Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.     

The detection of non-RoTat 1.2 Trypanosoma evansi

The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR amplification for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from different trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR amplification of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp amplification product was present in all five non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is specific for non-RoTat 1.2 T. evansi.     

Thiol-dependent proteases of African trypanosomes. Analysis by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels co-polymerized with fibrinogen

The proteases of several species of African trypanosomes were analysed by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels containing fibrinogen or collagen. After electrophoresis the gels were incubated in the presence of enzyme activators and/or inhibitors and then stained with Coomassie brilliant blue. The areas where the proteolytic activity had degraded the fibrinogen did not stain and so formed clear bands against a blue background. The proteases were found to have pH optima between 5 and 6, and required dithiothreitol or 2-mercaptoethanol for full expression of their activity. They were inhibited by amino acid chloromethanes, iodoacetamide, p-chloromercuribenzoate and other inhibitors of the thiol-dependent proteases, as well as by the trypanocidal drugs berenil (4,4'-diamidinodiazoaminobenzene-diacetamidoacetate) and pentamidine [1,5-di-(4-amidinophenoxy)pentane-di-(2- hydroxyethanesulphonate)]. Trypanosoma evansi, Trypanosoma brucei brucei and Trypanosoma brucei gambiense each have a protease with a relative molecular mass, Mr, of 28 000. In addition they occasionally exhibit activity at higher Mr values (up to 105000). Trypanosoma congolense has a low-Mr protease (31 000) and may exhibit higher-Mr proteases (up to 150000). The protease profiles of Trypanosoma vivax differ from the other species, T. brucei or T. congolense, and are present in lesser amounts. The proteases of the cultured procyclic forms are present in much smaller amounts than those of the metacyclic or mammalian blood stream forms of these parasites. The catalytic properties and inhibition characteristics of these thiol-dependent enzymes suggest that they resemble the mammalian lysosomal cathepsins B and L.     

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.     

Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense. These results further confirm our belief that the major pathways of methionine utilization, for polyamine synthesis, protein transmethylation and the trans-sulfuration pathway, are excellent targets for chemotherapeutic intervention against African trypanosomes.     

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.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

Predictive Computational Models of Substrate Binding by a Nucleoside Transporter

Transporters play a vital role in both the resistance mechanisms of existing drugs and effective targeting of their replacements. Melarsoprol and diamidine compounds similar to pentamidine and furamidine are primarily taken up by trypanosomes of the genus Trypanosoma brucei through the P2 aminopurine transporter. In standardized competition experiments with [³H]adenosine, P2 transporter inhibition constants (K_i) have been determined for a diverse dataset of adenosine analogs, diamidines, Food and Drug Administration-approved compounds and analogs thereof, and custom-designed trypanocidal compounds. Computational biology has been employed to investigate compound structure diversity in relation to P2 transporter interaction. These explorations have led to models for inhibition predictions of known and novel compounds to obtain information about the molecular basis for P2 transporter inhibition. A common pharmacophore for P2 transporter inhibition has been identified along with other key structural characteristics. Our model provides insight into P2 transporter interactions with known compounds and contributes to strategies for the design of novel antiparasitic compounds. This approach offers a quantitative and predictive tool for molecular recognition by specific transporters without the need for structural or even primary sequence information of the transport protein.     

Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populations and their cross-resistance

Resistance to melarsoprol and pentamidine was induced in bloodstream-form Trypanosoma brucei rhodesiense STIB 900 in vitro, and drug sensitivity was determined for melarsoprol, pentamidine and furamidine. The resistant populations were also inoculated into immunosuppressed mice to verify infectivity and to monitor whether rodent passage selects for clones with altered drug sensitivity. After proliferation in the mouse, trypanosomes were isolated and their IC(50) values to the three drugs were determined. To assess the stability of drug-induced resistance, drug pressure was ceased for 2 months and the drug sensitivity was determined again. Resistance was stable, with a few exceptions that are discussed. Drug IC(50)s indicated cross-resistance among all drugs, but to varying extents: resistance of the melarsoprol-selected and pentamidine-selected trypanosomes to pentamidine was the same, but the pentamidine-selected trypanosome population showed lower resistance to melarsoprol than the melarsoprol-selected trypanosomes. Interestingly, both resistant populations revealed the same intermediate cross-resistance to furamidine. Resistant trypanosome populations were characterised by molecular means, referring to the status of the TbAT1 gene. The melarsoprol-selected population apparently had lost TbAT1, whereas in the pentamidine-selected trypanosome population it was still present.     

What happens when Trypanosoma brucei leaves Africa

Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.     

Alternative oxidase (AOX) genes of African trypanosomes: phylogeny and evolution of AOX and plastid terminal oxidase families

To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (-E-, -EXXH-). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister-group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an alpha-proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.     

Natural and induced dyskinetoplastic trypanosomatids: how to live without mitochondrial DNA

Salivarian trypanosomes are the causative agents of several diseases of major social and economic impact. The most infamous parasites of this group are the African subspecies of the Trypanosoma brucei group, which cause sleeping sickness in humans and nagana in cattle. In terms of geographical distribution, however, Trypanosoma equiperdum and Trypanosoma evansi have been far more successful, causing disease in livestock in Africa, Asia, and South America. In these latter forms the mitochondrial DNA network, the kinetoplast, is altered or even completely lost. These natural dyskinetoplastic forms can be mimicked in bloodstream form T. brucei by inducing the loss of kinetoplast DNA (kDNA) with intercalating dyes. Dyskinetoplastic T. brucei are incapable of completing their usual developmental cycle in the insect vector, due to their inability to perform oxidative phosphorylation. Nevertheless, they are usually as virulent for their mammalian hosts as parasites with intact kDNA, thus questioning the therapeutic value of attempts to target mitochondrial gene expression with specific drugs. Recent experiments, however, have challenged this view. This review summarises the data available on dyskinetoplasty in trypanosomes and revisits the roles the mitochondrion and its genome play during the life cycle of T. brucei.     

Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library

RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.     

Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.     

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 evansi: cloning and expression in Spodoptera frugiperda [correction of fugiperda] insect cells of the diagnostic antigen RoTat1.2

A complementary DNA encoding the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigenic type (RoTat)1.2, currently used for experimental serological diagnosis of T. evansi infection in livestock, was cloned as a recombinant plasmid and sequenced. A recombinant baculovirus containing the coding region of RoTat1.2 VSG was constructed to express the protein in Spodoptera frugiperda [corrected] insect cells. From this, sufficient quantities of the recombinant protein are being produced for empirical and wide-scale objective assessment of the diagnostic potential of this antigen. The gene encoding the RoTat1.2 VSG was shown by PCR to be present in the genomes of many different cloned isolates of T. evansi, but not T. brucei, from geographically separate regions of Africa, Asia, and South America. With the recombinant RoTat1.2 at hand, it is now possible to investigate the extent to which epitopes on this VSG are conserved among different T. evansi isolates.