RESUMO
Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control.
Assuntos
DNA de Protozoário/genética , Evolução Molecular , Genes de Protozoários , Genoma de Protozoário , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Brasil/epidemiologia , Deleção de Genes , Leishmania infantum/patogenicidade , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/transmissão , Epidemiologia Molecular , Filogenia , Deleção de Sequência , Sequenciamento Completo do GenomaRESUMO
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.
Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Proteínas de Protozoários/genética , Animais , Leishmania infantum/metabolismo , Leishmania infantum/fisiologia , Proteínas de Protozoários/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Pathogen fitness landscapes change when transmission cycles establish in non-native environments or spill over into new vectors and hosts. The introduction of Leishmania infantum in the Americas into the Neotropics during European colonization represents a unique case study to investigate the mechanisms of ecological adaptation of this important parasite. Defining the evolutionary trajectories that drive L. infantum fitness in this new environment are of great public health importance as they will allow unique insight into pathways of host/pathogen co-evolution and their consequences for region-specific changes in disease manifestation. This review summarizes current knowledge on L. infantum genetic and phenotypic diversity in the Americas and its possible role in the unique epidemiology of visceral leishmaniasis (VL) in the New World. We highlight the importance of appreciating adaptive molecular mechanisms in L. infantum to understand the parasites' successful establishment on the continent.
Assuntos
Leishmania infantum/classificação , Leishmaniose Visceral/transmissão , Oceano Atlântico , Evolução Molecular , Aptidão Genética , Humanos , Leishmania infantum/genética , FenótipoRESUMO
Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.
Assuntos
Antimônio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/metabolismo , Fosfoproteínas/análise , Eletroforese em Gel Diferencial Bidimensional/métodos , Sequência de Aminoácidos , Simulação por Computador , Resistência a Medicamentos/efeitos dos fármacos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Reprodutibilidade dos TestesRESUMO
Mitogen-activated protein kinases (MAPKs) are involved in environmental signal sensing. They are thus expected to play key roles in the biology of Trypanosomatid parasites, which display complex life cycles and use extracellular cues to modulate cell differentiation. Despite their relevance, structural data of Trypanosomatid MAPKs is lacking. We have now determined the crystal structure of Leishmania major LmaMPK10, a stage-specifically activated MAPK, both alone and in complex with SB203580. LmaMPK10 was observed to be more similar to p38 than to other human MAPKs. However, significant differences could be identified in the catalytic pocket, as well as in potentially regulatory sites in the N-terminal lobe. The modified pocket architecture in LmaMPK10 precludes DFG-in/DFG-out regulatory flipping as observed in mammalian MAPKs. LmaMPK10-nucleotide association was also studied, revealing a potential C-terminal autoinhibitory mechanism. Overall, these data should speed the discovery of molecules interfering with LmaMPK10 functions, with relevance for antileishmanial drug development strategies.