The tritryps comparative repeatome: insights on repetitive element evolution in trypanosomatid pathogens

The major human pathogens Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major are collectively known as the Tritryps. The initial comparative analysis of their genomes has uncovered that Tritryps share a great number of genes, but repetitive DNA seems to be extremely variable between them. However, the in-depth characterization of repetitive DNA in these pathogens has been in part neglected, mainly due to the well-known technical challenges of studying repetitive sequences from de novo assemblies using short reads. Here, we compared the repetitive DNA repertories between the Tritryps genomes using genome-wide, low-coverage Illumina sequencing coupled to RepeatExplorer analysis. Our work demonstrates that this extensively implemented approach for studying higher eukaryote repeatomes is also useful for protozoan parasites like trypanosomatids, as we recovered previously observed differences in the presence and amount of repetitive DNA families. Additionally, our estimations of repetitive DNA abundance were comparable to those obtained from enhanced-quality assemblies using longer reads. Importantly, our methodology allowed us to describe a previously undescribed transposable element in Leishmania major (TATE element), highlighting its potential to accurately recover distinctive features from poorly characterized repeatomes. Together, our results support the application of this low-cost, low-coverage sequencing approach for the extensive characterization of repetitive DNA evolutionary dynamics in trypanosomatid and other protozoan genomes

Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences, and chromosomal rearrangements

Neospora caninum primarily infects cattle, causing abortions, with an estimated impact of a billion dollars on the worldwide economy annually. However, the study of its biology has been unheeded by the established paradigm that it is virtually identical to its close relative, the widely studied human pathogen Toxoplasma gondii. By revisiting the genome sequence, assembly, and annotation using third-generation sequencing technologies, here we show that the N. caninum genome was originally incorrectly assembled under the presumption of synteny with T. gondii. We show that major chromosomal rearrangements have occurred between these species. Importantly, we show that chromosomes originally named Chr VIIb and VIII are indeed fused, reducing the karyotype of both N. caninum and T. gondii to 13 chromosomes. We reannotate the N. caninum genome, revealing more than 500 new genes. We sequence and annotate the nonphotosynthetic plastid and mitochondrial genomes and show that although apicoplast genomes are virtually identical, high levels of gene fragmentation and reshuffling exist between species and strains. Our results correct assembly artifacts that are currently widely distributed in the genome database of N. caninum and T. gondii and, more importantly, highlight the mitochondria as a previously oversighted source of variability and pave the way for a change in the paradigm of synteny, encouraging rethinking the genome as basis of the comparative unique biology of these pathogens.INIA: FSSA_X_2014_1_10602

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi's genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a 'core compartment' and a 'disruptive compartment' which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes

Clinical and epidemiological features of tuberculosis isolated from critically ill patients

Human tuberculosis is still a major world health concern. In Uruguay, contrary to the world trend, an increase in cases has been observed since 2006. Although the incidence of MDR-resistant strains is low and no cases of XDR-TB were registered, an increase in the number of patients with severe tuberculosis requiring critical care admission was observed. As a first aim, we performed the analysis of the genetic structure of strains isolated from patients with severe tuberculosis admitted to an intensive care unit. We compared these results with those corresponding to the general population observing a statistically significant increase in the Haarlem genotypes among ICU patients (53.3% vs 34.7%; p < 0.05). In addition, we investigated the association of clinical outcomes with the genotype observing a major incidence of hepatic dysfunctions among patients infected with the Haarlem strain (p < 0.05). The cohort presented is one of the largest studied series of critically ill patients with tuberculosis

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi

Indigenous ancestry and admixture in the uruguayan population

The Amerindian group known as the Charrúas inhabited Uruguay at the timing of European colonial contact. Even though they were extinguished as an ethnic group as a result of a genocide, Charrúan heritage is part of the Uruguayan identity both culturally and genetically. While mitochondrial DNA studies have shown evidence of Amerindian ancestry in living Uruguayans, here we undertake whole-genome sequencing of 10 Uruguayan individuals with self-declared Charruan heritage. We detect chromosomal segments of Amerindian ancestry supporting the presence of indigenous genetic ancestry in living descendants. Specific haplotypes were found to be enriched in “Charrúas” and rare in the rest of the Amerindian groups studied. Some of these we interpret as the result of positive selection, as we identified selection signatures and they were located mostly within genes related to the infectivity of specific viruses. Historical records describe contacts of the Charrúas with other Amerindians, such as Guaraní, and patterns of genomic similarity observed here concur with genomic similarity between these groups. Less expected, we found a high genomic similarity of the Charrúas to Diaguita from Argentinian and Chile, which could be explained by geographically proximity. Finally, by fitting admixture models of Amerindian and European ancestry for the Uruguayan population, we were able to estimate the timing of the first pulse of admixture between European and Uruguayan indigenous peoples in approximately 1658 and the second migration pulse in 1683. Both dates roughly concurring with the Franciscan missions in 1662 and the foundation of the city of Colonia in 1680 by the Spanish.ANII: FSDA_1_2017_1_14364

A nature-inspired design yields a new class of steroids against trypanosomatids

This article belongs to the Special Issue Drug Discovery for Neglected DiseasesChagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as public health problems by the World Health Organization. These diseases affect millions of people around the world however, efficient and low-cost treatments are not available. Different steroid molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks, plants, fungi). These molecules have complex structures that make de novo synthesis extremely difficult. In this work, we designed new and simpler compounds with antiparasitic potential inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound 8 (ID_1260) was active in vitro (IC50 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD50 >2000 mg/kg) and no genotoxic effects, being a promising compound for anti-trypanosomatid drug development

El locus H de trypanosoma cruzi : caracterización de los genes pteridina-reductasa 1 y TCP17 / Carlos Alberto Robello Porto

Se realizó la caracterización de dos genes de Trypanosoma cruzi, tcp17 y pteridina reductasa 1, contiguos al gen tcpgp2. El gen tcp17 presenta un marco de lectura de 480 pb, y codifica para una proteína de 16,5 KDa. El análisis de homologías muestra que pertenece a una familia altamente conservada en la escala evolutiva, denominada YER057c. tcp17 es un gen de copia única que se localiza en dos cromosomas de 0,9 y 1,2 Mb, y se transcribe en los tres estadíos del parásito. Sin embargo la proteína se expresa diferencialmente siendo mayoritaria su expresión en el estadío epimastigota. Los estudios de localización por inmunofluorescencia y microscopía electrónica muestran que TCP17 es una proteína con una amplia distribución por toda la célula, pero su mayor concentración se observó en membrana plasmática, aparato de Golgi y estructuras vacuolares. El gen ptr1 (pteridina reductasa 1) es también un gen de copia única, presentando sus mayores homologías con los genes ptr1 de Leishmania y T. brucei. Las proteínas para las que codifican estos genes pertenecen a la familia de las deshidrogenasas/reductasas de cadena corta, ampliamente distribuídas en la escala evolutiva, y en ptr1 están presentes todos los motivos aminoacídicos conservados descritos para esta familia. Los estudios de expresión de PTR1 muestran que esta proteína también se expresa diferencialmente, siendo mayoritaria su expresión en el estadío epimastigota. Los estudios de transfección de este gen en T. cruzi muestran que su sobreexpresión confiere resistencia a los antifolatos aminopterina, trimetoprim y metotrexato, y no a pirimetamina. Para los ensayos enzimáticos la proteína se expresó en E. coli y se purificó por cromatografía de afinidad. PTR1 presentó actividad reductora dependiente de NADPH para los sustratos biopterina, dihidrobiopterina, folato y dihidrofolato, mientras que no se detectó ninguna actividad fTesis Universidad de Granada. Facultad de Ciencia

Nanopore Sequencing Significantly Improves Genome Assembly of the Protozoan Parasite Trypanosoma cruzi

Submitted by Nuzia Santos ([email protected]) on 2019-10-04T19:42:46Z No. of bitstreams: 1 Nanopore Sequencing Significantly.pdf: 446955 bytes, checksum: 8f6eb5397f33dccb221216fe8bbc8e43 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-10-04T19:45:25Z (GMT) No. of bitstreams: 1 Nanopore Sequencing Significantly.pdf: 446955 bytes, checksum: 8f6eb5397f33dccb221216fe8bbc8e43 (MD5)Made available in DSpace on 2019-10-04T19:45:25Z (GMT). No. of bitstreams: 1 Nanopore Sequencing Significantly.pdf: 446955 bytes, checksum: 8f6eb5397f33dccb221216fe8bbc8e43 (MD5) Previous issue date: 2019Laboratory of Host Pathogen Interactions – UBM. Institut Pasteur de Montevideo. Montevideo, Uruguay.Laboratory of Host Pathogen Interactions – UBM. Institut Pasteur de Montevideo. Montevideo, Uruguay / Seccion Genetica Evolutiva. Facultad de Ciencias. Universidad de la Republica. Montevideo, Uruguay.Laboratory of Host Pathogen Interactions – UBM. Institut Pasteur de Montevideo. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Rene Rachou. Grupo de Pesquisa Triatomíneos. Belo Horizonte, MG, Brasil.Microbial Genomics Laboratory. Institut Pasteur Montevideo. Montevideo, Uruguay / Center for Integrative Biology, Universidad Mayor, Santiago de Chile, Chile.Laboratory of Host Pathogen Interactions – UBM. Institut Pasteur de Montevideo. Montevideo, Uruguay / Departamento de Bioquımica. Facultad de Medicina. Universidad de la Republica. Montevideo, Uruguay.Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a 2-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation, and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ∼16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single-copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource because it now constitutes a high-quality assembly available for TcII (clade C), a prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of these parasites and reinforces the idea that T. cruzi is intraspecifically divided in three main clades. Finally, this work represents the introduction of Nanopore technology to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes

Informe final del proyecto: Generación de un banco de cepas y ADN estandarizado para estudios epidemiológicos de Mycobacterium bovis en Uruguay

La tuberculosis bovina (TBB) es una enfermedad infecto-contagiosa crónica del ganado que en ocasiones afecta a otras especies de mamíferos incluyendo al hombre. Recientemente se ha observado una duplicación en el número de establecimientos positivos a TB en Uruguay, generando preocupación en el sector ganadero, así como en las autoridades sanitarias pertinentes. El ganado detectado positivo a T. Bovis se mantiene aislado y es sacrificado. Algunas muestras son posteriormente cultivadas para confirmar el agente infeccioso. Sin embargo esto se realiza para un número reducido de muestras y no se realizan otro tipo de análisis de vanguardia que permiten estudiar la epidemiología de la enfermedad como ser la tipificación. Para poder evaluar la epidemiología de las enfermedades micobacterianas es fundamental poder discriminar a los miembros del complejo Mycobacterium tuberculosis. La diversidad genómica entre cepas de M. bovis es un factor importante en la determinación de la patogénesis que puede influir en el grado de virulencia, transmisibilidad, y respuesta del huésped. El diseño de estrategias e intervenciones en salud pública basadas en evidencias sobre la evolución, aparición y propagación de enfermedades infecciosas es uno de los principales objetivos de la epidemiología actual. El seguimiento que permita el trazado de las cepas de M. bovis circulantes en nuestro país con el fin de estudiar la dinámica de la TBB en Uruguay en relación a los datos genéticos/genómicos es de suma importancia. El objetivo de este proyecto es realizar un plan piloto para establecer un banco de islados y ADN que permita el seguimiento de la enfermedad a través de estudios bioquímicos y marcadores genéticos, que permita predecir el desarrollo de la enfermedad y mas importante ayude a establecer políticas concisas para el control de la enfermedad.Agencia Nacional de Investigación e Innovació