Filogenia de Pythium insidiosum pelos genes codificantes do fator de alongamento da tradução (Tef-1α), α e β-tubulina e análise do padrão de restrição por Pulse-Field Gel Electrophoresis (PFGE)
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Data
2020-02-28
Autores
Prado, Ana Carolina do
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Universidade Estadual Paulista (Unesp)
Resumo
Pythium insidiosum é o agente etiológico da pitiose, uma infecção granulomatosa crônica, com prevalência em regiões de clima tropical e subtropical que acomete mamíferos, principalmente equinos, cães e humanos. Este micro-organismo é um falso fungo que apresenta uma ampla distribuição geográfica, sendo muito prevalente na América do Sul (pitiose equina e canina) e na Tailândia (pitiose humana). Estudos moleculares têm permitido diagnóstico precoce e melhor compreensão das relações filogenéticas, dividindo o patógeno em três clados (I, II e III ou A, B e C). Entretanto essas informações são ainda bastante limitadas e novas regiões gênicas poderiam ajudar a esclarecer a história evolutiva dessa espécie. Assim sendo, este trabalho visou estabelecer as relações filogenéticas entre 52 isolados de P. insidiosum, americanos e asiáticos, por meio do sequenciamento de três novas regiões gênicas: a região do fator de alongamento da tradução (Tef-1α), α e β tubulina, além da padronização da técnica de Pulse Filed Gel Electrophoresis (PFGE) para esta espécie. A região do Tef-1α mostrou-se menos polimórfica em relação a α e β tubulina, entretanto separou as cepas aqui trabalhadas em dois clados distintos, sendo um composto apenas de cepas classificadas previamente como sendo do clado III (asiáticas), e agrupou todas as cepas americanas junto às cepas do clado II. A filogenia baseada no gene da β tubulina separou os isolados nos três clados esperados. Em relação à α tubulina houve diferenciação dos isolados em clados I e II, não observando amplificação do clado III. As cepas do clado II se subdividiram em dois clados monofiléticos. Concluímos que os resultados da filogenia dos genes aqui trabalhados são congruentes com os dados até então estudados para essa espécie, corroborando a hipótese de expansão clonal das cepas asiáticas para o continente americano. As análises de PFGE estão em fase de padronização dos perfis eletroforéticos e acreditamos que poderá complementar os dados sobre as relações evolutivas de Pythium insidiosum.
Pythium insidiosum is the etiological agent of pythiosis, a chronic granulomatous infection, prevalent in tropical and subtropical regions that affects mammals, especially horses, dogs and humans. This is a fungus-like microorganism that has a wide geographical distribution and is very prevalent in South America (equine and canine pythiosis) and Thailand (human pythiosis). Molecular studies have allowed early diagnosis and better understand of phylogenetic relationships, and the pathogen is currently divided into three clades (I, II and III or A, B and C). However, this information is still quite limited for this pathogen and new gene regions could help understanding the evolutionary history of this species. Therefore, this study aimed to establish the phylogenetic relationships among 52 american and asian P. insidiosum isolates by sequencing three new gene regions: the translation elongation factor (Tef-1α), α and β tubulin, in addition to the standardization of the Pulse Filed Gel Electrophoresis (PFGE) technique for this species. The Tef-1α region showed little polymorphic in relation to α and β tubulin, however it separated the strains here studied into two distinct clades, being composed only of strains previously classified as clade III (Asian), and grouped all the American strains next to the clade II strains. The β tubulin gene-based phylogeny separated the isolates into the three expected clades. Regarding α tubulin there was differentiation of isolates in clades I and II, not observing amplification of clade III. The clade II strains were subdivided into two monophyletic clades. We conclude that the results of the phylogeny of the genes here evaluated are consistent with the data previously studied for this species, corroborating the hypothesis of clonal expansion of Asian strains to the American continent. PFGE analyzes are in the phase of standardization of electrophoretic profiles and we believe it may complement the data on the evolutionary relationships of Pythium insidiosum.
Pythium insidiosum is the etiological agent of pythiosis, a chronic granulomatous infection, prevalent in tropical and subtropical regions that affects mammals, especially horses, dogs and humans. This is a fungus-like microorganism that has a wide geographical distribution and is very prevalent in South America (equine and canine pythiosis) and Thailand (human pythiosis). Molecular studies have allowed early diagnosis and better understand of phylogenetic relationships, and the pathogen is currently divided into three clades (I, II and III or A, B and C). However, this information is still quite limited for this pathogen and new gene regions could help understanding the evolutionary history of this species. Therefore, this study aimed to establish the phylogenetic relationships among 52 american and asian P. insidiosum isolates by sequencing three new gene regions: the translation elongation factor (Tef-1α), α and β tubulin, in addition to the standardization of the Pulse Filed Gel Electrophoresis (PFGE) technique for this species. The Tef-1α region showed little polymorphic in relation to α and β tubulin, however it separated the strains here studied into two distinct clades, being composed only of strains previously classified as clade III (Asian), and grouped all the American strains next to the clade II strains. The β tubulin gene-based phylogeny separated the isolates into the three expected clades. Regarding α tubulin there was differentiation of isolates in clades I and II, not observing amplification of clade III. The clade II strains were subdivided into two monophyletic clades. We conclude that the results of the phylogeny of the genes here evaluated are consistent with the data previously studied for this species, corroborating the hypothesis of clonal expansion of Asian strains to the American continent. PFGE analyzes are in the phase of standardization of electrophoretic profiles and we believe it may complement the data on the evolutionary relationships of Pythium insidiosum.
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Palavras-chave
Pitiose, Pythium insidiosum, Fator de alongamento da tradução (Tef-1α), α- tubulina, β-tubulina, Filogenia, Identificação molecular, Padrão de restrição, Pythiosis, Phylogeny, Molecular identification