Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone

Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.     

Mutational analysis of the Trypanosoma vivax alternative oxidase: the E(X)6Y motif is conserved in both mitochondrial alternative oxidase and plastid terminal oxidase and is indispensable for enzyme activity

Based on amino acid sequence similarity and the ability to catalyze the four-electron reduction of oxygen to water using a quinol substrate, mitochondrial alternative oxidase (AOX) and plastid terminal oxidase (PTOX) appear to be two closely related members of the membrane-bound diiron carboxylate group of proteins. In the current studies, we took advantage of the high activity of Trypanosoma vivax AOX (TvAOX) to examine the importance of the conserved Glu and the Tyr residues around the predicted third helix region of AOXs and PTOXs. We first compared the amino acid sequences of TvAOX with AOXs and PTOXs from various taxa and then performed alanine-scanning mutagenesis of TvAOX between amino acids Y(199) and Y(247). We found that the ubiquinol oxidase activity of TvAOX is completely lost in the E214A mutant, whereas mutants E215A and E216A retained more than 30% of the wild-type activity. Among the Tyr mutants, a complete loss of activity was also observed for the Y221A mutant, whereas the activities were equivalent to wild-type for the Y199A, Y212A, and Y247A mutants. Finally, residues Glu(214) and Tyr(221) were found to be strictly conserved among AOXs and PTOXs. Based on these findings, it appears that AOXs and PTOXs are a novel subclass of diiron carboxylate proteins that require the conserved motif E(X)(6)Y for enzyme activity.     

Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase

The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.     

Origins, evolutionary history, and taxonomic distribution of alternative oxidase and plastoquinol terminal oxidase

Alternative oxidase (AOX) and plastoquinol terminal oxidase (PTOX) are related quinol oxidases associated with respiratory and photosynthetic electron transport chains, respectively. Contrary to previous belief, AOX is present in numerous animal phyla, as well as heterotrophic and marine phototrophic proteobacteria. PTOX appears limited to organisms capable of oxygenic photosynthesis, including cyanobacteria, algae and plants. We propose that both oxidases originated in prokaryotes from a common ancestral di-iron carboxylate protein that diversified to AOX within ancient proteobacteria and PTOX within ancient cyanobacteria. Each then entered the eukaryotic lineage separately; AOX by the endosymbiotic event that gave rise to mitochondria and later PTOX by the endosymbiotic event that gave rise to chloroplasts. Both oxidases then spread through the eukaryotic domain by vertical inheritance, as well as by secondary and potentially tertiary endosymbiotic events.     

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.     

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.     

Alternative oxidases (AOX1a and AOX2) can functionally substitute for plastid terminal oxidase in Arabidopsis chloroplasts

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.     

Selection of susceptible and refractory lines of Glossina morsitans centralis for Trypanosoma congolense infection and their susceptibility to different pathogenic Trypanosoma species

Abstract .In a single generation of selection, two lines of Glossina morsitans centralis were established that differed significantly in susceptibility to Trypanosoma congolense clone IL 1180. Reciprocal crosses demonstrated that susceptibility was a maternally inherited trait. Differences between the lines, to all phases of the trypanosome infection, were maintained for eight generations, whereas differences in susceptibility to midgut infections were maintained for twenty-eight generations. Thereafter, the lines did not differ in susceptibility to Trypanosoma congolense IL 1180. Susceptibility to infections with Trypanosoma congolense IL 1180 was only a weak predictor of susceptibility to T. congolense clones IL 13-E3 and K60/1, as well as clone T. brucei brucei STIB 247-L. However, the susceptible and refractory lines displayed these phenotypes when tested with Trypanosoma vivax, indicating that the factors that affect susceptibility to trypanosomes are expressed both within and outside the midgut.     

The effect of starvation on the susceptibility of teneral and non-teneral tsetse flies to trypanosome infection

Transmission of vector-borne diseases depends largely on the ability of the insect vector to become infected with the parasite. In tsetse flies, newly emerged or teneral flies are considered the most likely to develop a mature, infective trypanosome infection. This was confirmed during experimental infections where laboratory-reared Glossina morsitans morsitans Westwood (Diptera: Glossinidae) were infected with Trypanosoma congolense or T. brucei brucei. The ability of mature adult tsetse flies to become infected with trypanosomes was significantly lower than that of newly emerged flies for both parasites. However, the nutritional status of the tsetse at the time of the infective bloodmeal affected its ability to acquire either a T. congolense or T. b. brucei infection. Indeed, an extreme period of starvation (3-4 days for teneral flies, 7 days for adult flies) lowers the developmental barrier for a trypanosome infection, especially at the midgut level of the tsetse fly. Adult G. m. morsitans became at least as susceptible as newly emerged flies to infection with T. congolense. Moreover, the susceptibility of adult flies, starved for 7 days, to an infection with T. b. brucei was also significantly increased, but only at the level of maturation of an established midgut infection to a salivary gland infection. The outcome of these experimental infections clearly suggests that, under natural conditions, nutritional stress in adult tsetse flies could contribute substantially to the epidemiology of tsetse-transmitted trypanosomiasis.     

Mechanical transmission of Trypanosoma evansi and T. congolense by Stomoxys niger and S. taeniatus in a laboratory mouse model

Abstract .Mechanical transmission of Trypanosoma evansi (South American origin) and T. congolense of Kilifi DNA type (Kenyan origin) was studied in laboratory mice using the African stable flies Stomoxys niger niger and S. taeniatus . Altogether, 355 flies were interrupted after feeding on infected blood and then transferred immediately to an uninfected mouse to complete feeding. Microscopy and subinoculation of triturated flies into uninfected mice demonstrated the survival of T. congolense in Stomoxys for up to 210 min and T. evansi for up to 480 min. Parasites survived for much longer periods in the digestive tract than inside or on the mouthparts. Trypanosoma congolense was transmitted only by S. n. niger , and only at low rates of 3, 8 and 10% using flies of different feeding histories: fed on blood the previous day, freshly caught, and teneral. Trypanosoma evansi was transmitted by both Stomoxys species at higher rates: S. taeniatus range 13–18%; S. n. niger range 17–35%. The highest transmission rate occurred with the combination of teneral S. n. niger and T. evansi.     

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.     

Comparison of the infection rate of tsetse, Glossina morsitans morsitans, fed in vitro or in vivo

Studies were made of infection rates of trypanosomes in the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae) when maintained in vivo (rabbits) or in vitro on high quality, gamma-irradiated, sterile defibrinated bovine blood, obtained from the Entomology Unit of the International Atomic Energy Agency (IAEA). For both Trypanosoma congolense Broden and T. b. brucei Plimmer & Bradford, in vitro maintenance significantly reduced the proportion of flies that developed mature metacyclic trypanosome infections.     

Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA

The genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major have been sequenced, but the phylogenetic relationships of these three protozoa remain uncertain. We have constructed trypanosomatid phylogenies based on genes for glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA). Trees based on gGAPDH nucleotide and amino acid sequences (51 taxa) robustly support monophyly of genus Trypanosoma, which is revealed to be a relatively late-evolving lineage of the family Trypanosomatidae. Other trypanosomatids, including genus Leishmania, branch paraphyletically at the base of the trypanosome clade. On the other hand, analysis of the SSU rRNA gene data produced equivocal results, as trees either robustly support or reject monophyly depending on the range of taxa included in the alignment. We conclude that the SSU rRNA gene is not a reliable marker for inferring deep level trypanosome phylogeny. The gGAPDH results support the hypothesis that trypanosomes evolved from an ancestral insect parasite, which adapted to a vertebrate/insect transmission cycle. This implies that the switch from terrestrial insect to aquatic leech vectors for fish and some amphibian trypanosomes was secondary. We conclude that the three sequenced pathogens, T. brucei, T. cruzi and L. major, are only distantly related and have distinct evolutionary histories.     

Trans-sialidase-like sequences from Trypanosoma congolense conserve most of the critical active site residues found in other trans-sialidases

Trypanosoma congolense is the agent of Nagana, the trypanosomiasis in African ruminants. Trypanosomes express an enzyme called trans-sialidase, which is believed to play an important role in maintaining pathogenicity of the parasites. Thus far, only two complete trans-sialidase sequences have been characterised, one from the American trypanosome T. cruzi and one from the African trypanosome T. brucei brucei. Although the crystal structure of T. cruzi trans-sialidase has recently been published [Buschiazzo et al., Mol. Cell 10 (2002), pp. 757-768], a number of questions concerning the exact transfer mechanism remain unanswered. The availability of further trans-sialidase sequences will ensure a better understanding of how transfer activity can be achieved and will provide the opportunity to develop highly specific, structure-based trans-sialidase inhibitors. Utilising a PCR-based approach two different trans-sialidase gene copies from T. congolense were identified, which share only 50% identity with each other, but show significant similarity with known viral, bacterial and trypanosomal sialidases and trans-sialidases. In both partial sequences most of the critical active site residues common to other trypanosomal sialidases and trans-sialidases are conserved. This is further illustrated by modelling the active site of the longer of the two partial gene sequences.     

Catabolism of Tryptophan by Trypanosoma evansi

ABSTRACT. Trypanosoma brucei gambiense , which causes human African trypanosomiasis, catabolizes the aromatic amino acid tryptophan via an initial aminotransferase catalyzed reaction to form several indole end products, which have been suggested to contribute to the pathogenesis of trypanosomiasis. To determine if this same pathway exists in T. evansi , the closely related trypanosome pathogen of domestic animals, tryptophan catabolism was examined in vitro and in vivo. As is the case with human African trypanosomes, T. evansi catabolized tryptophan to form indole-3-pyruvic acid and smaller amounts of indole-3-acetic acid and indole-3-lactic acid. Large concentrations of indole-3-pyruvic acid are excreted in urine of trypanosome-infected mice. However, indole-3-ethanol could not be detected in incubates of T. evansi or T. b. gambiense , even though the latter species had previously been reported to form this neutral metabolite. A new, previously unreported tryptophan metabolite was isolated and partially characterized from incubates of T. evansi and T. b. gambiense. Although the functional significance of tryptophan catabolism to trypanosomatids remains obscure, the pathway is quantitatively significant in all species examined thus far.     

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.     

Trypanosoma evansi: demonstration of a transferrin receptor derived from expression site-associated genes 6 and 7.

In Trypanosoma brucei, uptake of host transferrin is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor derived from the 2 expression site-associated genes 6 and 7 (ESAG6 and ESAG7). By using specific antibodies, it is shown here that T. evansi, a trypanosome species transmitted mechanically by biting flies, also expresses a transferrin receptor composed of ESAG6 and ESAG7. The cellular uptake of transferrin in T. evansi is completely inhibited with anti-T. brucei (ESAG6/7 heterodimer) antibodies. The demonstration of a functional ESAG6/7 transferrin receptor in T. evansi supports further its close relationship to T. brucei.     

Alternative oxidase present in procyclic Trypanosoma brucei may act to lower the mitochondrial production of superoxide

The mitochondrial electron transfer chain present in the procyclic form of the African trypanosome Trypanosoma brucei contains both cytochrome c oxidase and an alternative oxidase (TAO) as terminal oxidases that reduce oxygen to water. By contrast, the electron transfer chain of the primitive mitochondrion present in the bloodstream form of T. brucei contains only TAO as the terminal oxidase. TAO functions in the bloodstream forms to oxidize the ubiquinol produced by the glycerol-3-phosphate shuttle that results in the oxidation of the reduced nicotinamide adenine dinucleotide phosphate produced by glycolysis. The function, however, of TAO in the procyclic forms is unknown. In this study, we found that inhibition of TAO by the specific inhibitor salicylhydroxamic acid stimulates the formation of reactive oxygen species (ROS) in trypanosome mitochondria, resulting in mitochondrial alteration and increased oxidation of cellular proteins. Moreover, the activity and protein content of TAO in procyclic trypanosomes were increased when cells were incubated in the presence of hydrogen peroxide or antimycin A, the cytochrome bc1 complex inhibitor, which also results in increased ROS production. We suggest that one function of TAO in procyclic cells may be to prevent ROS production by removing excess reducing equivalents and transferring them to oxygen.     

Strain-specific difference in amino acid sequences of trypanosome alternative oxidase

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. The cDNA for TAO has been cloned from Trypanosoma brucei brucei EATRO110 strain and has been used for further characterization. In this study, we found amino acid sequence of the C-terminal part of TAO from the strain that we are using, T. b. brucei TC221, is considerably different from that of the EATRO110 strain.