Toxoplasma gondii: the model apicomplexan

Toxoplasma gondii is an obligate intracellular protozoan parasite which is a significant human and veterinary pathogen. Other members of the phylum Apicomplexa are also important pathogens including Plasmodium species (i.e. malaria), Eimeria species, Neospora, Babesia, Theileria and Cryptosporidium. Unlike most of these organisms, T. gondii is readily amenable to genetic manipulation in the laboratory. Cell biology studies are more readily performed in T. gondii due to the high efficiency of transient and stable transfection, the availability of many cell markers, and the relative ease with which the parasite can be studied using advanced microscopic techniques. Thus, for many experimental questions, T. gondii remains the best model system to study the biology of the Apicomplexa. Our understanding of the mechanisms of drug resistance, the biology of the apicoplast, and the process of host cell invasion has been advanced by studies in T. gondii. Heterologous expression of apicomplexan proteins in T. gondii has frequently facilitated further characterisation of proteins that could not be easily studied. Recent studies of Apicomplexa have been complemented by genome sequencing projects that have facilitated discovery of surprising differences in cell biology and metabolism between Apicomplexa. While results in T. gondii will not always be applicable to other Apicomplexa, T. gondii remains an important model system for understanding the biology of apicomplexan parasites.     

Transport of N-acetyl-D-galactosamine in Escherichia coli K92: effect on acetyl-amino sugar metabolism and polysialic acid production

The N-acetyl-D-galactosamine (GalNAc) transport system of Escherichia coli K92 was studied when the bacterium was grown in a chemically defined medium containing GalNAc as a carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.0. Under these conditions, the uptake rate was linear for at least 3 min and the calculated Km for GalNAc was 3 microM. The transport system was strongly inhibited by sodium arsenate (70%), potassium cyanide (62%) and 2,4-dinitrophenol (75%). Analysis of bacterial GalNAc phosphotransferase activity revealed in vitro GalNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that GalNAc uptake depends on a specific phosphotransferase system. Study of activity regulation showed that N-acetylglucosamine and mannosamine specifically inhibit the transport of GalNAc in this bacterium. Analysis of expression revealed that the GalNAc transport system is specifically induced by GalNAc but not by N-acetylglucosamine (GlcNAc) or N-acetylmannosamine (ManNAc), two intimately related sugars. Moreover, full induction of GalNAc transport required the presence of both cAMP and GalNAc. Comparative studies revealed that E. coli K92 has developed a regulation mechanism that specifically induces the appropriate permease based on the presence of each respective phospho-amino sugar (GlcNAc, ManNAc and GalNAc). In this regulation system, GlcNAc is the preferred amino sugar as the carbon source. Finally, when E. coli K92 was grown using GalNAc, capsular polysialic acid production was strongly affected. The presence of intracellular phosphoderivative acetylamino sugars, generated by the action of the phosphotransferase transport system, can be responsible for this effect.     

Purine nucleobase transport in the intraerythrocytic malaria parasite

Hypoxanthine, a nucleobase, serves as the major source of the essential purine group for the intraerythrocytic malaria parasite. In this study we have measured the uptake of hypoxanthine, and that of the related purine nucleobase adenine, by mature blood-stage Plasmodium falciparum parasites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. The uptake of both [3H]hypoxanthine and [3H]adenine was comprised of at least two components; in each case there was a rapid equilibration of the radiolabel between the intra- and extracellular solutions via a low-affinity transport mechanism, and an accumulation of radiolabel (such that the estimated intracellular concentration exceeded the extracellular concentration) via a higher-affinity process. The uptake of [3H]adenine was studied in more detail. The rapid, low-affinity equilibration of [3H]adenine between the intra-and extracellular solution was independent of the energy status of the parasite whereas the higher-affinity accumulation of the radiolabel was ATP-dependent. A kinetic analysis of adenine uptake revealed that the low-affinity (equilibrative) process had a Km of approximately 1.2mM, similar to the value of 0.82 mM estimated here (using the Xenopus laevis oocyte expression system) for the Km for the transport of adenine by PfENT1, a parasite-encoded member of the 'equilibrative nucleoside/nucleobase transporter' family. The results indicate that nucleobases enter the intraerythrocytic parasite via a rapid, equilibrative process that has kinetic characteristics similar to those of PfENT1.     

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C₁₆. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C₁₆ incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts.     

Entobdella soleae--pointers to the future

The biology of the monogenean skin parasite Entobdella soleae and its relationship with its host, the common sole (Solea solea), are probably better known than those of any other monogeneans. The author describes his early involvement with this parasite and the special features of parasite and host that make this relationship so suitable for parasitological studies. Aspects of the biology of E. soleae that have been investigated are briefly mentioned, but most of the paper is concerned with areas of the parasite's biology that remain a challenge to determine. Unresolved areas are as follows: (1) the identity of the factor (or factors) in host skin mucus that stimulates hatching of the parasite's eggs; (2) whether or not the larvae of the parasite are attracted to their host; (3) the nature of factors controlling the contrasting behaviour of adult parasites on the upper and lower surfaces of the host; (4) how nutrients are supplied to the remote regions of the haptor; (5) whether the host has any control via its immune system over parasite invasion success and survival; (6) how the parasite copes with the migratory habits of some sole populations, assuming that such populations are infested with the parasite. The intimacy of this parasite/host relationship is its most remarkable feature, the reflection of which, not surprisingly, is the greatly restricted host range of the parasite. E. soleae has been reported from only three host species, all highly specialised bottom-dwelling members of Solea. It is all the more surprising that relatives of E. soleae, such as Neobenedenia melleni, retain the ability to parasitise an enormous range of hosts. How this versatility is achieved remains to be seen.     

Assembly and export of a Toxoplasma microneme complex in Giardia lamblia

The microneme proteins of Toxoplasma gondii belong to a large family of adhesins of apicomplexan parasites involved in motility and host cell invasion. During secretory transport, soluble micronemes associate with membrane-bound carriers/escorters and become exposed on the parasite surface as complexes with an array of adhesive domains. Previously, we have exploited the intestinal protozoan Giardia lamblia as an expression system to produce correctly folded and unglycosylated monomeric surface proteins of T. gondii. Here, we report assembly and export of a trimeric microneme (MIC1/4/6) adhesin complex from Toxoplasma. Co-expressed, recombinant microneme proteins were used to investigate structural requirements for microneme complex formation. In addition, export of a microneme subunit induced development of novel Golgi-like compartments demonstrating the existence of post endoplasmic reticulum structures involved in constitutive secretion in this 'Golgi-less' cell. Recreation of the trimeric microneme escorter-cargo system in Giardia is a versatile tool to analyse universal requirements for complex assembly, receptor-ligand interactions and Golgi neogenesis in the basal Giardia secretory system.     

Diseases of Nephrops and Metanephrops: a review

Nephrops and Metanephrops are commercially exploited genera within the family Nephropidae (clawed lobsters). Commercial fisheries for each genus exist in the Northern and Southern Hemispheres and utilise trawling or trapping for capture. Despite a relative lack of dedicated disease surveys on lobsters from these fisheries, several important symbionts and pathogens have been described. The most significant known pathogen of Metanephrops (challengeri) is a microsporidian parasite (Myospora metanephrops) which causes destruction of the skeletal and heart muscles of infected lobsters while the most significant known pathogen of Nephrops (norvegicus) is a dinoflagellate parasite assigned to the genus Hematodinium. This parasite has been responsible for an ongoing epidemic in fished populations of N. norvegicus in Northern Europe since at least the early 1980s and since then extensive studies on its life history and pathogenesis have occurred. Despite these research efforts significant gaps exist in our knowledge of the effects of parasites such as Hematodinium on the fished and non-fished portions of Nephrops populations and on the effect of fishery practices on the spread of infection. Furthermore, little is known about the effect of this (and other) pathogens on cohort survivability and the likelihood that early life stages will be effectively recruited to the fishery. This review summarises the available literature on diseases of these two lobster genera and provides an assessment of future research needs in this discipline.     

New clinical and experimental insights into Old World and neotropical ocular toxoplasmosis

Retinal lesions or other ocular manifestations are serious consequences of infection with the protozoan parasite Toxoplasma gondii. Whilst classically considered a consequence of congenital transmission, recent screening studies estimated that 2% of T. gondii seropositive persons in Europe and North America have retinal lesions, most of them persisting unnoticed. The situation is more dramatic in South America, probably due to the predominance of virulent strains. Some of these strains seem to exhibit ocular or neuronal tropism and are responsible for severe ocular lesions. Despite the medical importance, the physiopathological mechanisms have only recently begun to be elucidated. The particular immune-privileged situation in the eye has to be considered. Studies on French patients showed low or undetectable ocular parasite loads, but a clear Th1/Th17 type immune reaction. Suitable mouse models have appeared in the last few years. Using such a model, IL-17A proved to impair parasite control and induce pathology. In contrast, in South American patients, the parasite seems to be much less efficiently controlled through a Th2 type or suppressive immune response that favors parasite replication. Finally, several host genetic markers controlling immune response factors have been associated with ocular involvement of T. gondii infection, mainly in South America.     

Plasmodium falciparum-induced channels

To survive within a red blood cell, the malaria parasite alters dramatically the permeability of the host's plasma membrane (allowing the uptake of essential nutrients and the removal of potentially hazardous metabolites). The pathway(s) responsible for the increased permeability have been proposed as putative chemotherapeutic targets and/or selective routes for antimalarial agents that target the internal parasite. This review covers our current understanding of this parasite-induced phenomenon in Plasmodium falciparum-infected human red blood cells. In particular, recent electrophysiological studies, using the patch-clamp technique, are reviewed.     

Trypanosoma cruzi epimastigotes: regulation of myo-inositol transport by effectors of protein kinases A and C

Inositol is the precursor for most Trypanosoma cruzi surface molecules, including phosphoinositides, glycosylinositolphospholipids and glycosylphosphatidylinositol anchors. As the parasite is an inositol auxotroph, the inositol transport system might be a potential target for new trypanocide drugs, as some of its properties are different from its mammalian counterpart. Here, we investigated the modulation exerted by effectors of PKA and PKC on this transport system to comply with the parasite physiology. Pre-incubation of the cells with either dibutyryl-cyclic AMP (25 microM) or forskolin (30 microM) decreased the myo-inositol uptake by half, this effect being reversed by KT5720 (PKA inhibitor). Conversely, pre-incubation of the cells with PMA (2.8 microg/ml) or serum (5%) had a approximately 50% stimulation in myo-inositol uptake, being this effect reversed by staurosporine (0.5 microM) or sphingosine (10 microM). These results allow us to conclude that the myo-inositol transport system in T. cruzi epimastigotes is inhibited by PKA and stimulated by PKC effectors.     

Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis

Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.     

Gene expression analysis during liver stage development of Plasmodium

The complex life cycle of malaria parasites requires significant changes in gene expression as the parasites move from vector to host and back to the vector. Although recognised as an important vaccine and drug target, the liver stage parasite has remained difficult to study. One of the major impediments in identifying parasite gene expression at the liver stage has remained the large number of uninfected hepatocytes relative to the number of infected hepatocytes in the liver after sporozoite inoculation. This article describes several of the approaches that have been utilised to overcome this difficulty in rodent models of malaria. While significant progress has been made to identify genes that are expressed during liver stage parasite development, a great deal more work remains to be done.     

How does Anopheles gambiae kill malaria parasites?

The malaria parasite's lifecycle in the mosquito vector Anopheles gambiae involves several translocations within and between tissues during which large parasite losses have been documented. Interestingly, during the critical transition stages of midgut invasion and relocation of sporozoites from the oocysts to the salivary glands the mosquito innate immune system is activated. These defense reactions could, at least partially, be responsible for the parasite killing in the mosquito. This important question is now being approached by the dissection of the mosquito innate immune system as well as genetic and genomic studies.     

Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives

Chromatin structure plays a vital role in epigenetic regulation of protozoan parasite gene expression. Epigenetic gene regulation impacts upon parasite virulence, differentiation and cell-cycle control. Recent work in many laboratories has elucidated the functions of proteins that regulate parasite gene expression by chemical modification of constituent nucleosomes. A major focus of investigation has been the characterization of post-translational modifications (PTMs) of histones and the identification of the enzymes responsible. Despite conserved features and specificity common to all eukaryotes, parasite enzymes involved in chromatin modification have unique functions that regulate unique aspects of parasite biology.     

Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source

Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.