Trypanosoma brucei s.l.: characterisation of stocks from Central Africa by PCR analysis of mobile genetic elements

To better understand the epidemiology of sleeping sickness in the Central African sub-region, notably the heterogeneity of Human African Trypanosomiasis (HAT) foci, the mobile genetic element PCR (MGE-PCR) technique was used to genotype Trypanosoma brucei s.l. (T. brucei s.l.) isolates from this sub-region. Using a single primer REV B, which detects positional variation of the mobile genetic element RIME, via amplification of flanking regions, MGE-PCR revealed a micro genetic variability between Trypanosoma brucei gambiense (T. b. gambiense) isolates from Central Africa. The technique also revealed the presence of several T. b. gambiense genotypes and allowed the identification of minor and major ubiquitous genotypes in HAT foci. The presence of several T. b. gambiense genotypes in HAT foci may explain the persistence and the resurgence phenomena of the disease and also the epidemic and the endemic status of some Central African sleeping sickness foci. The MGE-PCR technique represents a simple, rapid, and specific method to differentiate Central African T. brucei s.l. isolates.     

Use of mobile genetic elements as tools for molecular epidemiology

Trypanosomiasis is a complex zoonotic disease where human-infective and non-human-infective strains of Trypanosoma brucei interact in the same transmission cycles. Differentiating these strains is paramount to understanding disease epidemiology. Restriction fragment length polymorphism analysis of repetitive DNA has provided such a method for distinguishing human and non-human isolates. Unfortunately, this approach requires large amounts of material and a more rapid approach is required. We have developed a novel technique, mobile genetic element-PCR, for assaying for positional variation of the mobile genetic element, RIME. The trypanosome genome contains up to 400 copies of RIME. Using this approach we have observed considerable variation between strains of T. brucei. Such a technique may offer potential as a method for differentiating non-human- and human-infective trypanosomes and shows promise as a rapid sensitive tool for investigating the epidemiology of sleeping sickness.     

Genetic analysis of the human infective trypanosome <Emphasis Type="Italic">Trypanosoma brucei gambiense</Emphasis>: chromosomal segregation,crossing over,and the construction of a genetic map

Background  

Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense.     

Evidence for Multiple Origins of Human Infectivity in Trypanosoma brucei Revealed by Minisatellite Variant Repeat Mapping

In recent years a wide variety of biochemical and molecular typing systems has been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationship between different species and subspecies. However, such methods have failed to differentiate between two of the classically defined subspecies of the protozoan parasite Trypanosoma brucei: the human infective, T. b. rhodesiense, which causes African sleeping sickness, and the non-human infective T. b. brucei. This has led to the hypothesis that T. b. rhodesiense is a host range variant of T. b. brucei. In this paper we test this hypothesis by examining highly polymorphic tandemly repeated regions of the trypanosome genome, i.e., minisatellite loci. We have employed the technique of minisatellite variant repeat mapping by PCR (MVR-PCR), which determines the distribution of variant repeat units along the tandem array of one minisatellite, MS42. The maps generated by this technique not only allow unequivocal allele identification but also contain within them cladistic information which we used to determine the possible genetic relationship between the different subspecies of T. brucei. Our findings revealed that human infective (T. b. rhodesiense) isolates from Uganda are more closely related to the local non-human infective isolates (T. b. brucei) than they are to other human infective stocks from different regions, suggesting that human infectivity has originated independently in these different geographical regions. This would infer that the separate classification of all human infective stocks from East Africa into the subspecies T. b. rhodesiense is genetically inappropriate and it would be better to consider geographically separate populations as host range variants of T. brucei brucei or perhaps as a series of different subspecies. Based on these data, it is clear that MVR mapping is a very useful tool for the analysis of zoonotic eukaryotic pathogens where delineation of the origins of outbreaks of disease and definition of human infective strains are key questions.     

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.     

Trypanosoma brucei gambiense: study of population genetic structure of Central African stocks using amplified fragment length polymorphism (AFLP)

To understand the maintenance and resurgence of historical Human African Trypanosomiasis (HAT) foci, AFLP was used to genotype 100 Central African Trypanosoma brucei s.l. stocks. This technique confirmed the high genetic stability of T. b. gambiense group 1 stocks and the micro genetic variability within Central African T. b. gambiense stocks. It revealed several T. b. gambiense genotypes and allowed the identification of minor and major genotypes in HAT foci. The coexistence of these genotypes in the same focus suggests that clustering of stocks according to HAT focus does not provide the true genetic picture of trypanosome circulating within the disease focus because the minor genotypes are generally underestimated. The presence of minor and major genotypes in HAT foci may explain the persistence and the resurgence of Central African sleeping sickness foci.     

Trypanosoma brucei rhodesiense: characterisation of stocks from Zambia, Kenya, and Uganda using repetitive DNA probes

We have previously described a system for characterising the relationships between trypanosome stocks of the T.brucei group based on Southern blotting with repetitive DNA probes followed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Parasitol. 39, 213-226, 1990). In this study, we extend this analysis to examine the relationships between trypanosome stocks isolated from major sleeping sickness foci in Zambia, Kenya, and Uganda. We show that the trypanosome strains responsible for disease in Zambia are quite distinct from those sampled from the Kenya/Uganda foci. Furthermore, the human serum resistant stocks isolated from the Kenya/Uganda foci which were isolated from man (or from animals) were found to form a tight group in the cluster analysis, while stocks isolated from nonhuman sources in the same area or stocks from elsewhere were found in separate groups. Thus, the human infective trypanosome strains found in these foci may have common origins and have, perhaps, arisen by clonal selection from a common source.     

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.     

Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element

An ordered restriction map of the ribosomal RNA genes of Trypanosoma brucei brucei is presented. Bgl II fragments of T.b.brucei genomic DNA were cloned into pAT 153, and the clones containing rDNA identified. Restriction maps were established and the sense strands identified. One clone was shown by heteroduplex mapping to contain a 1.1 kb inserted sequence which was demonstrated to be widely distributed throughout the genomes of members of the subgenus Trypanozoon. However, in two other subgenera of Trypanosoma, Nannomonas and Schizotrypanum, the sequence is far less abundant. Analysis of the genomic DNA from two serodemes of T.b.brucei showed that the sequence was present in the rRNA of only one of them, implying that the sequence is a mobile element and that its appearance in rDNA is a comparitively recent occurrence.     

MGE-PCR: a novel approach to the analysis of Toxoplasma gondii strain differentiation using mobile genetic elements

The position of mobile genetic elements (MGE) within eukaryotic genomes is often highly variable and we have exploited this phenomenon to develop a novel approach to strain differentiation in Toxoplasma gondii. Two PCR based strategies were designed in which specific primers were used to amplify T. gondii MGE's revealing information on element size and positional variation. The first PCR strategy involved the use of a standard two primer PCR while the second strategy used a single specific primer in a step-up PCR protocol. This approach was applied to T. gondii reference strains which were either acute virulent or avirulent to mice. The use of a standard two primer PCR reaction revealed the presence of a virulence related marker in which all avirulent strains possessed an additional 688 bp band. The single primer PCR strategy demonstrated that all virulent strains had identical banding patterns suggesting invariance within this group of strains. However, all avirulent strains had different banding patterns indicating the presence of a number of individual lineages within this group. The applicability and sensitivity of MGE-PCR in epidemiological studies was demonstrated by direct amplification of T. gondii from sheep tissue samples. All sheep isolates, tested in this way, gave identical banding patterns suggesting the presence of an endemic Toxoplasma strain on this farm.     

A novel DNA nucleotide in Trypanosoma brucei only present in the mammalian phase of the life-cycle.

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.     

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.     

Further studies on difluoromethylornithine in African trypanosomes

DL-alpha-Difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) was previously shown to cure mice infected with Trypanosoma brucei brucei, a parasite of game and cattle in Africa and Trypanosoma brucei rhodesiense, a human African Sleeping Sickness pathogen. Our studies now indicate that DFMO blocks ornithine decarboxylase and lowers trypanosome polyamine levels in vivo. Polyamine uptake in T.b. brucei also resembles that previously described for mammalian cells. The therapeutic potential of DFMO can now also be extended to another human pathogen, Trypanosoma brucei gambiense. Finally, DFMO acts synergistically with another drug, bleomycin, to cure acute trypanosome infections, and furthermore, this same drug combination provides a new approach to the treatment of trypanosomal infections of the central nervous system.     

The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.     

Sex and evolution in trypanosomes

Trypanosoma brucei is still the only kinetoplastid known to undergo genetic exchange, but it seems unreasonable to suppose that it evolved this process all by itself. The position of T. brucei on a molecular phylogenetic tree constructed from 18S ribosomal RNA gene sequences offers no clues to the likely existence of genetic exchange in trypanosome species other than the Salivaria, because this group of trypanosomes appears to have diverged from the rest a very long time ago. Antigenic variation is one characteristic shared by the Salivaria, which has been particularly well-studied in T. brucei. The large proportion of the genome devoted to variant antigen genes and related sequences in T. brucei, suggests a possible role for genetic exchange in enhancing the diversity of the repertoire. Alternatively, genetic exchange may counter potential excessive double-strand DNA damage brought about by the DNA rearrangements associated with antigenic variation. The remarkable biparental inheritance of organelle DNA (=kinetoplast DNA) in T. brucei is without precedent in other eukaryotes. The result of genetic exchange is to enhance the heterogeneity of the kinetoplast DNA minicircles.     

Dicationic biphenyl benzimidazole derivatives as antiprotozoal agents

A series of biphenyl benzimidazoles diamidines 6a-i were synthesized from their respective diamidoximes, through the bis-O-acetoxyamidoxime followed by hydrogenation in glacial acetic acid/ethanol in the presence of Pd-C. The target compounds contain hydroxy and/or methoxy substituted 1,3-phenyl groups as the central spacer between the two amidino bearing aryl groups. All of the diamidines showed strong DNA affinities as judged by high DeltaTm values with poly(dA.dT)2, which varied with structure and is discussed. Seven of the nine new diamidines gave in vitro IC50 values of approximately 30 nM or less versus Trypanosoma brucei rhodesiense (T.b.r.). Generally the diamidines were less active versus Plasmodium falciparum (P.f.), however one compound exhibited excellent activity with an IC50 value of 2.1 nM. Five of the nine diamidines exhibited excellent in vivo activity in the trypanosomal STIB900 mouse model giving 3/4 or 4/4 cures at dosage of 20 mg/kg i.p. and three showed similar efficacy at dosage of 10 mg/kg or lower.     

Mitochondrial initiation factor 2 of Trypanosoma brucei binds imported formylated elongator-type tRNA(Met)

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA(Met) present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA(Met) carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA(Met). In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA(Met). Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA(Met), suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knock-down of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyl-tRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.