Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase

The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 microg ml(-1) was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vitro. The gene encoding blasticidin S deaminase (BSD) was therefore incorporated into cassettes for targeting to the cysteine proteinase C locus of L. mexicana (CPC::BSD) and the tubulin locus of T. brucei (tub::RAD51-BSR). Following transfection of mutant parasites that contained other well-established selectable marker genes (HYG, NEO, BLE, PAC and SAT), clones resistant to 10 microg ml(-1) blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results confirm that BSD can be used as a selectable marker gene for targeted chromosomal integration during genetic manipulations of trypanosomatids.     

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.     

RNA interference in Trypanosoma brucei: a high-throughput engine for functional genomics in trypanosomatids?

RNA interference (RNAi) is the technique of choice for down-regulating the gene function of suitable genes in African trypanosomes. A recent report by Subramanian and co-workers describes a high-throughput method for gene function discovery using RNAi in Trypanosoma brucei. The phenotype of most of the Open Reading Frames from chromosome 1 of T. brucei was analysed using a battery test of standard protocols. The authors propose that this technique could be used to mine the full genome of T. brucei and to reveal the core proteomic map of the other two major trypanosomatids, Trypanosoma cruzi and Leishmania major, despite the lack of a homologous mechanism of genetic silencing.     

Genetic engineering of Trypanosoma (Dutonella) vivax and in vitro differentiation under axenic conditions

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.     

VSG 117 gene is conservatively present and early expressed in Trypanosma evansi YNB stock

African trypanosomes, including Trypanosoma brucei and the closely related species Trypanosoma evansi, are flagellated unicellular parasites that proliferate extracellularly in the mammalian bloodstream and tissue spaces. They evade host immune system by periodically switching their variant surface glycoprotein (VSG) coat. Each trypanosome possesses a vast archive of VSGs with distinct sequence identity and different strains contain different archive of VSGs. VSG 117 was reported as a widespread VSG detected in the genomes of all the T. brucei strains. In this study, the presence and expression of VSG 117 gene was observed in T. evansi YNB stock by RT-PCR with VSG-specific primers. We further confirmed that this VSG tends to be expressed in the early stage of T. evansi infections (on day 12-15) by immuno-screening the previously isolated infected blood samples. It is possible that the VSG 117 gene evolved and spread through the African trypanosome population via genetic exchange, before T. evansi lost its ability to infect tsetse fly. Our finding provided an evidence of the close evolutionary relationship between T. evansi and T. brucei, in the terms of VSG genes.     

Programmed cell death in procyclic form Trypanosoma brucei rhodesiense --identification of differentially expressed genes during con A induced death

Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR) to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described). Evidence for an ancestral death machinery, 'proto-apoptosis' in single celled organisms is discussed.     

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.     

An expanded family of proteins with BPI/LBP/PLUNC-like domains in trypanosome parasites: an association with pathogenicity?

Trypanosomatids are protozoan parasites that cause human and animal disease. Trypanosoma brucei telomeric ESs (expression sites) contain genes that are critical for parasite survival in the bloodstream, including the VSG (variant surface glycoprotein) genes, used for antigenic variation, and the SRA (serum-resistance-associated) gene, which confers resistance to lysis by human serum. In addition, ESs contain ESAGs (expression-site-associated genes), whose functions, with few exceptions, have remained elusive. A bioinformatic analysis of the ESAG5 gene of T. brucei showed that it encodes a protein with two BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelium clone)-like domains and that it belongs to a multigene family termed (GR)ESAG5 (gene related to ESAG5). Members of this family are found with various copy number in different members of the Trypanosomatidae family. T. brucei has an expanded repertoire, with multiple ESAG5 copies and at least five GRESAG5 genes. In contrast, the parasites of the genus Leishmania, which are intracellular parasites, have only a single GRESAG5 gene. Although the amino acid sequence identity between the (GR)ESAG5 gene products between species is as low as 15-25%, the BPI/LBP/PLUNC-like domain organization and the length of the proteins are highly conserved, and the proteins are predicted to be membrane-anchored or secreted. Current work focuses on the elucidation of possible roles for this gene family in infection. This is likely to provide novel insights into the evolution of the BPI/LBP/PLUNC-like domains.     

Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme,NADH-dependent fumarate reductase

In all trypanosomatids, including Trypanosoma brucei, glycolysis takes place in peroxisome-like organelles called glycosomes. These are closed compartments wherein the energy and redox (NAD(+)/NADH) balances need to be maintained. We have characterized a T. brucei gene called FRDg encoding a protein 35% identical to Saccharomyces cerevisiae fumarate reductases. Microsequencing of FRDg purified from glycosome preparations, immunofluorescence, and Western blot analyses clearly identified this enzyme as a glycosomal protein that is only expressed in the procyclic form of T. brucei but is present in all the other trypanosomatids studied, i.e. Trypanosoma congolense, Crithidia fasciculata and Leishmania amazonensis. The specific inactivation of FRDg gene expression by RNA interference showed that FRDg is responsible for the NADH-dependent fumarate reductase activity detected in glycosomal fractions and that at least 60% of the succinate secreted by the T. brucei procyclic form (in the presence of d-glucose as the sole carbon source) is produced in the glycosome by FRDg. We conclude that FRDg plays a key role in the energy metabolism by participating in the maintenance of the glycosomal NAD(+)/NADH balance. We have also detected a significant pyruvate kinase activity in the cytosol of the T. brucei procyclic cells that was not observed previously. Consequently, we propose a revised model of glucose metabolism in procyclic trypanosomes that may also be valid for all other trypanosomatids except the T. brucei bloodstream form. Interestingly, H. Gest has hypothesized previously (Gest, H. (1980) FEMS Microbiol. Lett. 7, 73-77) that a soluble NADH-dependent fumarate reductase has been present in primitive organisms and evolved into the present day fumarate reductases, which are quinol-dependent. FRDg may have the characteristics of such an ancestral enzyme and is the only NADH-dependent fumarate reductase characterized to date.     

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX(2)CX(4)HX(4)C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.     

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.     

The population genetics of Trypanosoma brucei and the origin of human infectivity

The African trypanosome, Trypanosoma brucei, is a zoonotic parasite transmitted by tsetse flies. Two of the three subspecies, T. brucei gambiense and T.b. rhodesiense, cause sleeping sickness in humans whereas the third subspecies, T.b. brucei, is not infective to humans. We propose that the key to understanding genetic relationships within this species is the analysis of gene flow to determine the importance of genetic exchange within populations and the relatedness of populations. T.brucei parasites undergo genetic exchange when present in infections of mixed genotypes in tsetse flies in the laboratory, although this is not an obligatory process. Infections of mixed genotype are surprisingly common in field isolates from tsetse flies such that there is opportunity for genetic exchange to occur. Population genetic analyses, taking into account geographical and host species of origin, show that genetic exchange occurs sufficiently frequently in the field to be an important determinant of genetic diversity, except where particular clones have acquired the ability to infect humans. Thus, T. brucei populations have an 'epidemic' genetic structure, but the better-characterized human-infective populations have a 'clonal' structure. Remarkably, the ability to infect humans appears to have arisen on multiple occasions in different geographical locations in sub-Saharan Africa. Our data indicate that the classical subspecies terminology for T. brucei is genetically inappropriate. It is an implicit assumption in most infectious disease biology that when a zoonotic pathogen acquires the capability to infect humans, it does so once and then spreads through the human population from that single-source event. For at least one major pathogen in tropical medicine, T. brucei, this assumption is invalid.