Protozoan genomes: gene identification and annotation

The draft sequence of several complete protozoan genomes is now available and genome projects are ongoing for a number of other species. Different strategies are being implemented to identify and annotate protein coding and RNA genes in these genomes, as well as study their genomic architecture. Since the genomes vary greatly in size, GC-content, nucleotide composition, and degree of repetitiveness, genome structure is often a factor in choosing the methodology utilised for annotation. In addition, the approach taken is dictated, to a greater or lesser extent, by the particular reasons for carrying out genome-wide analyses and the level of funding available for projects. Nevertheless, these projects have provided a plethora of material that will aid in understanding the biology and evolution of these parasites, as well as identifying new targets that can be used to design urgently required drug treatments for the diseases they cause.     

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.     

Genomics and post-genomics in parasitology: genome babble or a real opportunity?

The genome projects represent one of the most important developments in our knowledge of parasites. However, translation of this knowledge into an understanding of parasite biology and then on to drugs, vaccines and other healthcare developments for the diseases will need some élan and clarity of thought by scientists and funding organizations. Only then will the activity associated with post-genomics be turned from what I have termed 'genome babble' to real opportunities in understanding these 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.     

Metabolic reconstruction and analysis for parasite genomes

With the completion of sequencing projects for several parasite genomes, efforts are ongoing to make sense of this mass of information in terms of the gene products encoded and their interactions in the growth, development and survival of parasites. The emerging science of systems biology aims to explain the complex relationship between genotype and phenotype by using network models. One area in which this approach has been particularly successful is in the modeling of metabolism. With an accurate picture of the set of metabolic reactions encoded in a genome, it is now possible to identify enzymes or transporters that might be viable targets for new drugs. Because these predictions greatly depend on the quality and completeness of the genome annotation, there are substantial efforts in the scientific community to increase the numbers of metabolic enzymes identified. In this review, we discuss the opportunities for using metabolic reconstruction and analysis tools in parasitology research, and their applications to protozoan parasites.     

Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling

Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein.     

Genetics and evolution of triatomines: from phylogeny to vector control

Triatomines are hemipteran bugs acting as vectors of the protozoan parasite Trypanosoma cruzi. This parasite causes Chagas disease, one of the major parasitic diseases in the Americas. Studies of triatomine genetics and evolution have been particularly useful in the design of rational vector control strategies, and are reviewed here. The phylogeography of several triatomine species is now slowly emerging, and the struggle to reconcile the phenotypic, phylogenetic, ecological and epidemiological species concepts makes for a very dynamic field. Population genetic studies using different markers indicate a wide range of population structures, depending on the triatomine species, ranging from highly fragmented to mobile, interbreeding populations. Triatomines transmit T. cruzi in the context of complex interactions between the insect vectors, their bacterial symbionts and the parasites; however, an integrated view of the significance of these interactions in triatomine biology, evolution and in disease transmission is still lacking. The development of novel genetic markers, together with the ongoing sequencing of the Rhodnius prolixus genome and more integrative studies, will provide key tools to expanding our understanding of these important insect vectors and allow the design of improved vector control strategies.     

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.     

The role of extracellular vesicles in Plasmodium and other protozoan parasites

Protozoan parasites and other microorganisms use various pathways to communicate within their own populations and to manipulate their outside environments, with the ultimate goal of balancing the rate of growth and transmission. In higher eukaryotes, including humans, circulating extracellular vesicles are increasingly recognized as key mediators of physiological and pathological processes. Recent evidence suggests that protozoan parasites, including those responsible for major human diseases such as malaria and Chagas disease, use similar machinery. Indeed, intracellular and extracellular protozoan parasites secrete extracellular vesicles to promote growth and induce transmission, to evade the host immune system, and to manipulate the microenvironment. In this review we will discuss the general pathways of extracellular vesicle biogenesis and their functions in protozoan infections.     

Interactions of Cryptosporidium parvum, Giardia lamblia, vaccinal poliovirus type 1, and bacteriophages phiX174 and MS2 with a drinking water biofilm and a wastewater biofilm

Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, phiX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination.     

Establishing a liquid-phase IEF in combination with 2-DE for the analysis of Leishmania proteins

The recent completion of genome sequencing projects for Leishmania major and near completion for two other species, L. infantum and L. braziliensis, has provided the needed genomic information for investigating the proteomes of Leishmania parasites. However, the design of effective 2-DE-based proteome mapping for complex protozoan parasites like Leishmania has proven to be severely compromised due to extensive overcrowding of spots especially in the acidic regions, coupled to a relatively low representation of basic proteins. In the present study, we optimized a liquid-phase IEF in combination with 2-DE for L. amazonensis promastigote as a way of reducing protein complexity and enhancing representation for low-abundance proteins on gels. Of 20 pH-based fractions eluted from Rotofor cells, 5 representative fractions selected from acidic, basic or neutral regions of the proteome and with adequate protein concentration were further analyzed by 2-DE using medium-range IPG strips. On this basis, we were able to generate high-resolution 2-DE maps encompassing both the acidic and basic ends of the proteome with enhanced spot representation.     

Assessing the impact of Plasmodium falciparum genome sequencing

With the publication of the complete sequences for chromosomes 2 and 3 and the increasing availability of shotgun sequence covering most of its genome, Plasmodium falciparum biology is entering its post-genomic era. Analysis of the results generated to date has identified higher-order organisation of gene families involved in parasite pathology, provided information regarding the unique biology of this parasite and allowed the identification of potential chemotherapeutic drug targets. Continuing efforts to complete the P. falciparum genome and the availability of sequences from other protozoan parasites will facilitate a broader understanding of their biology, particularly with respect to their pathogenicity.     

The Truman Show for protozoan parasites: A review of in vitro cultivation platforms

Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host–parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases.     

Trypanosomatid comparative genomics: Contributions to the study of parasite biology and different parasitic diseases

In 2005, draft sequences of the genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, also known as the Tri-Tryp genomes, were published. These protozoan parasites are the causative agents of three distinct insect-borne diseases, namely sleeping sickness, Chagas disease and leishmaniasis, all with a worldwide distribution. Despite the large estimated evolutionary distance among them, a conserved core of ~6,200 trypanosomatid genes was found among the Tri-Tryp genomes. Extensive analysis of these genomic sequences has greatly increased our understanding of the biology of these parasites and their host-parasite interactions. In this article, we review the recent advances in the comparative genomics of these three species. This analysis also includes data on additional sequences derived from other trypanosmatid species, as well as recent data on gene expression and functional genomics. In addition to facilitating the identification of key parasite molecules that may provide a better understanding of these complex diseases, genome studies offer a rich source of new information that can be used to define potential new drug targets and vaccine candidates for controlling these parasitic infections.     

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

Leishmania spp., protozoan parasites with a digenetic life cycle, cause a spectrum of diseases in humans. Recently several Leishmania spp. have been sequenced which significantly boosted the number and quality of proteomic studies conducted. Here a historic review will summarize work of the pre-genomic era and then focus on studies after genome information became available. Firstly works comparing the different life cycle stages, in order to identify stage specific proteins, will be discussed. Identifying post-translational modifications by proteomics especially phosphorylation events will be discussed. Further the contribution of proteomics to the understanding of the molecular mechanism of drug resistance and the investigation of immunogenic proteins for the identification of vaccine candidates will be summarized. Approaches of how potentially secreted proteins were identified are discussed. So far 30-35% of the total predicted proteome of Leishmania spp. have been identified. This comprises mainly the abundant proteins, therefore the last section will look into technological approaches on how this coverage may be increased and what the gel-free and gel-based proteomics have to offer will be compared.     

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.