoptry protein kinases and pseudokinases) and Plasmodium (FIKKs).     

Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites

Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.     

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.     

Location, location, location: trafficking and function of secreted proteases of Toxoplasma and Plasmodium

The Apicomplexan parasites Toxoplasma gondii and Plasmodium species are obligate intracellular parasites that rely upon unique secretory organelles for invasion and other specialized functions. Data is emerging that proteases are critical for the biogenesis of micronemes and rhoptries, regulated secretory organelles reminiscent of dense core granules and secretory lysosomes of higher eukaryotes. Proteases targeted to the Plasmodium food vacuole, a unique organelle dedicated to hemoglobin degradation, are also critical to parasite survival. Thus study of the targeting and function of the proteases of the Apicomplexa provides a fascinating model system to understand regulated secretion and secretory organelle biogenesis.     

Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.     

Functional genetics in Apicomplexa: potentials and limits

The Apicomplexans are obligate intracellular protozoan parasites and the causative agents of severe diseases in humans and animals. Although complete genome sequences are available since many years and for several parasites, they are replete with putative genes of unassigned function. Forward and reverse genetic approaches are limited only to a few Apicomplexans that can either be propagated in vitro or in a convenient animal model. This review will compare and contrast the most recent strategies developed for the genetic manipulation of Plasmodium falciparum, Plasmodium berghei and Toxoplasma gondii that have taken advantage of the intrinsic features of their respective genomes. Efforts towards the improvement of the transfection efficiencies in malaria parasites, the development of approaches to study essential genes and the elaboration of high-throughput methods for the identification of gene function will be discussed.     

Plasmodium sex determination and transmission to mosquitoes

In order to be transmitted by their mosquito vector, malaria parasites undergo sexual reproduction, which occurs between specialized male and female parasites (gametes) within the blood meal in the mosquito. Nothing was known about how Plasmodium determines the sex of its gametocytes (gamete precursors), which are produced in the vertebrate host. Recently, erythropoietin, the vertebrate hormone controlling erythropoiesis in response to anaemia, was implicated in Plasmodium sex determination in animal models of malaria. This review examines the available information and addresses the relevance of such a sex determining mechanism for Plasmodium falciparum transmission to mosquitoes, with special reference to low gametocytaemias.     

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.     

High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei

This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10(-3)-10(-4). It provides methods for: (i) in vitro cultivation and purification of the schizont stage;(ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two (antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10-15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.     

Density-dependent impact of the human malaria parasite Plasmodium falciparum gametocyte sex ratio on mosquito infection rates

Malaria parasites produce male and female life cycle stages (gametocytes) that must fertilize to achieve successful colonization of the mosquito. Gametocyte sex ratios have been shown to be under strong selection pressure both as an adaptive response to a worsening blood environment for transmission and according to the number of co-infecting clones in the vertebrate. Evidence for an impact of sex ratio on the transmission success of Plasmodium falciparum has, however, been more controversial. Theoretical models of fertilization predict that increasingly male sex ratios will be favoured at low gametocyte densities to ensure fertilization. Here, we analyse in vitro transmission studies of P. falciparum to Anopheles gambiae mosquitoes and test this prediction. We find that there is a discernible effect of sex ratio on transmission but which is dependent upon the gametocyte density. While increasingly male sex ratios do give higher transmission success at low gametocyte densities, they reduce success at higher densities. This therefore provides empirical confirmation that sex ratio has an immediate impact on transmission success and that it is density-dependent. Identifying the signals used by the parasite to alter its sex ratio is essential to determine the success of transmission-blocking vaccines that aim to impede the fertilization process.     

Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites?

Together with micronemes and rhoptries, dense granules are specialised secretory organelles of Apicomplexa parasites. Among Apicomplexa, Plasmodium represents a model of parasites propagated by way of an insect vector, whereas Toxoplasma is a model of food borne protozoa forming cysts. Through comparison of both models, this review summarises data accumulated over recent years on alternative strategies chosen by these parasites to develop within a parasitophorous vacuole and explores the role of dense granules in this process. One of the characteristics of the Plasmodium erythrocyte stages is to export numerous parasite proteins into both the host cell cytoplasm and/or plasma membrane via the vacuole used as a step trafficking compartment. Whether this feature can be correlated to few storage granules and a restricted number of dense granule proteins, is not yet clear. By contrast, the Toxoplasma developing vacuole is decorated by abundantly expressed dense granule proteins and is characterised by a network of membranous nanotubes. Although the exact function of most of these proteins remains currently unknown, recent data suggest that some of these dense granule proteins could be involved in building the intravacuolar membranous network. Conserved expression of the Toxoplasma dense granule proteins throughout most of the parasite stages suggests that they could also be key elements of the cyst formation.     

Transmission stages of Plasmodium: does the parasite use the one same signal, provided both by the host and the vector, for gametocytogenesis and sporozoite maturation?

Among the microorganisms that strictly depend upon other organisms (hosts or vectors) for achieving their life cycle, protozoan and metazoan parasites have been often primarily distinguished through the major pathogenic processes they could induce. A variety of different mechanisms linked to parasitism can indeed systemically (e.g. Plasmodium falciparum) or locally (e.g. Toxoplasma gondii) induce important alterations of tissue homeostasis. But more than obvious pathogenicity, it is the capacity to be transmitted that is essential for parasite survival and there is increasing evidence that certain parasites can achieve their life cycle to the point of transmission in the absence of clinically detectable processes. For this, constitutive microenvironments of the host or vector can be exploited. Moreover, parasites are sometimes able to highjack effectors of the host's immune response towards conditioning the microenvironments which are permissive to differentiation of transmissible developmental stages. Based on a few examples taken from studies on the transmission stages of Leishmania, Toxoplasma and Plasmodium, we have here attempted to formulate a few hypothesis on the biology of the transmission stages of P. falciparum, i.e. on gametocytogenesis and sporozoite maturation. As discussants, we may have been somewhat dwarfed by issues evoked by the organizers of this meeting in the title of the session, i.e. 'Vector-parasite-man interactions'!... In reaction, we may have taken refuge in somewhat over-selective comments, biased by the objects of our personal research....     

Plasmodium motility: actin not actin' like actin

Apicomplexan parasites such as Plasmodium and Toxoplasma display actomyosin-dependent motility in the absence of readily detectable actin polymers. Three recent studies indicate that parasite actin polymers, either harvested from parasites or formed from purified recombinant proteins, are exceptionally short ( approximately 100 nm). We propose that parasite motility could be directed by the transient formation of short actin filament scaffolds. Parasite actin polymers that support transmembrane receptors are pulled, by myosin interaction, backwards along the parasite periphery, resulting in forward movement.     

Role of ATG3 in the parasite Toxoplasma gondii: Autophagy in an early branching eukaryote

Toxoplasma gondii belongs to the phylum Apicomplexa, a diverse group of early branching unicellular eukaryotes related to dinoflagellates and ciliates. Like several other Apicomplexa such as Plasmodium (the causative agent of malaria), T. gondii is a human pathogen responsible for a potentially lethal disease called toxoplasmosis. Most Apicomplexa have complex life cycles, involving intermediate hosts and vectors, which include obligatory intracellular developmental stages. In the case of malaria and toxoplasmosis, it is that replicative process, leading to the ultimate lysis of the host cell, which is causing the symptoms of the disease. For Toxoplasma, the invasive and fast-replicating form of the parasite is called the tachyzoite. While autophagy has been a fast-growing field of research in recent years, not much was known about the relevance of this catabolic process in medically important apicomplexan parasites. Vesicles resembling autophagosomes had been described in drug-treated Plasmodium parasites in the early 1970s and a potential role for autophagy in organelle recycling during differentiation between Plasmodium life stages has also been recently described. Interestingly, recent database searches have identified putative orthologs of the core machinery responsible for the formation of autophagosomes in several protists, including Toxoplasma. In spite of an apparently reduced machinery (only about one-third of the yeast ATG genes appear to be conserved), T. gondii seemed thus able to perform macroautophagy, but the cellular functions of the pathway for this parasite remained to be demonstrated.