Protein kinases as targets for antimalarial intervention: Kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes

The surge of interest in protein kinases as targets for chemotherapeutic intervention in a number of diseases such as cancer and neurodegenerative disorders has stimulated research aimed at determining whether enzymes of this class might also be considered as targets in the context of diseases caused by parasitic protists. Here, we present an overview of recent developments in this field, concentrating (i) on the benefits gained from the availability of genomic databases for a number of parasitic protozoa, (ii) on the emerging field of structure-aided design of inhibitors targeting protein kinases of parasitic protists, (iii) on the concept known as transmission-blockade, whereby kinases implicated in the development of the parasite in their arthropod vector might be targeted to interfere with disease transmission, and (iv) on the possibility of controlling parasitic diseases through the inhibition of host cell protein kinases that are required for the establishment of infection by the parasites.     

Molecular biology techniques in parasite ecology

Molecular techniques are increasingly being used to study the ecology of a variety of organisms. These techniques represent important tools for the study of the systematics, population genetics, biogeography and ecology of parasites. Here, we review the techniques that have been employed to study the ecology and systematics of parasites (including bacteria and viruses). Particular emphasis is placed on the techniques of isoenzyme electrophoresis, in situ hybridisation and nucleic acid amplification to characterise parasite/microbial communities. The application of these techniques will be exemplified using ticks, bacterial endosymbionts and parasitic protozoa.     

New perspectives on protozoa

The interest of the British research community in protozoa has in the past emphasized the parasitic organisms-especially those causing diseases of economic importance in Africa. The last 20 years have witnessed dramatic changes in protozoology, and the purpose of this meeting was to indicate the nature and significance of these changes.
Three areas were discussed. The first was our present understanding of protozoan diversity and the evolution of protozoa (by David Patterson), the second (by Bland Finlay) presented some novel and ecological important symbiotic relationships between protozoa and other microorganisms), and finally Graham Coombs discussed the problems still being posed by parasitic protozoa and the application of new technologies to those problems.     

Clan CD cysteine peptidases of parasitic protozoa

Parasitic protozoa contain an abundance of cysteine peptidases that are crucial for a range of important biological processes. The most studied cysteine peptidases of parasitic protozoa belong to the group of papain-like enzymes known as clan CA. However, several more recently identified cysteine peptidases differ fundamentally from the clan CA enzymes and have been included together in clan CD. Enzymes of this clan have now been identified in parasitic protozoa. Many have important roles and also differ significantly from known mammalian counterparts. The main characteristics of clan CD enzymes are outlined here, in particular glycosylphosphatidylinositol (GPI):protein transamidase, metacaspase and separase, and their differences from the clan CA enzymes are described.     

Protein kinases as targets for anti-parasitic chemotherapy

Parasitic protozoa infecting humans have a staggering impact on public health, especially in the developing world. Furthermore, several protozoan species are major pathogens of domestic animals and have a considerable impact on food production. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in protein kinases inhibitors as potential drugs against a variety of diseases, the possibility that protein kinases may represent targets for novel anti-parasitic agents is being explored. Research into parasite protein kinases has benefited greatly from genome and EST sequencing projects, with the genomes of a few species fully sequenced (notably that of the human malaria parasite Plasmodium falciparum) and several more under way. The overall picture that emerged from research in this area shows that the phylogenetic isolation of parasitic protozoa is reflected by atypical structural and functional properties of many of their protein kinase homologues. Likewise, evidence is emerging, which suggests that the organisation of some otherwise well-conserved signal transduction pathways is divergent in some parasitic species. The differences between protein kinases of a parasite and their homologues in its host cell suggest that specific inhibition of the former can be achieved. The development of anti-parasitic drugs based on protein kinase inhibition is being pursued following two avenues: one consists of screening chemical libraries on recombinant enzymes; several protein kinases from parasitic protozoa are now available for this approach. The second approach relies on the identification of the molecular targets of kinase inhibitors which display anti-parasitic properties. This has led to promising developments in a few instances, in particular regarding PKG as a drug target against Eimeria and Toxoplasma, and purvalanol B, a purine-based CDK inhibitor which appears to affect unexpected targets in several protozoan parasites. The recent resolution of the structure of a Plasmodium protein kinase complexed with small inhibitory molecules opens the way to a rational approach towards the design of anti-parasitic drugs based on kinase inhibition.     

Reproductive clonality in protozoan pathogens—truth or artefact?

The debate around the frequency and importance of genetic exchange in parasitic protozoa is now several decades old. Recently, fresh assertions have been made that predominant clonal evolution explains the population structures of several key protozoan pathogens. Here, we present an alternative perspective. On the assumption that much apparent clonality may be an artefact of inadequate sampling and study design, we review current research to define why sex might be so difficult to detect in protozoan parasite populations. In doing so, we contrast laboratory models of genetic exchange in parasitic protozoa with natural patterns of genetic diversity and consider the fitness advantage of sex at different evolutionary scales. We discuss approaches to improve the accuracy of efforts to characterize genetic exchange in the field. We also examine the implications of the first population genomic studies for the debate around sex and clonality in parasitic protozoa and discuss caveats for the future.     

Towards a population genetics of microorganisms: The clonal theory of parasitic protozoa

Over the past 15 years, molecular investigations, including the study of isozymes and DNA markers, have provided much information on the genetic variation, population structure, breeding system and other population characteristics of parasitic protozoa. For some parasitic protozoa, but not for others, the evidence indicates that their reproduction is prevailingly clonal. In this article, Michel Tibayrenc and Francisco Ayala propose that the issue of whether the predominant mode of reproduction of a given micro-organism is clonal or sexual can only be settled by population genetics information, and they summarize evidence favoring a clonal population structure for a number of parasitic protozoa.     

Amphibian antimicrobial peptides and Protozoa: Lessons from parasites

Antimicrobial peptides (AMPs) from amphibians and other eukaryotes recognize pathogenicity patterns mostly related to differences in membrane composition between the host and a variety of bacterial, fungal and protozoan pathogens. Compared to the other two groups, protozoa are fairly neglected targets in antimicrobial chemotherapy, despite their role as causative agents for scourges such as malaria, amoebiasis, Chagas' disease or leishmaniasis. Herein we review the scarce but growing body of knowledge addressing the use of amphibian AMPs on parasitic protozoa, the adaptations of the protozoan to AMP pressure and their impact on AMP efficacy and specificity, and the current and foreseeable strategies for developing AMPs into practical therapeutic alternatives against parasitic disease.     

Autophagy in Apicomplexa: a life sustaining death mechanism?

Programmed cell death (PCD) pathways remain understudied in parasitic protozoa in spite of the fact that they provide potential targets for the development of new therapy. The best understood PCD pathway in higher eukaryotes is apoptosis although emerging evidence also points to autophagy as a mediator of death in certain physiological contexts. Bioinformatic analyses coupled with biochemical and cell biological studies suggest that parasitic protozoa possess the capacity for PCD including a primordial form of apoptosis. Recent work in Toxoplasma and emerging data from Plasmodium suggest that autophagy-related processes may serve as an additional death promoting pathway in Apicomplexa. Detailed mechanistic studies into the molecular basis for PCD in parasitic protozoa represent a fertile area for investigation and drug development.     

Parasite cryopreservation by vitrification

Parasitic protozoa and helminths and parasitic/vector insects each have distinct requirements for cryopreservation. Most parasitic protozoa respond to cryopreservation stresses similarly to other single cell suspensions, but few species are currently routinely cryopreserved by protocols specifically designed for vitrification. With slow equilibrium cooling, some protozoa osmotically dehydrated by solutes concentrated in the residual unfrozen fraction will survive by vitrifying. Several species of helminths, together with insect embryos cannot be cryopreserved by slow cooling protocols and have an absolute requirement for vitrification. Studies incorporating slow cooling and stepped cooling of both protozoa and helminths, particularly the intraerythrocytic stages of malaria and the schistosomula larvae of Schistosoma mansoni have aided in the design of vitrification protocols for parasites. For helminths, the most widely used cryopreservation protocol, originally successful for cryopreserving S. mansoni schistosomula, consists of the addition of ethanediol in two steps, followed by rapid cooling (approximately 5100 degrees C min(-1)) to -196 degrees C. This technique exploits the temperature-dependent differential in permeability of the cryoprotectant additive (CPA) to first permeate into the organism at 37 degrees C followed by a dehydration-mediated internal CPA increase in concentration resulting from incubation in a second higher CPA concentration at 0 degree C. Samples are rapidly warmed/diluted (approximately 14,000 degrees C min(-1)) to recover the organisms from liquid nitrogen storage. Variations on this technique have also been successful in cryopreserving the larvae and adult worms of filariae, muscle stage larvae of Trichinella spp., the infective stages of gastro-intestinal nematode parasites and insect embryos. Other protocols where the dehydration step precedes CPA addition have been used to cryopreserve entomogenous nematode larvae by vitrification. Techniques that utilize high concentrations of CPA cocktails and slower cooling, developed for the vitrification of mammalian embryos, have been applied to the cryopreservation of parasitic protozoa, but with limited success to date. Where cryopreservation by classical slow cooling methods is possible, vitrification has enhanced the levels of survival obtained. And vitrification has enabled the successful cryopreservation of those parasitic species not able to be cryopreserved by traditional methods. Since a limited number of parasitic organisms has been cryopreserved using vitrification protocols, there is considerable scope for further improvement in the cryopreservation techniques used for many parasitic species.     

Stress response pathways in protozoan parasites

Diseases caused by protozoan parasites have a dramatic impact on world health. Emerging drug resistance and a general lack of experimental understanding has created a void in the medicine cabinet used to treat these widespread infections. A novel therapeutic idea that is receiving more attention is centred on targeting the microbe's response to the multitude of environmental stresses it encounters. Protozoan pathogens have complex life cycles, often having to transition from one host to another, or survive in a cyst form in the environment until a new host arrives. The need to respond to environmental cues and stress, and endure in less than optimal conditions, is paramount to their viability and successful progression through their life cycle. This review summarizes the research on parasitic stress responses for Apicomplexa, kinetoplastids and anaerobic protozoa, with an eye towards how these processes may be exploited therapeutically.     

Genetics and biochemistry of Leishmania membrane transporters

The ability to clone and functionally express genes encoding membrane transporters in Leishmania and related parasitic protozoa has illuminated the processes whereby these parasites acquire nutrients from their hosts. It is now possible to probe the physiological functions of these permeases and investigate their role in drug delivery and resistance.     

钙酸体的结构特征和功能

钙酸体是一类存在于原生寄生虫中的特殊的细胞器。它的结构及元素成分特殊,在寄生虫的生长、生存及毒性方面具有重要作用。本文主要介绍了钙酸体的发现和命名、结构特征及其在寄生虫中的功能。     

Cell death and human intestinal protozoa: a brief overview

Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.     

Targeting polyamines of parasitic protozoa in chemotherapy

All parasitic protozoa contain polyamines and in recent years they, and their associated enzymes, have attracted attention as drug targets because they might reveal novel antiparasite therapies. How justified is this approach to drug discovery? In this review, Sylke Müller, Graham Coombs and Rolf Walter summarize the current status of research into drugs that exploit polyamine metabolism of trypanosomatid and malaria parasites, and propose priorities for research into such drugs. This review was inspired by an Expert Meeting entitled 'Polyamine Metabolism of Parasitic Protozoa as a Drug Target'.     

Symbionts and organelles in ancrobic protozoa and fungi

The discovery of methanogenic bacteria as endosymbionts of free-living anaerobic protozoa opened new fields of research in microbial ecology, cell physiology and molecular biology. Recent information from 16S rRNA sequence studies has shown in three cases that endosymbiotic methanogenic bacteria differ from free-living species. Frequently, endosymbiotic methanogens are localized in anaerobic protozoa near hydrogenosomes - organelles that produce H2, C02 and acetate, all of which are substrates for methanogenesis. Hydrogenosomes are also present in anaerobic fungi. The current view is that the organelles are endosymbllont-derived and were probably acquired on several distinct occasions during evolution.     

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.     

Multidrug resistance and ABC transporters in parasitic protozoa

Drug resistance is an important problem in parasitic protozoa. We review here the role of ABC transporters in drug resistance in parasites. We have concentrated on gene and gene products for which there is a strong evidence for their role in resistance.     

Frontiers in research on parasitic protozoa

Frontiers in research on parasitic protozoa was the theme of the Autumn Symposium of the British Section of the Society of Protozoologists, held 2 September 2002, in London, UK.     

Crystalloid body, refractile body and virus-like particles in Apicomplexa: what is in there?

The phylum of Apicomplexa comprises parasitic protozoa that share distinctive features such as the apical complex, the apicoplast, specialized cytoskeletal components and secretory organelles. Other unique cytoplasmic inclusions sharing similar features have been described in some representatives of Apicomplexa, although under different denominations. These are the crystalloid body, present for example in Cryptosporidium, Plasmodium and Cystoisospora; the refractile body in Eimeria and Lankesterella; and virus-like particles, also present in Eimeria and Cryptosporidium. Yet, the specific role of these cytoplasmic inclusions in the cell cycle of these protozoa is still unknown. Here, we discuss their morphology, possible inter-relatedness and speculate upon their function to bring these organelles back to the attention of the scientific community and promote new interest towards original research on these elusive structures.