Transcriptomic analysis of benznidazole-resistant and susceptible Trypanosoma cruzi populations.

BACKGROUND: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in the chronic phase of the disease and several toxic side effects. Trypanosoma cruzi strains that are naturally resistant to both drugs have been reported. We performed a comparative transcriptomic analysis of wild-type and BZ-resistant T. cruzi populations using high-throughput RNA sequencing to elucidate the metabolic pathways related to clinical drug resistance and identify promising molecular targets for the development of new drugs for treating CD. METHODS: All complementary DNA (cDNA) libraries were constructed from the epimastigote forms of each line, sequenced and analysed using the Prinseq and Trimmomatic tools for the quality analysis, STAR as the aligner for mapping the reads against the reference genome (T. cruzi Dm28c-2018), the Bioconductor package EdgeR for statistical analysis of differential expression and the Python-based library GOATools for the functional enrichment analysis. RESULTS: The analytical pipeline with an adjusted P-value of < 0.05 and fold-change > 1.5 identified 1819 transcripts that were differentially expressed (DE) between wild-type and BZ-resistant T. cruzi populations. Of these, 1522 (83.7%) presented functional annotations and 297 (16.2%) were assigned as hypothetical proteins. In total, 1067 transcripts were upregulated and 752 were downregulated in the BZ-resistant T. cruzi population. Functional enrichment analysis of the DE transcripts identified 10 and 111 functional categories enriched for the up- and downregulated transcripts, respectively. Through functional analysis we identified several biological processes potentially associated with the BZ-resistant phenotype: cellular amino acid metabolic processes, translation, proteolysis, protein phosphorylation, RNA modification, DNA repair, generation of precursor metabolites and energy, oxidation-reduction processes, protein folding, purine nucleotide metabolic processes and lipid biosynthetic processes. CONCLUSIONS: The transcriptomic profile of T. cruzi revealed a robust set of genes from different metabolic pathways associated with the BZ-resistant phenotype, proving that T. cruzi resistance mechanisms are multifactorial and complex. Biological processes associated with parasite drug resistance include antioxidant defenses and RNA processing. The identified transcripts, such as ascorbate peroxidase (APX) and iron superoxide dismutase (Fe-SOD), provide important information on the resistant phenotype. These DE transcripts can be further evaluated as molecular targets for new drugs against CD.

Expression Profile of Genes Related to the Th17 Pathway in Macrophages Infected by Leishmania major and Leishmania amazonensis: The Use of Gene Regulatory Networks in Modeling This Pathway.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania-macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4-24 h was observed in the former. However, there was a decrease in basal levels at 48-72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania-macrophage interaction over time that extends beyond the analysis of single-gene expression.

Identification and characterization of microsatellite markers for population genetic studies of Panstrongylus megistus (Burmeister, 1835) (Triatominae: Reduviidae).

BACKGROUND: Panstrongylus megistus is the most important vector of Chagas disease in Brazil. Studies show that the principal factor hindering the control of triatomines is reinfestation of houses previously treated with insecticides. Studies at the microgeographic level are therefore necessary to better understand these events. However, an efficient molecular marker is not yet available for carrying out such analyses in this species. The aim of the present study was to identify and characterize microsatellite loci for future population genetic studies of P. megistus. METHODS: This study work consisted of five stages: (i) sequencing of genomic DNA; (ii) assembly and selection of contigs containing microsatellites; (iii) validation of amplification and evaluation of polymorphic loci; (iv) standardization of the polymorphic loci; and (v) verification of cross-amplification with other triatomine species. RESULTS: Sequencing of males and females generated 7,908,463 contigs with a total length of 2,043,422,613 bp. A total of 2,043,690 regions with microsatellites in 1,441,091 contigs were obtained, with mononucleotide repeats being the most abundant class. From a panel of 96 loci it was possible to visualize polymorphisms in 64.55% of the loci. Of the 20 loci genotyped, the number of alleles varied from two to nine with an average of 4.9. Cross-amplification with other species of triatomines was observed in 13 of the loci. CONCLUSIONS: Due to the high number of alleles encountered, polymorphism and the capacity to amplify from geographically distant populations, the microsatellites described here show promise for utilization in population genetic studies of P. megistus.

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines.

BACKGROUND: One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing. METHODS: All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search. RESULTS: The analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation. CONCLUSIONS: The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis.

Diversity and genome mapping assessment of disordered and functional domains in trypanosomatids.

The proteins that have structural disorder exemplify a class of proteins which is part of a new frontier in structural biology that demands a new understanding of the paradigm of structure/function correlations. In order to address the location, relative distances and the functional/structural correlation between disordered and conserved domains, consensus disordered predictions were mapped together with CDD domains in Leishmania braziliensis M2904, Leishmania infantum JPCM5, Trypanosoma cruzi CL-Brener Esmeraldo-like, Trypanosoma cruzi Dm28c, Trypanosoma cruzi Sylvio X10, Blechomonas ayalai B08-376 and Paratrypanosoma confusum CUL13 predicted proteomes. Our results depicts the role of protein disorder in key aspects of parasites biology highlighting: a) statistical significant association between genome structural location of protein disordered consensus stretches and functional domains; b) that disordered protein stretches appear in greater percentage at upstream or downstream position of the predicted domain; c) a possible role of structural disorder in several gene expression, control points that includes but are not limited to: i) protein folding; ii) protein transport and degradation; and iii) protein modification. In addition, for values of protein with disorder content greater than 40%, a small percentage of protein binding sites in IDPs/IDRs, a higher hypothetical protein annotation frequency was observed than expected by chance and trypanosomatid multigene families linked with virulence are rich in protein with disorder content. SIGNIFICANCE: T. cruzi and Leishmania spp are the etiological agents of Chagas disease and leishmaniasis, respectively. Currently, no vaccine or effective drug treatment is available against these neglected diseases and the knowledge about the post-transcriptional and post-translational mechanisms of these organisms, which are key for this scenario, remain scarce. This study depicts the potential impact of the proximity between protein structural disorder and functional domains in the post-transcriptional regulation of pathogenic versus human non-pathogenic trypanosomatids. Our results revealed a significant statistical relationship between the genome structural locations of these two variables and disordered regions appearing more frequently at upstream or downstream positions of the CDD locus domain. This flexibility feature would maintain structural accessibility of functional sites for post-translational modifications, shedding light into this important aspect of parasite biology. This hypothesis is corroborated by the functional enrichment analysis of disordered proteins subset that highlight the involvement of this class of proteins in protein folding, protein transport and degradation and protein modification. Furthermore, our results pointed out: a) the impact of protein disorder in the process of genome annotation (proteins tend to be annotated as hypothetical when the disorder content reaches ~40%); b) that trypanosomatid multigenic families linked with virulence have a key protein disorder content.

The molecular sensory machinery of a Chagas disease vector: expression changes through imaginal moult and sexually dimorphic features.

The triatomine bug Rhodnius prolixus is a main vector of Chagas disease, which affects several million people, mostly in Latin-America. Host searching, pheromone communication, and microclimatic preferences are aspects of its behaviour that depend on multimodal sensory inputs. The molecular bases of these sensory processes are largely unknown. The expression levels of genes transcribed in antennae were compared between 5th instar larvae, and female and male adults by means of RNA-Seq. The antennae of R. prolixus showed increased expression of several chemosensory-related genes in imaginal bugs, while both sexes had similar expression patterns for most target genes. Few cases suggest involvement of target genes in sexually dimorphic functions. Most odorant and ionotropic receptor genes seemed to be expressed in all libraries. OBPs and CSPs showed very high expression levels. Other sensory-related genes such as TRPs, PPKs and mechanoreceptors had consistent levels of expression in all libraries. Our study characterises most of the sensory gene repertoire of these insects, opening an avenue for functional genetics studies. The increase in expression of chemosensory genes suggests an enhanced role in adult bugs. This knowledge allows developing new behaviour interfering strategies, increasing the options for translational research in the vector control field.

Análise de dados de sequenciamento de RNA voltados à comparação de expressão gênica visando a um melhor entendimento dos mecanismos de resistência aos antimoniais

A origem do que hoje chamamos de Sequenciamento de Nova Geração foi impulsionada pelo sequenciamento do genoma humano e pela necessidade de inovações técnicas, tecnológicas e computacionais que reduzissem os custos e o tempo de análise. Essa necessidade possibilitou de uma maneira sem precedentes o aumento dos estudos de genômica e transcriptômica. O melhor entendimento do transcriptoma de um organismo é essencial para identificar e interpretar como a maquinaria gênica atua nos processos biológicos. Um grupo de organismos de grande interesse em saúde pública e causadores do conjunto de doenças negligenciadas conhecidas como leishmanioses são os parasitos protozoários do gênero Leishmania. Anualmente, estima-se que aproximadamente 300 mil novos casos e 20 mil mortes relacionadas à leishmaniose visceral são registrados. O tratamento das leishmanioses é problemático devido principalmente à alta toxicidade dos antimoniais pentavalentes e o surgimento de parasitos resistentes a esses compostos. Por fim, considerando que esses parasitas são agentes etiológicos de uma importante doença negligenciada, pesquisas que tragam novas perspectivas para o entendimento dos mecanismos de resistência aos compostos antimoniais são de particular importância. Neste contexto, analisamos comparativamente o transcriptoma de duas linhagens de Leishmania infantum (MHOM/BR/74/PP75), uma selvagem (LiWTS) e outra resistente ao antimônio trivalente (SbIII) (LiSbR) com o objetivo de identificar genes diferencialmente expressos que possam estar associados aos mecanismos de resistência

Para tanto, utilizamos a plataforma de sequenciamento Illumina HiSeq 2000 para o sequenciamento das amostras. Parte do processo analítico envolveu a reanotação funcional dos genes de L. infantum JPCM5 que foi utilizada como cepa referência para o processo de mapeamento das leituras geradas. As amostras foram avaliadas quanto à sua qualidade com os programas Prinseq e FastQC. As sequências adaptadoras e de baixa qualidade foram removidas utilizando o Trimmomatic. O TopHat2 foi utilizado para o processo de mapeamento das leituras no genoma de L. infantum JPCM5 e o DESeq2 para a realização das análises estatísticas

Esse pipeline analítico e a busca por genes que possuíssem um p-valor ajustado 1.2 possibilitaram a identificação de 719 genes diferencialmente expressos (699 com regulação positiva e 20 com regulação negativa) quando comparamos a linhagem resistente tratada 0.06 mg de SbIII (LiSbR 0.06) com a linhagem LiWTS , e 779 genes diferencialmente expressos (749 com regulação positiva e 30 com regulação negativa) quando comparamos as linhagens LiSbR 0.06 e LiWTS 0.06, ambas tratadas com baixa dose de antimônio. No entanto, não observamos nenhum gene diferencialmente expresso quando comparamos as linhagens selvagens tratadas e não tratadas com SbIII, indicando ausência de transcrição gênica diferencial devido ao estresse da droga. Além disso, realizamos a classificação funcional dos produtos proteicos destes genes de acordo com o Pfam. Observamos que grande parte desses genes codificam chaperonas e proteínas relacionadas a estresse, transportadores, proteínas estruturais, proteínas envolvidas nos processos de ubiquitinação e processamento de DNA e RNA, enzimas metabólicas (envolvidas nos processos de proteólise, metabolismo de ácidos graxos, carboidratos e proteínas, entre outras), controle do ciclo celular, proteínas que atuam na mediação da interação com outras proteínas, proteínas com função desconhecida na biologia de Leishmania spp. e proteínas hipotéticas. Neste estudo observamos um conjunto de genes diferencialmente expressos em L. infantum evidenciando que o fenômeno de resistência desse parasito aos compostos antimoniais é multigênico e complexo