Evolução das espécies do gênero sporothrix baseado nas sequências dos inteins vma e prp8
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Data
2020-10-30
Autores
Oliveira, Alana Lucena
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Universidade Estadual Paulista (Unesp)
Resumo
A esporotricose é uma micose causada principalmente pelas espécies do clado patogênico do gênero Sporothrix. Estas espécies compreendem S. schenckii, S. brasiliensis, S. globosa e S. luriei. S. schenckii tem uma ampla distribuição mundial enquanto S. globosa é uma sapronose comum na Ásia e S. luriei é uma espécie bem restrita e com pouquíssimos relatos. A esporotricose causada por S. brasiliensis é uma zoonose que afeta principalmente os gatos, cuja incidência aumentou no Brasil na última década, sendo considerada uma das micoses emergentes mais importantes na atualidade. Visando a eliminação de surtos, junto a campanhas de prevenção e medidas de vigilância, é muito importante contar com métodos diagnósticos de baixo custo, rápidos e precisos compreendendo as corretas espécies envolvidas. Foi realizada a padronização e diferenciação das espécies do clado patogênico do gênero Sporothrix por meio de biologia molecular utilizando regiões gênicas específicas dos inteins VMA e PRP8 que ainda não foram estudas para essas espécies. A região dos inteins VMA e PRP8 já foram utilizadas com finalidades de diagnóstico e estudos filogenéticos em outros fungos, sendo, portanto, uma região promissora para esses estudos no gênero Sporothrix Inteins são sequencias de proteínas presentes em alguns organismos que têm a capacidade de realizar um processo de auto-splicing logo após a tradução da sua proteína hospedeira. Sendo comprovada a existência dos dois inteins (VMA e PRP8) na maioria das espécies do clado patogênico de Sporothrix spp., o objetivo foi caracterizar os inteins PRP8 e VMA visando inferências diagnósticas e evolutivas, incluindo amostras de DNA a partir de isolados em cultivo . Até então, analisamos 22 cepas de isolados clínicos (5 S. globosa, 5 S. schenckii, 12 S. brasiliensis) pelo sequenciamento das regiões dos inteins com primers por nós desenhados e analisamos por bioinformática uma cepa de S. luriei. O intein PRP8 foi encontrado em todas as espécies enquanto o VMA foi ausente em S. globosa e S. luriei. Os inteins sempre foram encontrados na forma de Full-length intein. As análises filogenéticas permitiram a diferenciação das espécies de S. schenckii próximo a S. brasiliensis e S. globosa mais próxima de S. luriei ao analisar as sequencias do intein PRP8. Igualmente, o intein VMA evidenciou um agrupamento separado das espécies S. schenckii e S. brasiliensis. A presença ou ausência e análise do polimorfismo encontrado no intein VMA permitiu desenhar um par de primers adicional para diferenciar as espécies S. schenckii, S. brasiliensis e S. globosa por meio de PCR/eletroforese, sem necessidade de sequenciamento de DNA.
Sporotrichosis is a fungal disease caused mainly by species of the pathogenic clade of the genus Sporothrix. These species comprise S. schenckii, S. brasiliensis, S. globosa and S. luriei. S. schenckii has a broad worldwide distribution while S. globosa is a common sapronosis in Asia and S. luriei is a very restricted species with very few reports associated with disease. S. brasiliensis is a zoonosis that mainly affects cats and has had a great incidence in Brazil in the last decade, being considered one of the most important emerging mycoses in recent times. In order to eliminate outbreaks, along with prevention campaigns and surveillance measures, it is very important to have low-cost, fast and accurate diagnostic methods, identifying the correct species involved. Standardization and differentiation of the species from the pathogenic clade of the complex genus Sporothrix were carried out by means of molecular biology using specific gene regions of the VMA and PRP8 inteins that have not yet been studied within these species. The VMA and PRP8 intein regions have already been used for the purposes of diagnosis and phylogenetic studies in other fungi, and are thus considered promising regions for the genus Sporothrix. Inteins are protein sequences present in some organisms that have the ability to perform a self-splicing process just after their host protein is translated. Since the existence of both inteins is proven in the majority of all species of the Sporothrix pathogenic clade, we aim to characterize the PRP8 and VMA inteins in order to diagnose these species molecularly and to determine the evolutionary history of the group, including DNA samples from cultivated isolates. Currently, we have 22 strains of clinical isolates (5 S. globosa, 5 S. schenckii, 12 S. brasiliensis) by sequencing the regions with designed primers and one strain of S. luriei that was analyzed by bioinformatics. The PRP8 intein was found in all species while the VMA intein was absent in S. globosa and S. luriei. Inteins was always found in full-length form. Phylogenetic analyses allowed species differentiation by grouping S. schenckii close to S. brasiliensis and S. globosa close to S. luriei when analyzing the sequences of the PRP8 intein. Likewise, the VMA intein showed a separate clustering of the species S. schenckii and S. brasiliensis. The absence of inteins in S. globosa and the polymorphism found in the VMA intein allowed the designing of an additional primer set to differentiate the species S. schenkii, S. brasiliensis and S. globosa thought only a PCR/Eletrophoresis technique without the need for sequencing.
Sporotrichosis is a fungal disease caused mainly by species of the pathogenic clade of the genus Sporothrix. These species comprise S. schenckii, S. brasiliensis, S. globosa and S. luriei. S. schenckii has a broad worldwide distribution while S. globosa is a common sapronosis in Asia and S. luriei is a very restricted species with very few reports associated with disease. S. brasiliensis is a zoonosis that mainly affects cats and has had a great incidence in Brazil in the last decade, being considered one of the most important emerging mycoses in recent times. In order to eliminate outbreaks, along with prevention campaigns and surveillance measures, it is very important to have low-cost, fast and accurate diagnostic methods, identifying the correct species involved. Standardization and differentiation of the species from the pathogenic clade of the complex genus Sporothrix were carried out by means of molecular biology using specific gene regions of the VMA and PRP8 inteins that have not yet been studied within these species. The VMA and PRP8 intein regions have already been used for the purposes of diagnosis and phylogenetic studies in other fungi, and are thus considered promising regions for the genus Sporothrix. Inteins are protein sequences present in some organisms that have the ability to perform a self-splicing process just after their host protein is translated. Since the existence of both inteins is proven in the majority of all species of the Sporothrix pathogenic clade, we aim to characterize the PRP8 and VMA inteins in order to diagnose these species molecularly and to determine the evolutionary history of the group, including DNA samples from cultivated isolates. Currently, we have 22 strains of clinical isolates (5 S. globosa, 5 S. schenckii, 12 S. brasiliensis) by sequencing the regions with designed primers and one strain of S. luriei that was analyzed by bioinformatics. The PRP8 intein was found in all species while the VMA intein was absent in S. globosa and S. luriei. Inteins was always found in full-length form. Phylogenetic analyses allowed species differentiation by grouping S. schenckii close to S. brasiliensis and S. globosa close to S. luriei when analyzing the sequences of the PRP8 intein. Likewise, the VMA intein showed a separate clustering of the species S. schenckii and S. brasiliensis. The absence of inteins in S. globosa and the polymorphism found in the VMA intein allowed the designing of an additional primer set to differentiate the species S. schenkii, S. brasiliensis and S. globosa thought only a PCR/Eletrophoresis technique without the need for sequencing.
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Palavras-chave
Sporotrix spp., Intein VMA, Intein PRP8, Diagnóstico molecular, Filogenia, Molecular diagnosis, Phylogen