Cloning and functional expression of a gene encoding a P1 type nucleoside transporter from Trypanosoma brucei.

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purines de novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of the TbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites. TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.     

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.     

Calcium regulation in protozoan parasites

The calcium ion (Ca(2+)) is used as a major signaling molecule in a diverse range of eukaryotic cells including several human parasitic protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, Leishmania spp, Plasmodium spp, Toxoplasma gondii, Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis. Ca(2+) is critical for invasion of intracellular parasites, and its cytosolic concentration is regulated by the concerted operation of several transporters present in the plasma membrane, endoplasmic reticulum, mitochondria and acidocalcisomes. Recent findings have shed light on the function of these transporters, the roles that they play in cellular metabolism and their potential use for targeting them for new therapies.     

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.     

Nucleoside transporters of parasitic protozoa

Protozoan parasites are incapable of synthesizing purine nucleotides de novo and so must salvage preformed purines from their hosts. This process of purine acquisition is initiated by the translocation of preformed host purines across parasite or host membranes. Here, we report upon the identification and isolation of DNAs encoding parasite nucleoside transporters and the functional characterization of these proteins in various expression systems. These potential approaches provide a powerful approach for a thorough molecular and biochemical dissection of nucleoside transport in protozoan parasites.     

Social parasites

Protozoan parasites cause tremendous human suffering worldwide, but strategies for therapeutic intervention are limited. Recent studies illustrate that the paradigm of microbes as social organisms can be brought to bear on questions about parasite biology, transmission and pathogenesis. This review discusses recent work demonstrating adaptation of social behaviors by parasitic protozoa that cause African sleeping sickness and malaria. The recognition of social behavior and cell-cell communication as a ubiquitous property of bacteria has transformed our view of microbiology, but protozoan parasites have not generally been considered in this context. Works discussed illustrate the potential for concepts of sociomicrobiology to provide insight into parasite biology and should stimulate new approaches for thinking about parasites and parasite-host interactions.     

Latitudinal gradients of parasite species richness in primates

Infectious disease risk is thought to increase in the tropics, but little is known about latitudinal gradients of parasite diversity. We used a comparative data set encompassing 330 parasite species reported from 119 primate hosts to examine latitudinal gradients in the diversity of micro and macroparasites per primate host species. Analyses conducted with and without controlling for host phylogeny showed that parasite species richness increased closer to the equator for protozoan parasites, but not for viruses or helminths. Relative to other major parasite groups, protozoa reported from wild primates were transmitted disproportionately by arthropod vectors. Within the protozoa, our results revealed that vector‐borne parasites showed a highly significant latitudinal gradient in species richness. This higher diversity of vector‐borne protozoa near the tropics could be influenced by a greater abundance or diversity of biting arthropods in the tropics, or by climatic effects on vector behaviour and parasite development. Many vector‐borne diseases, such as leishmaniasis, trypanosomiasis, and malaria pose risks to both humans and wildlife, and nearly one‐third of the protozoan parasites from free‐living primates in our data set have been reported to infect humans. Because the geographical distribution and prevalence of many vector‐borne parasites are expected to increase because of global warming, these results are important for predicting future parasite‐mediated threats to biodiversity and human health.     

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.     

The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

Background  

The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these.     

Six related nucleoside/nucleobase transporters from Trypanosoma brucei exhibit distinct biochemical functions

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.     

Nucleobase transporters (review)

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

Mammalian apoptotic signalling pathways: multiple targets of protozoan parasites to activate or deactivate host cell death

Programmed cell death is an essential mechanism of the host to combat infectious agents and to regulate immunity during infection. Consequently, activation and deactivation of the hosts' cell death pathways by protozoan parasites play critical roles in parasite control, pathogenesis, immune evasion and parasite dissemination within the host. Here, we discuss advances in the understanding of these fascinating host-parasite interactions with special emphasis on how protozoa can modulate the cell death apparatus of its host.     

Nucleobase transporters

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members fromarchea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

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.     

Secretory pathway of trypanosomatid parasites.

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs.     

The occurrence of blood-inhabiting protozoa in captive and free-living penguins

While the susceptibility to infection with blood protozoa, particularly Plasmodium spp. and Leucocytozoon, of a wide range of species of penguins held in captivity is well established, the parasitism of penguins in the wild has been less studied. It appears, however, that free-living penguins from temperate locations exhibit infrequent parasitism, with the exception of Spheniscus demersus in Southern Africa in which infection with Babesia peircei is endemic, while the few available reports suggest that Antarctic and sub-antarctic species exhibit a generalised absence of blood-borne parasites. We have extended these studies by examining 194 thin blood smears for blood protozoa obtained from 4 species of free-living penguins from 5 sites, and have detected no blood parasites in penguins from either Antarctic (Aptenodytes forsteri, Pygoscelis adeliae) or temperate (Eudyptulaminor, Spheniscus humboldti) locations. We discuss the possible reasons for the relative scarcity of blood protozoa in wild penguins, which include biting preferences or absence of suitable vectors, host specificity of the parasites, and long periods spent at sea by most penguin species, resulting in reduced opportunity for maintenance of parasite life-cycles. Accepted: 6 July 1998     

Disease Ecology Meets Ecosystem Science

Growing evidence indicates that parasites—when considered—can play influential roles in ecosystem structure and function, highlighting the need to integrate disease ecology and ecosystem science. To strengthen links between these traditionally disparate fields, we identified mechanisms through which parasites can affect ecosystems and used empirical literature searches to explore how commonly such mechanisms have been documented, the ecosystem properties affected, and the types of ecosystems in which they occur. Our results indicate that ecosystem-disease research has remained consistently rare, comprising less than 2% of disease ecology publications. Existing studies from terrestrial, freshwater, and marine systems, however, demonstrate that parasites can strongly affect (1) biogeochemical cycles of water, carbon, nutrients, and trace elements, (2) fluxes of biomass and energy, and (3) temporal ecosystem dynamics including disturbance, succession, and stability. Mechanistically, most studies have demonstrated density-mediated indirect effects, rather than trait-mediated effects, or direct effects of parasites, although whether this is representative remains unclear. Looking forward, we highlight the importance of applying traits-based approaches to predict when parasites are most likely to exert ecosystem-level effects. Future research should include efforts to extend host–parasite studies across levels of ecological organization, large-scale manipulations to experimentally quantify ecosystem roles of parasites, and the integration of parasites and disease into models of ecosystem functioning.     

Comparative tests of parasite species richness in primates

Some hosts harbor diverse parasite communities, whereas others are relatively parasite free. Many factors have been proposed to account for patterns of parasite species richness, but few studies have investigated competing hypotheses among multiple parasite communities in the same host clade. We used a comparative data set of 941 host-parasite combinations, representing 101 anthropoid primate species and 231 parasite taxa, to test the relative importance of four sets of variables that have been proposed as determinants of parasite community diversity in primates: host body mass and life history, social contact and population density, diet, and habitat diversity. We defined parasites broadly to include not only parasitic helminths and arthropods but also viruses, bacteria, fungi, and protozoa, and we controlled for effects of uneven sampling effort on per-host measures of parasite diversity. In nonphylogenetic tests, body mass was correlated with total parasite diversity and the diversity of helminths and viruses. When phylogeny was taken into account, however, body mass became nonsignificant. Host population density, a key determinant of parasite spread in many epidemiological models, was associated consistently with total parasite species richness and the diversity of helminths, protozoa, and viruses tested separately. Geographic range size and day range length explained significant variation in the diversity of viruses.     

Glycoconjugates in Leishmania infectivity

Leishmaniasis is a major health problem to humans and is caused by one of the world's major pathogens, the Leishmania parasite. These protozoa have the remarkable ability to avoid destruction in hostile environments they encounter throughout their life cycle. That Leishmania parasites have adapted to not only survive, but to proliferate largely is due to the protection conferred by unique glycoconjugates that are either on the parasites' cell surface or secreted. Most of these specialized molecules are members of a family of phosphoglycans while others are a family of glycosylinositol phospholipids. Together they have been implicated in a surprisingly large number of functions for the parasites throughout their life cycle and, therefore, are key players in their pathogenesis. This review summarizes the biological roles of these glycoconjugates and how they are believed to contribute to Leishmania survival in destructive surroundings.