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Trypanosoma vivax displays distinct mitochondrial genomes: intact in Africa and degraded in South America. This finding, formerly reported from four isolates, suggested an association between mitochondrial mutations and adaptation to exclusive mechanical transmission outside Africa, circumventing mitochondrial activity essential for cyclical transmission by tsetse flies. Here, we characterized the mitochondrial ND7 and COIII genes in 25 South American isolates of T. vivax from livestock from Argentina, Brazil, Colombia, Ecuador, and Venezuela, as well as in 11 African isolates from Mozambique, Ethiopia, Uganda, Nigeria, and Burkina Faso from tsetse flies, antelopes, and cattle. Phylogenetic inference supported the lineages TVV (Trypanosoma vivax vivax) and TVL (Trypanosoma vivax-like), predominant in South America/West Africa and East Africa, respectively. Both ND7 and COIII genes were intact in all African TVV and TVL isolates and, for the first time, in 10 South American isolates from Brazil. Remaining South American isolates exhibited some degree of gene erosion, including ~142 bp deletion at the 3′ end of ND7 disclosed in Venezuela and Colombia, ~163 bp deletion at the 3′ end of ND7 in Brazil and Argentina, and ~750 bp deletion from the 5′ end of ND7 to the 3′ end of COIII in Venezuela. Comprehensive analyses of whole mitochondrial genomes from additional South American and African isolates are crucial for formulating reliable hypotheses regarding the origin, absence, and rapid mitochondrial degradation observed in South American T. vivax.

The Leishmaniinae subfamily of the Trypanosomatidae contains both genus Zelonia (monoxenous) and Endotrypanum (dixenous). They are amongst the nearest known relatives of Leishmania, which comprises many human pathogens widespread in the developing world. These closely related lineages are models for the genomic biology of monoxenous and dixenous parasites. Herein, we used comparative genomics to identify the orthologous groups (OGs) shared among 26 Leishmaniinae species to investigate gene family expansion/contraction and applied two phylogenomic approaches to confirm relationships within the subfamily. The Endotrypanum monterogeii and Zelonia costaricensis genomes were assembled, with sizes of 29.9 Mb and 38.0 Mb and 9.711 and 12.201 predicted protein-coding genes, respectively. The genome of E. monterogeii displayed a higher number of multicopy cell surface protein families, including glycoprotein 63 and glycoprotein 46, compared to Leishmania spp. The genome of Z. costaricensis presents expansions of BT1 and amino acid transporters and proteins containing leucine-rich repeat domains, as well as a loss of ABC-type transporters. In total, 415 and 85 lineage-specific OGs were identified in Z. costaricensis and E. monterogeii. The evolutionary relationships within the subfamily were confirmed using the supermatrix (3384 protein-coding genes) and supertree methods. Overall, this study showed new expansions of multigene families in monoxenous and dixenous parasites of the subfamily Leishmaniinae.

Abstract Background The subgenus Megatrypanum Hoare, 1964 of Trypanosoma Gruby, 1843 comprises trypanosomes of cervids and bovids from around the world. Here, the white-tailed deer Odocoileus virginianus (Zimmermann) and its ectoparasite, the deer ked Lipoptena mazamae Rondani, 1878 (hippoboscid fly), were surveyed for trypanosomes in Venezuela. Results Haemoculturing unveiled 20% infected WTD, while 47% (7/15) of blood samples and 38% (11/29) of ked guts tested positive for the Megatrypanum-specific TthCATL-PCR. CATL and SSU rRNA sequences uncovered a single species of trypanosome. Phylogeny based on SSU rRNA and gGAPDH sequences tightly cluster WTD trypanosomes from Venezuela and the USA, which were strongly supported as geographical variants of the herein described Trypanosoma (Megatrypanum) trinaperronei n. sp. In our analyses, the new species was closest to Trypanosoma sp. D30 from fallow deer (Germany), both nested into TthII alongside other trypanosomes from cervids (North American elk and European fallow, red and sika deer), and bovids (cattle, antelopes and sheep). Insights into the life-cycle of T. trinaperronei n. sp. were obtained from early haemocultures of deer blood and co-culture with mammalian and insect cells showing flagellates resembling Megatrypanum trypanosomes previously reported in deer blood, and deer ked guts. For the first time, a trypanosome from a cervid was cultured and phylogenetically and morphologically (light and electron microscopy) characterised. Conclusions In the analyses based on SSU rRNA, gGAPDH, CATL and ITS rDNA sequences, neither cervids nor bovids trypanosomes were monophyletic but intertwined within TthI and TthII major phylogenetic lineages. One host species can harbour more than one species/genotype of trypanosome, but each trypanosome species/genotype was found in a single host species or in phylogenetically closely related hosts. Molecular evidence that L. mazamae may transmit T. trinaperronei n. sp. suggests important evolutionary constraints making tight the tripartite T. trinaperronei-WTD-deer ked association. In a plausible evolutionary scenario, T. trinaperronei n. sp. entered South America with North American white-tailed deer at the Pliocene-Pleistocene boundary following the closure of the Panama Isthmus.

Trypanosoma rely on variant surface glycoprotein (VSG) to escape host immunity, but mechanisms generating antigenic diversity of VSG are poorly understood. Here, Silva-Pereira et al. show that T. vivax has a limited antigenic repertoire compared to T. brucei and that recombination plays little role in diversifying T. vivax VSG sequences.

Abstract Background The genus Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown. Results Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade (terrestrial lineage) forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi (trypanosomes from caimans, African and Brazilian tabanids and tsetse flies); Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp. Conclusions Prior to this description of Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.

The symbiosis in trypanosomatids is a mutualistic relationship characterized by extensive metabolic exchanges between the bacterium and the protozoan. The symbiotic bacterium can complete host essential metabolic pathways, such as those for heme, amino acid, and vitamin production. Experimental assays indicate that the symbiont acquires phospholipids from the host trypanosomatid, especially phosphatidylcholine, which is often present in bacteria that have a close association with eukaryotic cells. In this work, an in-silico study was performed to find genes involved in the glycerophospholipid (GPL) production of Symbiont Harboring Trypanosomatids (SHTs) and their respective bacteria, also extending the search for trypanosomatids that naturally do not have symbionts. Results showed that most genes for GPL synthesis are only present in the SHT. The bacterium has an exclusive sequence related to phosphatidylglycerol production and contains genes for phosphatidic acid production, which may enhance SHT phosphatidic acid production. Phylogenetic data did not indicate gene transfers from the bacterium to the SHT nucleus, proposing that enzymes participating in GPL route have eukaryotic characteristics. Taken together, our data indicate that, differently from other metabolic pathways described so far, the symbiont contributes little to the production of GPLs and acquires most of these molecules from the SHT.

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of Angomonas ambiguus, which is sister to A. desouzai, but bears an endosymbiont genetically close to that of A. deanei. Based on phylogenetic inferences, it was proposed that the bacterium from A. deanei had been horizontally transferred to A. ambiguus. Here, we sequenced the bacterial genomes from two A. ambiguus isolates, including a new one from Papua New Guinea, and compared them with the published genome of the A. deanei endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of A. ambiguus were obtained from A. deanei and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.

Trypanosoma rangeli is a generalist hemoflagellate that infects mammals and is transmitted by triatomines around Latin America. Due to its high genetic diversity, it can be classified into two to five lineages. In Brazil, its distribution outside the Amazon region is virtually unknown, and knowledge on the ecology of its lineages and on host species diversity requires further investigation. Here, we analyzed 57 T. rangeli samples obtained from hemocultures and blood clots of 1392 mammals captured in different Brazilian biomes. The samples were subjected to small subunit (SSU) rDNA amplification and sequencing to confirm T. rangeli infection. Phylogenetic inferences and haplotype networks were reconstructed to classify T. rangeli lineages and to infer the genetic diversity of the samples. The results obtained in our study highlighted both the mammalian host range and distribution of T. rangeli in Brazil: infection was observed in five new species (Procyon cancrivorous, Priodontes maximum, Alouatta belzebul, Sapajus libidinosus, and Trinomys dimidiatus), and transmission was observed in the Caatinga biome. The coati (Nasua nasua) and capuchin monkey (S. libidinosus) are the key hosts of T. rangeli. We identified all four T. rangeli lineages previously reported in Brazil (A, B, D, and E) and possibly two new genotypes.

Abstract Background Bat trypanosomes are implicated in the evolution of the T. cruzi clade, which harbours most African, European and American trypanosomes from bats and other trypanosomes from African, Australian and American terrestrial mammals, including T. cruzi and T. rangeli, the agents of the American human trypanosomiasis. The diversity of bat trypanosomes globally is still poorly understood, and the common ancestor, geographical origin, and evolution of species within the T. cruzi clade remain largely unresolved. Methods Trypanosome sequences were obtained from cultured parasites and from museum archived liver/blood samples of bats captured from Guatemala (Central America) to the Brazilian Atlantic Coast. Phylogenies were inferred using Small Subunit (SSU) rRNA, glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), and Spliced Leader (SL) RNA genes. Results Here, we described Trypanosoma wauwau n. sp. from Pteronotus bats (Mormoopidae) placed in the T. cruzi clade, then supporting the bat-seeding hypothesis whereby the common ancestor of this clade likely was a bat trypanosome. T. wauwau was sister to the clade T. spp-Neobats from phyllostomid bats forming an assemblage of trypanosome species exclusively of Noctilionoidea Neotropical bats, which was sister to an Australian clade of trypanosomes from indigenous marsupials and rodents, which possibly evolved from a bat trypanosome. T. wauwau was found in 26.5 % of the Pteronotus bats examined, and phylogeographical analysis evidenced the wide geographical range of this species. To date, this species was not detected in other bats, including those that were sympatric or shared shelters with Pteronotus. T. wauwau did not develop within mammalian cells, and was not infective to Balb/c mice or to triatomine vectors of T. cruzi and T. rangeli. Conclusions Trypanosoma wauwau n. sp. was linked to Pteronotus bats. The positioning of the clade T. wauwau/T.spp-Neobats as the most basal Neotropical bat trypanosomes and closely related to an Australian lineage of trypanosomes provides additional evidence that the T. cruzi clade trypanosomes likely evolved from bats, and were dispersed in bats within and between continents from ancient to unexpectedly recent times.

Abstract Background Several Trypanosoma species transmitted by leeches infect marine and freshwater fish worldwide. To date, all South American fish trypanosome species identified have been based on unreliable morphological parameters. We recently isolated and cultured trypanosomes from the Brazilian armoured catfishes Hypostomus luetkeni and H. affinis. Here, we report the first phylogenetic analyses of South American (Brazilian) trypanosomes isolated from fish, and from leeches removed from these fish. We also analysed morphologically and morphometrically the different forms of fish, leech and cultured trypanosomes. Methods V7V8 SSU rRNA and gGAPDH sequences were used for phylogenetic analysis of Brazilian fish and leech trypanosomes. Trypanosomes from cultures, fish blood and leech samples were also characterized morphologically and morphometrically by light and electron microscopy. Results In blood smears from fish high trypanosome prevalence (90–100 %) and parasitemia (0.9-1.0x102) were observed. Phylogenetic relationships using SSU rRNA and gGAPDH showed that, despite relevant sequence divergence, all Brazilian fish (and derived cultures) and leech trypanosomes clustered together into a single clade. The Brazilian clade clustered with European, North American and African fish trypanosomes. Based on sequence analysis, we uncovered a new species of Brazilian fish trypanosome, Trypanosoma abeli n. sp. Trypanosoma abeli cultures contained pleomorphic epimastigotes, small trypomastigotes and rare sphaeromastigotes. Ultrastructural features of T. abeli included a cytostome-cytopharynx complex in epi- and trypomastigotes, a compact rod-like kinetoplast, lysosome-related organelles (LROs) and multivesicular bodies. Trypanosomes found in fish blood smears and leech samples were highly pleomorphic, in agreement with sequence data suggesting that catfishes and leeches often have mixed trypanosome infections. Conclusions Trypanosoma abeli n. sp. is the first trypanosome from South American fishes isolated in culture, positioned in phylogenetic trees and characterized at the ultrastructural level. Trypanosoma abeli n. sp. is highly prevalent in H. luetkeni and H. affinis armoured catfish from the Atlantic Forest biome, and in other catfish species from the Amazon and the Pantanal. Sequencing data suggested that Brazilian catfish often have mixed trypanosome infections, highlighting the importance of molecular characterization to identify trypanosome species in fishes and leeches.

This study is about the inter- and intra-specific genetic diversity of trypanosomatids of the genus Angomonas, and their association with Calliphoridae (blowflies) in Neotropical and Afrotropical regions. Microscopic examination of 3,900 flies of various families, mostly Calliphoridae, revealed that 31% of them harbored trypanosomatids. Small subunit rRNA (SSU rRNA) barcoding showed that Angomonas predominated (46%) over the other common trypanosomatids of blowflies of genera Herpetomonas and Wallacemonas. Among Angomonas spp., A. deanei was much more common than the two-other species, A. desouzai and A. ambiguus. Phylogenetic analyses based on SSU rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and internal transcribed spacer rDNA (ITS rDNA) sequences revealed a marked genetic diversity within A. deanei, which comprised four infraspecific genotypes (Dea1–Dea4), and four corresponding symbiont genotypes (Kcr1–Kcr4). Host and symbiont phylogenies were highly congruent corroborating their co-divergence, consistent with host-symbiont interdependent metabolism and symbiont reduced genomes shaped by a long coevolutionary history. We compared the diversity of Angomonas/symbionts from three genera of blowflies, Lucilia, Chrysomya and Cochliomyia. A. deanei, A. desouzai, and A. ambiguus were found in the three genera of blowflies in South America. In Africa, A. deanei and A. ambiguus were identified in Chrysomya. The absence of A. desouzai in Africa and its presence in Neotropical Cochliomyia and Lucilia suggests parasite spillback of A. desouzai into Chrysomya, which was most likely introduced four decades ago from Africa into the Neotropic. The absence of correlation between parasite diversity and geographic and genetic distances, with identical genotypes of A. deanei found in the Neotropic and Afrotropic, is consistent with disjunct distribution due to the recent human-mediated transoceanic dispersal of Angomonas by Chrysomya. This study provides the most comprehensive data gathered so far on the genetic repertoires of a genus of trypanosomatids found in flies from a wide geographical range.

Descreve-se, neste trabalho, as alterações hematológicas e o proteinograma de um cão naturalmente infectado por Trypanosoma evansi. Este animal apresentou anemia normocítica normocrômica, leucopenia com linfopenia seguida por neutropenia e linfocitose; trombocitopenia; hiperproteinemia com aumento das frações beta e gama globulinas e hipoalbuminemia. Por ser este o primeiro relato de infecção por T. evansi em cães no município de Santa Maria, RS, destaca-se a sua importância epidemiológica, alertando os médicos veterinários para a existência de reservatórios do parasita na região e para a possibilidade de novos achados laboratoriais.This paper describes the hematological alterations and proteinogram of a dog naturally infected by Trypanosoma evansi. This dog was presented with normochromic-normocytic anemia, leucopenia with lymphopenia followed for neutrophenia and lymphocitosis; and trombocitopenia. Hyperproteinemia with an increase of beta and gamma globulin fractions and hypoalbuminemia. By being the first case reported of T. evansi infection in dogs in Santa Maria, RS, Brazil, the epidemiological significance of such findings will alert the veterinarians to the existence of a possible parasite's reservoir in the region warning to the possibility of new laboratory findings.

Abstract Background Trypanosoma conorhini and Trypanosoma rangeli, like Trypanosoma cruzi, are kinetoplastid protist parasites of mammals displaying divergent hosts, geographic ranges and lifestyles. Largely nonpathogenic T. rangeli and T. conorhini represent clades that are phylogenetically closely related to the T. cruzi and T. cruzi-like taxa and provide insights into the evolution of pathogenicity in those parasites. T. rangeli, like T. cruzi is endemic in many Latin American countries, whereas T. conorhini is tropicopolitan. T. rangeli and T. conorhini are exclusively extracellular, while T. cruzi has an intracellular stage in the mammalian host. Results Here we provide the first comprehensive sequence analysis of T. rangeli AM80 and T. conorhini 025E, and provide a comparison of their genomes to those of T. cruzi G and T. cruzi CL, respectively members of T. cruzi lineages TcI and TcVI. We report de novo assembled genome sequences of the low-virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E ranging from ~ 21–25 Mbp, with ~ 10,000 to 13,000 genes, and for the highly virulent and hybrid T. cruzi CL we present a ~ 65 Mbp in-house assembled haplotyped genome with ~ 12,500 genes per haplotype. Single copy orthologs of the two T. cruzi strains exhibited ~ 97% amino acid identity, and ~ 78% identity to proteins of T. rangeli or T. conorhini. Proteins of the latter two organisms exhibited ~ 84% identity. T. cruzi CL exhibited the highest heterozygosity. T. rangeli and T. conorhini displayed greater metabolic capabilities for utilization of complex carbohydrates, and contained fewer retrotransposons and multigene family copies, i.e. trans-sialidases, mucins, DGF-1, and MASP, compared to T. cruzi. Conclusions Our analyses of the T. rangeli and T. conorhini genomes closely reflected their phylogenetic proximity to the T. cruzi clade, and were largely consistent with their divergent life cycles. Our results provide a greater context for understanding the life cycles, host range expansion, immunity evasion, and pathogenesis of these trypanosomatids.

Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2.The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms.This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.

Abstract Background Proline racemase (PRAC) enzymes of Trypanosoma cruzi (TcPRAC), the agent of Chagas disease, and Trypanosoma vivax (TvPRAC), the agent of livestock trypanosomosis, have been implicated in the B-cells polyclonal activation contributing to immunosuppression and the evasion of host defences. The similarity to prokaryotic PRAC and the absence in Trypanosoma brucei and Trypanosoma congolense have raised many questions about the origin, evolution, and functions of trypanosome PRAC (TryPRAC) enzymes. Findings We identified TryPRAC homologs as single copy genes per haploid genome in 12 of 15 Trypanosoma species, including T. cruzi and T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini and T. lewisi, all parasites of mammals. Polymorphisms in TcPRAC genes matched T. cruzi genotypes: TcI-TcIV and Tcbat have unique genes, while the hybrids TcV and TcVI contain TcPRACA and TcPRACB from parental TcII and TcIII, respectively. PRAC homologs were identified in trypanosomes from anurans, snakes, crocodiles, lizards, and birds. Most trypanosomes have intact PRAC genes. T. rangeli possesses only pseudogenes, maybe in the process of being lost. T. brucei, T. congolense and their allied species, except the more distantly related T. vivax, have completely lost PRAC genes. Conclusions The genealogy of TryPRAC homologs supports an evolutionary history congruent with the Trypanosoma phylogeny. This finding, together with the synteny of PRAC loci, the relationships with prokaryotic PRAC inferred by taxon-rich phylogenetic analysis, and the absence in trypanosomatids of any other genera or in bodonids or euglenids suggest that a common ancestor of Trypanosoma gained PRAC gene by a single and ancient horizontal gene transfer (HGT) from a Firmicutes bacterium more closely related to Gemella and other species of Bacilli than to Clostridium as previously suggested. Our broad phylogenetic study allowed investigation of TryPRAC evolution over long and short timescales. TryPRAC genes diverged to become species-specific and genotype-specific for T. cruzi and T. rangeli, with resulting genealogies congruent with those obtained using vertically inherited genes. The inventory of TryPRAC genes described here is the first step toward the understanding of the roles of PRAC enzymes in trypanosomes differing in life cycles, virulence, and infection and immune evasion strategies.

Small Heat-Shock Proteins (sHSPs) and other proteins bearing alpha-crystallin domains (ACD) participate in defense against heat and oxidative stress and play important roles in cell cycle, cytoskeleton dynamics, and immunological and pathological mechanisms in eukaryotes. However, little is known about these proteins in early-diverging lineages of protists such as the kinetoplastids. Here, ACD-like proteins (ACDp) were investigated in genomes of 61 species of 12 kinetoplastid genera, including Trypanosoma spp. (23 species of mammals, reptiles and frogs), Leishmania spp. (mammals and lizards), trypanosomatids of insects, Phytomonas spp. of plants, and bodonids. Comparison of ACDps based on domain architecture, predicted tertiary structure, phylogeny and genome organization reveals a kinetoplastid evolutionarily conserved repertoire, which diversified prior to trypanosomatid adaptation to parasitic life. We identified 9 ACDp orthologs classified in 8 families of TryACD: four previously recognized (HSP20, Tryp23A, Tryp23B and ATOM69), and four characterized for the first time in kinetoplastids (TryACDP, TrySGT1, TryDYX1C1 and TryNudC). A single copy of each ortholog was identified in each genome alongside TryNudC1/TrypNudC2 homologs and, overall, ACDPs were under strong selection pressures at main phylogenetic lineages. Transcripts of all ACDPs were identified across the life stages of T. cruzi, T. brucei and Leishmania spp., but proteomic profiles suggested that most ACDPs may be species- and stage-regulated. Our findings establish the basis for functional studies, and provided evolutionary and structural support for an underestimated repertoire of ACDps in the kinetoplastids.

In trypanosomatids, the kinetoplast is the portion of the single mitochondrion that is connected to the basal body and contains the kDNA, a network composed by circular and interlocked DNA. The kDNA packing is conducted by Kinetoplast Associated Proteins (KAPs), which are similar to eukaryotic histone H1. In symbiont-harboring trypanosomatids (SHTs) such as Angomonas deanei and Strigomonas culicis, a ß-proteobacterium co-evolves with the host in a mutualistic relationship. The prokaryote confers nutritional benefits to the host and affects its cell structure. Atomic force microscopy showed that the topology of isolated kDNA networks is quite similar in the two SHT species. Ultrastructural analysis using high-resolution microscopy techniques revealed that the DNA fibrils are more compact in the kinetoplast region that faces the basal body and that the presence of the symbiotic bacterium does not interfere with kDNA topology. However, RT-PCR data revealed differences in the expression of KAPs in wild-type protozoa as compared to aposymbiotic cells. Immunolocalization showed that different KAPs present distinct distributions that are coincident in symbiont-bearing and in symbiont-free cells. Although KAP4 and KAP7 are shared by all trypanosomatid species, the expanded repertoire of KAPs in SHTs can be used as phylogenetic markers to distinguish different genera.

The generalist parasite Trypanosoma cruzi has two phylogenetic lineages associated almost exclusively with bats-Trypanosoma cruzi Tcbat and the subspecies T. c. marinkellei. We present new information on the genetic variation, geographic distribution, host associations, and potential vectors of these lineages. We conducted field surveys of bats and triatomines in southern Ecuador, a country endemic for Chagas disease, and screened for trypanosomes by microscopy and PCR. We identified parasites at species and genotype levels through phylogenetic approaches based on 18S ribosomal RNA (18S rRNA) and cytochrome b (cytb) genes and conducted a comparison of nucleotide diversity of the cytb gene. We document for the first time T. cruzi Tcbat and T. c. marinkellei in Ecuador, expanding their distribution in South America to the western side of the Andes. In addition, we found the triatomines Cavernicola pilosa and Triatoma dispar sharing shelters with bats. The comparisons of nucleotide diversity revealed a higher diversity for T. c. marinkellei than any of the T. c. cruzi genotypes associated with Chagas disease. Findings from this study increased both the number of host species and known geographical ranges of both parasites and suggest potential vectors for these two trypanosomes associated with bats in rural areas of southern Ecuador. The higher nucleotide diversity of T. c. marinkellei supports a long evolutionary relationship between T. cruzi and bats, implying that bats are the original hosts of this important parasite.

Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:1.1.1.169) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

BackgroundThe Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.Methodology/principal findingsThe genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (José-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively.ConclusionsDespite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.

It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellate's cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.