Neospora caninum: a cause of immune-mediated failure of pregnancy?

Resistance to many intracellular protozoan parasites is dependent on T helper cell 1 cytokine responses. This has important repercussions for pregnant females because strong T helper cell 1 cytokine responses are incompatible with successful pregnancy. Thus, there are two possible consequences of infection with protozoans such as Leishmania major, Plasmodium falciparum and Toxoplasma gondii during pregnancy: (1) pregnancy is compromised; or (2) resistance to the parasite is compromised. The apicomplexan Neospora caninum is a parasite renowned for its association with abortion in cattle. Furthermore, a major route of transmission for this parasite is congenital. The evidence for the hypothesis that T helper cell 1 cytokines play a role in these events is reviewed here.     

Cysteine protease inhibitors as chemotherapy for parasitic infections.

Analysis of the evolution, localization and biologic function of papain family cysteine proteases in metazoan and protozoan parasites has provided important and often surprising insights into the biochemistry and cellular function of this diverse enzyme family. Furthermore, the relative lack of redundancy of cysteine proteases in parasites compared to their mammalian hosts makes them attractive targets for the development of new antiparasitic chemotherapy. The treatment of experimental models of parasitic diseases with cysteine protease inhibitors has provided an important 'proof of concept' for the use of cysteine protease inhibitors in vivo. Evidence has now accumulated that cysteine protease inhibitors can selectively arrest replication of a microbial pathogen without untoward toxicity to the host. Furthermore, this can be achieved with reasonable dosing schedules and oral administration of the drug. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. Work on Trypanosoma brucei, the agent of African trypanosomiasis, is preliminary but also promising. Target validation studies have shown that biotinylated or radiolabeled irreversible inhibitors specifically bind to the cysteine protease targets thought to represent the major activity within the parasite. In the case of T. cruzi, the effect of inhibitors appears to be predominantly in blocking protease processing. Transfection studies using variant constructs have supported this model. Finally, the generation of null mutants for the multiple protease genes in Leishmania mexicana has provided the first genetic support for the key role of this enzyme family in parasite virulence. Safety studies in rodents and analysis of uptake of inhibitors by parasites and host cells suggest that the selectivity of inhibitors for the parasite targets may reside in the lack of redundancy of parasite proteases, the higher concentration of host proteases in intracellular compartments, and differential uptake of inhibitors by parasites. Attempts to elicit resistance to cysteine protease inhibitors in parasite cultures suggest that mechanisms of induced resistance are independent of resistance to the traditional antiparasitic agents. This suggests that cysteine protease inhibitors may provide an alternative to traditional therapy in drug-resistant organisms.     

Characterization and role of protozoan parasite proteasomes

The proteasome, a large non-lysosomal multi-subunit protease complex, is ubiquitous in eukaryotic cells. In protozoan parasites, the proteasome is involved in cell differentiation and replication, and could therefore be a promising therapeutic target. This article reviews the present knowledge of proteasomes in protozoan parasites of medical importance such as Giardia, Entamoeba, Leishmania, Trypanosoma, Plasmodium and Toxoplasma spp.     

Analysis of Monensin Sensitivity in Toxoplasma gondii Reveals Autophagy as a Mechanism for Drug Induced Death

Understanding the mechanisms by which anti-parasitic drugs alter the physiology and ultimately kill is an important area of investigation. Development of novel parasitic drugs, as well as the continued utilization of existing drugs in the face of resistant parasite populations, requires such knowledge. Here we show that the anti-coccidial drug monensin kills Toxoplasma gondii by inducing autophagy in the parasites, a novel mechanism of cell death in response to an antimicrobial drug. Monensin treatment results autophagy, as shown by translocation of ATG8 to autophagosomes, as well as causing marked morphological changes in the parasites' mitochondria. Use of the autophagy inhibitor 3-methyladenine blocks autophagy and mitochondrial alterations, and enhances parasite survival, in monensin-exposed parasites, although it does not block other monensin-induced effects on the parasites, such as late S-phase cell cycle arrest. Monensin does not induce autophagy in a parasite strain deficient in the mitochondrial DNA repair enzyme TgMSH-1 an enzyme that mediates monensin-induced late S-phase arrest. TgMSH-1 therefore either mediates cell cycle arrest and autophagy independently, or autophagy occurs downstream of cell cycle arrest in a manner analogous to apoptosis of cells arrested in G(2) of the cell cycle. Overall, our results point to autophagy as a potentially important mode of cell death of protozoan parasites in response to antimicrobial drugs and indicate that disruption of the autophagy pathway could result in drug resistance.     

Modulation of host cell intracellular Ca2+

During the course of evolution, protozoan parasites have developed strategies to subvert the immune response of their host in order to multiply, reproduce and survive. One of these inherited strategies is their capacity to modulate the host cell transductional mechanisms in their favor. Alteration of host cells Ca(2-) homeostasis following interaction and/or invasion by protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Plasmodium falciparum or Entamoeba histolytica has been reported. There is direct evidence that such disturbances are responsible for pathogenesis observed during parasitic infections. This homeostatic imbalance of Ca(2+) in the host cell is an early inducible event whose underlying mechanisms needs further investigation, as discussed here by Martin Olivier.     

Plasmodium falciparum: erythrocytic stages die by autophagic-like cell death under drug pressure

It has been reported that an apoptotic cell death process can occur with protozoans, but no consensus on Plasmodium susceptibility to apoptosis was reached till now. Thus, we evaluated if Plasmodium falciparum blood forms undergo apoptosis after in vitro pressure with chloroquine, S-nitroso-N-acetyl-penicillamine (SNAP) or staurosporine. Inhibition of parasite growth and loss of viability were observed in treated cultures by both light microscopy and flow cytometry. When DNA fragmentation was verified, only a small number of TUNEL-positive parasites was detected in treated cultures and pretreatment of parasite with a general caspase inhibitor was not able to prevent parasite death. Considering the lack of apoptotic characteristics and the observation of parasites with cytoplasmatic vacuolization by electron microscopy, we conclude that P. falciparum parasites under chloroquine, SNAP or staurosporine pressures do not die by apoptosis but by a process similar to autophagy. The autophagic pathway could be explored as an alternative target for the development of new antimalarial drugs.     

Mechanisms of drug resistance in Leishmania

The emergence of drug resistance in protozoan parasites is a major obstacle to their control. Since vaccines are not yet in sight for several of these parasites, there is on urgent need to develop new and better drugs. These antimicrobial agents will possibly be more expensive, and will therefore impose on additional burden in health-care costs and in the planning of public health policies of the developing countries. A better understanding of drug resistance, to try to circumvent or overcome it, and the search for new specific cellular targets of parasites are warranted. The development, in vitro, of drug-resistant parasite cell lines has been instrumental in our understanding of the mechanisms of drug resistance in parasitic protozoans. Marc Ouellette and Barbara Popodopoulou here present on overview of the recent progress on the elucidation of mechanisms of drug resistance in the protozoan parasite Leishmania, selected under laboratory conditions.     

Pentamidine uptake and resistance in pathogenic protozoa: past,present and future

Diamidines, and pentamidine in particular, have a long history as valuable chemotherapeutic agents against infectious disease. Their selectivity is due mostly to selective accumulation by the pathogen, rather than the host cell; and acquired resistance is frequently the result of changes in transmembrane transport of the drug. Here, recent progress in elucidating the mechanisms of diamidine transport in three important protozoan pathogens, Trypanosoma brucei, Leishmania and Plasmodium falciparum, is reviewed, and the implications for drug resistance are discussed.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Comparative protein modeling of spermidine synthase from Plasmodium falciparum: A potential target for anti-malarial drug therapy

Malaria, caused by protozoan parasites of the genus Plasmodium, affects up to 500 million individuals and kills over 1 million people every year. The increasing resistance of the malaria parasites has enforced strategies for finding new drug targets. In recent years, enzymes associated with the polyamine metabolism have attracted attention as drug targets. Cytosolic Plasmodium falciparum spermidine synthase (PfPAPT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite polyamine amine biosynthesis, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper I propose a theoretical low-resolution 3D model for PfPAPT based on crystal structure of the Arabidopsis thaliana, by multiple alignment followed by intensive optimization; validation and dynamic simulations in water. Comparison between the active sites of PfPAPT and human PAPT revealed key differences that could be useful for the design of new selective inhibitors of Plasmodium PAPT.     

Invasion by P. falciparum merozoites suggests a hierarchy of molecular interactions

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.     

Isolation of genes mediating resistance to inhibitors of nucleoside and ergosterol metabolism in Leishmania by overexpression/selection

We tested a general method for the identification of drug resistance loci in the trypanosomatid protozoan parasite Leishmania major. Genomic libraries in a multicopy episomal cosmid vector were transfected into susceptible parasites, and drug selections of these transfectant libraries yielded parasites bearing cosmids mediating resistance. Tests with two antifolates led to the recovery of cosmids encoding DHFR-TS or PTR1, two known resistance genes. Overexpression/selection using the toxic nucleoside tubercidin similarly yielded the TOR (toxic nucleoside resistance) locus, as well as a new locus (TUB2) conferring collateral hypersensitivity to allopurinol. Leishmania synthesize ergosterol rather than cholesterol, making this pathway attractive as a chemotherapeutic target. Overexpression/selection using the sterol synthesis inhibitors terbinafine (TBF, targeting squalene epoxidase) and itraconazole (ITZ, targeting lanosterol C(14)-demethylase) yielded nine new resistance loci. Several conferred resistance to both drugs; several were drug-specific, and two TBF-resistant cosmids induced hypersensitivity to ITZ. One TBF-resistant cosmid encoded squalene synthase (SQS1), which is located upstream of the sites of TBF and ITZ action in the ergosterol biosynthetic pathway. This suggests that resistance to "downstream" inhibitors can be mediated by increased expression of ergosterol biosynthetic intermediates. Our studies establish the feasibility of overexpression/selection in parasites and suggest that many Leishmania drug resistance loci are amenable to identification in this manner.     

Can a single “powerless” mitochondrion in the malaria parasite contribute to parasite programmed cell death in the asexual stages?

The protozoan pathogens responsible for malaria are from the Plasmodium genus, with Plasmodium falciparum and Plasmodium vivax accounting for almost all clinical infections. With recent estimates of mortality exceeding 800,000 annually, malaria continues to take a terrible toll on lives and the early promises of medicine to eradicate the disease have yet to approach realization, in part due to the spread of drug resistant parasites. Recent reports of artemisinin-resistance have prompted renewed efforts to identify novel therapeutic options, and one such pathway being considered for antimalarial exploit is the parasite's programmed cell death (PCD) pathway. In this mini-review, we will discuss the roles of the plasmodium mitochondria in cell death and as a target of antimalarial compounds, taking into account recent data suggesting that PCD pathways involving the mitochondria may be attractive antimalarial targets.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

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....     

Programmed cell death in trypanosomatids: is it an altruistic mechanism for survival of the fittest?

The protozoan parasites Leishmania, Trypanosoma cruzi and Trypanosoma brucei show multiple features consistent with a form of programmed cell death (PCD). Despite some similarities with apoptosis of mammalian cells, PCD in trypanosomatid protozoans appears to be significantly different. In these unicellular organisms, PCD could represent an altruistic mechanism for the selection of cells, from the parasite population, that are fit to be transmitted to the next host. Alternatively, PCD could help in controlling the population of parasites in the host, thereby increasing host survival and favoring parasite transmission, as proposed by Seed and Wenk. Therefore, PCD in trypanosomatid parasites may represent a pathway involved both in survival and propagation of the species.     

Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of the SDPM pathway in the parasite's growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites.     

The role of programmed cell death in Plasmodium-mosquito interactions

Many host-parasite interactions are regulated in part by the programmed cell death of host cells or the parasite. Here we review evidence suggesting that programmed cell death occurs during the early stages of the development of the malaria parasite in its vector. Zygotes and ookinetes of Plasmodium berghei have been shown to die by programmed cell death (apoptosis) in the midgut lumen of the vector Anopheles stephensi, or whilst developing in vitro. Several morphological markers, indicative of apoptosis, are described and evidence for the involvement of a biochemical pathway involving cysteine proteases discussed in relationship to other protozoan parasites. Malaria infection induces apoptosis in the cells of two mosquito tissues, the midgut and the follicular epithelium. Observations on cell death in both these tissues are reviewed including the role of caspases as effector molecules and the rescue of resorbing follicles resulting from inhibition of caspases. Putative signal molecules that might induce parasite and vector apoptosis are suggested including nitric oxide, reactive nitrogen intermediates, oxygen radicals and endocrine balances. Finally, we suggest that programmed cell death may play a critical role in regulation of infection by the parasite and the host, and contribute to the success or not of parasite establishment and host survival.     

Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms

Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.