Different kinetoplast degradation patterns in American Trypanosoma vivax strains: Multiple independent origins or fast evolution?

We analyzed the kinetoplast (mitochondrial genome) of Trypanosoma vivax strains from America and Africa to determine their precise architecture and to understand their adaptive response to mechanical transmission. The use of long-read based assemblies that retain individuality of tandem repeats, without erasing inter-copy variability, allowed us to investigate the evolutionary dynamics of repetitive kinetoplast-DNA. This analysis revealed that repeat elements located in edges of repeat clusters are less active in terms of renewal, whereas internal copies appear to undergo a permanent process of birth-and-death. Comparing different American strains with the African Y486 strain, we found that in the former, protein coding genes from the maxicircle contain several function disrupting mutations that with very few exceptions are present in one or the other American strain but not in both, suggesting the absence of common ancestry for most of the genomic changes that led to their loss of oxidative phosphorylation capacity. Analysis of another component of kinetoplast, the minicircles, revealed great loss of diversity, and loss of their encoded guideRNAs. Both groups of American strains retain minimal sets required to edit the still functional A6-APTase and RPS12 genes. The extensive maxi- and minicircle divergence suggests a history of multiple introduction events in America of strains that probably started to degrade their kinetoplast in Africa. The notion that kinetoplast degradation began after incursion in America would imply a pace of accumulation of genetic changes considerably faster than other trypanosomatids.

First report and molecular identification of Trypanosoma (Duttonella) vivax outbreak in cattle population from Ecuador.

The bovine trypanosomosis is responsible for economic losses from tropical and subtropical areas of Africa and Latin America. This disease is characterized by fever, anaemia, loss of production and even death. Few studies have been carried out in Ecuador regarding Trypanosoma spp. presence but the species has not been determined in cattle and those have only determined the presence of genus, but not the species. The aim of this study was to identify and characterize the trypanosome species involved in the suspected bovine trypanosomosis outbreak reported in Convento Village in Manabí Province located in the coastal region of Ecuador. Twenty cattle from three farms were sampled. Three samples were positive for T. vivax, using an end-point polymerase chain reaction (PCR) to amplify a fragment of the cathepsin L-like cysteine protease (CatL-like) gene. A phylogenetic tree analysis of these three Ecuadorian isolates showed a close relationship with isolates from South America (Colombia, Brazil and Venezuela) and West Africa (Nigeria). This is the first report of T. vivax in Ecuadorian cattle.

Comparison of conventional and molecular techniques for Trypanosoma vivax diagnosis in experimentally infected cattle / Comparação de técnicas convencionais e moleculares para diagnóstico em bovinos experimentalmente infectados por Trypanosoma vivax

Abstract Livestock infections by Trypanosoma vivax have been occurring with increasing frequency, mainly due to the presence of animals with subclinical infections and without apparent parasitaemia, making diagnosis challenging. The aim of the present study was to evaluate several techniques used for T. vivax diagnosis in order to assess the best way of using them during the course of the disease. Molecular methods demonstrated higher rates of detection than parasitological methods, detecting 33 of the 54 (61.1%) known positive samples, while the hematocrit centrifugation technique (best parasitological test) detected only 44.4%. The serological methods, IFAT and ELISA, detected seropositivity in 51 of the 54 (94.4%) and 49 of the 54 (90.7%) known positive samples, respectively. Despite being highly sensitive, the latter only demonstrates exposure to the infectious agent and does not indicate whether the infection is active. The present study was the first to use the qPCR for a South American isolate, improving disease detection and quantification. Furthermore, the analyses revealed that the patent phase of the disease may extend up to 42 days, longer than previously reported. The combination of several diagnostic techniques can lower the frequency of false negative results and contributes toward better disease control.

Resumo Infecções por Trypanosoma vivax têm ocorrido com frequência crescente em animais de produção, principalmente pela aquisição de animais com infecções subclínicas e sem aparente parasitemia, o que dificulta o diagnóstico. O objetivo do presente estudo foi avaliar várias técnicas empregadas para o diagnóstico de T. vivax, a fim de verificar a melhor maneira de utilizá-las durante o curso da doença. Os métodos moleculares demonstraram maiores taxas de detecção que os métodos parasitológicos, detectando 33 das 54 (61,1%) amostras sabidamente positivas, enquanto a técnica de hemoconcentração (melhor teste parasitológico) detectou apenas 44,4%. Os métodos sorológicos, RIFI e ELISA, detectaram soropositividade em 51 das 54 (94,4%) e 49 das 54 (90,7%) amostras sabidamente positivas, respectivamente. Apesar de serem altamente sensíveis, estes testes apenas demonstram a exposição ao agente infeccioso, e não indicam se a infecção permanece ativa. O presente estudo foi o primeiro a utilizar a qPCR para um isolado sul-americano, melhorando sua detecção e quantificação. Além disso, as análises revelaram que a fase patente da doença pode se estender por até 42 dias após a infecção, sendo maior que anteriormente relatado. A combinação de várias técnicas de diagnóstico pode evitar a frequência de resultados falso-negativos e contribuir para um melhor controle da doença.

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing.

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.

Recombinant and native TviCATL from Trypanosoma vivax: Enzymatic characterisation and evaluation as a diagnostic target for animal African trypanosomosis.

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat, was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket. Leucine was preferred in P2 and basic and non-bulky, hydrophobic residues accepted in P1 and P3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA.

Detection and molecular characterization of Trypanosoma (Duttonella) vivax in dairy cattle in the state of Sergipe, northeastern Brazil / Detecção e caracterização molecular de Trypanosoma (Duttonella) vivax em gado leiteiro no estado de Sergipe, no Nordeste do Brasil

Abstract Trypanosoma (Duttonella) vivax is an important cause of economic losses among feedlot cattle. These losses are related to the morbidity, mortality, reproductive issues and decreased production. It is known that the clinical signs observed in infections by this protozoon are similar to other hemoparasitosis, which difficult the diagnosis. Therefore, the aim of this study was to detect and molecularly characterize an outbreak of trypanosomiasis caused by T. (D.) vivax in dairy cattle in the municipality of São Miguel Aleixo, state of Sergipe, Brazil. Blood samples from cattle (n = 15) presenting clinical signs compatible with trypanosomiasis were collected and parasitological and molecular evaluated. Among the samples analyzed, 34% (5/15) were positive from blood smears, 60% (9/15) from the buffy coat method and 80% (12/15) from the molecular method. The DNA sequence obtained (659 bp) showed 99% similarity to T. (D.) vivax sequences that are available in the GenBank database. The presence of this protozoon in cattle herds is a problem for producers. Diagnosing trypanosomiasis is problematic because its evolution is similar to that of other parasitic blood diseases. In addition, this is the first report of infection by T. (D.) vivax in cattle in the state of Sergipe, northeastern Brazil.

Resumo Trypanosoma (Duttonella) vivax é responsável por consideráveis perdas econômicas na bovinocultura. Estas perdas estão relacionados à morbidade, mortalidade, problemas reprodutivos e declínio na produção. Sabe-se que os sinais clínicos apresentados em infecções por este protozoário se assemelha a outras hemoparasitoses, dificultando muitas vezes o diagnóstico. Portanto, objetivou-se com este estudo detectar a ocorrência de T. (D.) vivax em bovinos leiteiros no município de São Miguel Aleixo, Estado de Sergipe, Brasil. Para tanto, amostras de sangue (n = 15) foram coletadas e avaliadas através de métodos parasitológicos e moleculares. Do total das amostras analisadas, 34% (5/15) foram positivas no esfregaço sanguíneo, 60% (9/15) pelo método do Buffy Coat, enquanto na biologia molecular 80% (12/15) amplificaram um fragmento de DNA (659 pb) compatível com T. (D.) vivax (GenBank). Em conclusão a presença de T. (D.) vivax nos rebanhos bovinos caracteriza-se como um problema para os pecuaristas, como também para o diagnóstico, uma vez que essa tripanossomíase apresenta evolução semelhante a outras hemoparasitoses. Ademais, este é o primeiro relato de infecção por T. (D.) vivax em bovinos do estado de Sergipe, nordeste do Brasil.

Kinetoplast adaptations in American strains from Trypanosoma vivax.

The mitochondrion role changes during the digenetic life cycle of African trypanosomes. Owing to the low abundance of glucose in the insect vector (tsetse flies) the parasites are dependent upon a fully functional mitochondrion, capable of performing oxidative phosphorylation. Nevertheless, inside the mammalian host (bloodstream forms), which is rich in nutrients, parasite proliferation relies on glycolysis, and the mitochondrion is partially redundant. In this work we perform a comparative study of the mitochondrial genome (kinetoplast) in different strains of Trypanosoma vivax. The comparison was conducted between a West African strain that goes through a complete life cycle and two American strains that are mechanically transmitted (by different vectors) and remain as bloodstream forms only. It was found that while the African strain has a complete and apparently fully functional kinetoplast, the American T. vivax strains have undergone a drastic process of mitochondrial genome degradation, in spite of the recent introduction of these parasites in America. Many of their genes exhibit different types of mutations that are disruptive of function such as major deletions, frameshift causing indels and missense mutations. Moreover, all but three genes (A6-ATPase, RPS12 and MURF2) are not edited in the American strains, whereas editing takes place normally in all (editable) genes from the African strain. Two of these genes, A6-ATPase and RPS12, are known to play an essential function during bloodstream stage. Analysis of the minicircle population shows that its diversity has been greatly reduced, remaining mostly those minicircles that carry guide RNAs necessary for the editing of A6-ATPase and RPS12. The fact that these two genes remain functioning normally, as opposed to that reported in Trypanosoma brucei-like trypanosomes that restrict their life cycle to the bloodstream forms, along with other differences, is indicative that the American T. vivax strains are following a novel evolutionary pathway.

Cathepsin L-like genes of Trypanosoma vivax from Africa and South America--characterization, relationships and diagnostic implications.

We characterized sequences from genes encoding cathepsin L-like (CatL-like) cysteine proteases from African and South American isolates of Trypanosoma vivax and T. vivax-like organisms, and evaluated their suitability as genetic markers for population structure analysis and diagnosis. Phylogenetic analysis of sequences corresponding to CatL-like catalytic domains revealed substantial polymorphism, and clades of sequences (TviCatL1-9) were separated by large genetic distances. TviCatL1-4 sequences were from cattle isolates from West Africa (Nigeria and Burkina Faso) and South America (Brazil and Venezuela), which belonged to the same T. vivax genotype. T. vivax-like genotypes from East Africa showed divergent sequences, including TviCatL5-7 for isolates from Mozambique and TviCatL8-9 for an isolate from Kenya. Phylogenetic analysis of CatL-like gene data supported the relationships among trypanosome species reflected in the phylogenies based on the analysis of small subunit (SSU) of ribosomal RNA gene sequence data. The discovery of different CatL-like sequences for each genotype, defined previously by ribosomal DNA data, indicate that these sequences provide useful targets for epidemiological and population genetic studies. Regions in CatL-like sequences shared by all T. vivax genotypes but not by other trypanosomes allowed the establishment of a specific and sensitive diagnostic PCR for epidemiological studies in South America and Africa.

Trypanosoma (Duttonella) vivax: its biology, epidemiology, pathogenesis, and introduction in the New World - a review

The biology, epidemiology, pathogenesis, diagnostic techniques, and history of the introduction of Trypanosoma (Duttonella) vivax in the New World are reviewed. The two main immunological responses of trypanosome-infected animals - antibody production and immunodepression - are discussed in the context of how these responses play a role in disease tolerance or susceptibility. Isolation and purification of T. vivax are briefly discussed. The recent reports of bovine trypanosomiasis diagnosed in cattle on farms located in the Pantanal region of the states of Mato Grosso do Sul and Mato Grosso, Brazil, are also discussed.

The VIPER elements of trypanosomes constitute a novel group of tyrosine recombinase-enconding retrotransposons.

VIPER was initially characterized as a 2326bp LTR-like retroelement associated to SIRE, a short interspersed repetitive element specific of Trypanosoma cruzi. It carried a single ORF that coded for a putative reverse transcriptase-RNAse H protein, suggesting that it could be a truncated copy of a longer retroelement. Herein we report the identification and characterization of a complete 4480bp long VIPER in the T. cruzi genome. The complete VIPER harbored three non-overlapped domains encoding for a GAG-like, a tyrosine recombinase and a reverse transcriptase-RNAse H proteins. VIPER elements were also found in the genomes of Trypanosoma brucei and Trypanosoma vivax, but not in Leishmania sp. On the basis of its reverse transcriptase phylogeny, VIPER was classified as an LTR retroelement. However, VIPER was structurally related to the tyrosine recombinase encoding retroelements, DIRS and Ngaro. Phylogenetic analysis showed that VIPER's tyrosine recombinase grouped with the transposases RCI1 of Escherichia coli and Ye24 and Ye72 of Haemophilus influenzae within a major branch of prokaryotic recombinases. Taken together, VIPER's structure, the nature of its tyrosine recombinase, the unique features of its reverse transcriptase catalytic consensus motif and the fact that it was found in Trypanosomes, an early branching eukaryote, suggest that VIPER may be the closest relative of the founder element of the tyrosine recombinase encoding retrotransposons known up to date. Our analysis revealed that tyrosine recombinase-encoding retroelements were originated as early in evolution as non-LTR retroelements and suggests that VIPER, Ngaro and DIRS elements may constitute a third group of retrotransposons, distinct from both LTR and non-LTR retroelements.

Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax.

The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.

Glucose uptake in Trypanosoma vivax and molecular characterization of its transporter gene.

A gene, TvHT1, encoding a glucose transporter protein, has been cloned from the haemoflagellate protozoon, Trypanosoma vivax, which has an active Kreb's cycle in the mammalian stage. The deduced polypeptide is similar in amino acid sequence to other kinetoplastid hexose transporters from Trypanosoma brucei (THT1 and THT2), Trypanosoma cruzi (TcrHT1) and Leishmania (Pro-1). The similarity is higher with THT2 (expressed in T. brucei insect forms) than with the other isoforms. The kinetic properties of glucose uptake in Chinese Hamster Ovary (CHO) cells expressing TvHT1 and in trypanosomes show s a saturable transport mechanism typical of a facilitated carrier system, with a similar affinity for D-glucose as that of the T. brucei bloodstream form carrier, THT1 (Km = 0.548 +/- 0.01 mM, Vmax = 4.26 +/- 0.12 nmol.min-1.mg protein-1 in CHO cells and Km = 0.585 +/- 0.068 mM, Vmax = 88.5 +/- 6.2 nmol.min-1.mg protein-1 in T. vivax). The specificity of the TvHT1 protein for various D-glucose analogues, as judged by inhibition of 2-deoxy-D-arabinose-hexose transport, shows properties that are intermediate between those of THT1 on the one hand and TcrHT1 and THT2 on the other. As with the hexose transporters in the other members of Kinetoplastida, the TvHT1-encoded system differs from erythrocyte-type glucose transport by its moderate sensitivity to cytochalasin B and its capacity to transport fructose.