Avaliação de estruturas bacterianas envolvidas no estabelecimento do padrão de aderência híbrido localizado/difuso em amostras de Escherichia coli enteropatogênica atípica pertencentes ao sorotipo O2:H16
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Data
2020-02-19
Autores
Vieira, Melissa Arruda
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Universidade Estadual Paulista (Unesp)
Resumo
O principal mecanismo de virulência de Escherichia coli enteropatogênica (EPEC) é a capacidade de causar uma lesão histopatológica na mucosa intestinal denominada attaching and effacing (AE), caracterizada pela aderência íntima das bactérias, destruição das microvilosidades e formação de estruturas semelhantes a pedestais, ricos em F-actina, nos enterócitos infectados. Genes do locus of enterocyte effacement (região LEE) codificam todas as proteínas necessárias para a formação da lesão AE. As EPEC são divididas em típicas (tEPEC) e atípicas (aEPEC), com base na presença do EPEC adherence factor plasmid no primeiro grupo. A partir de um conjunto de sete amostras de aEPEC pertencentes ao sorotipo O2:H16, obtidos de surtos e casos esporádicos de diarreia, mostramos que cinco deles produziram uma adesão híbrida localizada/difusa (AL/AD) em células HeLa. Neste estudo, uma amostra de aEPEC deste sorotipo (282/14), que produziu o padrão AL/AD, foi selecionada para investigar as estruturas bacterianas envolvidas em seu fenótipo adesivo. Para este propósito, a amostra de aEPEC 282/14 foi mutagenizada usando o kit EZ::TN <R6Kyori/KAN-2> Tnp transposome kit, gerando uma biblioteca de inserções Tn5. Esses mutantes de inserção Tn5 foram testados quanto a perda ou redução da capacidade aderente, em ensaios realizados em 6 h de incubação com células HeLa. Dentre 320 clones pesquisados, nove foram considerados deficientes em sua capacidade de interagir com células epiteliais e quatro deles apresentaram a inserção Tn5 em genes da região LEE, como: tir, escV e grlR. Com o intuito de confirmar o papel da interação Intimina-Tir no estabelecimento deste fenótipo híbrido, foram construídos mutantes isogênicos nos genes eae e escN, que codificam a adesina intimina e a ATPase necessária para a montagem do SST3 (sistema de secreção tipo III), respectivamente. Os mutantes isogênicos gerados na intimina (eae) e no translocon do T3SS (escN) mostraram uma diminuição no número de bactérias associadas a células (P<0,05), em comparação com a amostra selvagem. A complementação in trans restaurou a capacidade dos mutantes eae e escN em aderirem eficientemente às células HeLa. A busca de polimorfismos em domínios funcionais do SST3 mostrou não interferir na adesão da amostra escolhida para este estudo. Em conclusão, demonstrou-se que o estabelecimento do fenótipo híbrido de adesão AL/AD na amostra de aEPEC 282/14 é dependente exclusivamente da interação Intimina-Tir.
The main virulence mechanism of enteropathogenic Escherichia coli (EPEC) is the capacity to cause a histopathological lesion on the intestinal mucosa, termed Attaching and Effacing (AE); characterized by intimate bacterial adherence, microvillus destruction and formation of F-actin rich pedestal-like structures, in infected enterocytes. Genes of the locus of enterocyte effacement (LEE region) encode all proteins necessary for AE lesion formation. EPEC are divided in typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in the former group. From a collection of seven aEPEC O2:H16, obtained from outbreak and sporadic cases of diarrhea, we showed that five of them produced a hybrid localized/diffuse adherence (LA/DA) in HeLa cells. In this study, we selected one aEPEC isolate of this serotype (282/14) that produced the LA/DA pattern, to investigate the bacterial structures involved in its adhesive phenotype. For this purpose, aEPEC 282/14 was mutagenized using the EZ::TN < R6Kyori/KAN-2 > Tnp transposome kit, generating a library of Tn5 insertions. These Tn5 insertion mutants were screened for non-adherent or less adherent mutants, in assays performed in 6 h of incubation with HeLa cells. Among the 320 clones screened, nine were considered deficient in their ability to interact with epithelial cells, and four of them presented the Tn5 insertion in genes within the LEE region, such as tir, escV, and grlR. In order to confirm the role of Intimin-Tir interaction in the establishment of this hybrid phenotype, we constructed isogenic mutants in the eae and escN genes, which encode the adhesin Intimin and the ATPase necessary for the T3SS (type III secretion system) assembly, respectively. The isogenic mutants generated in the Intimin (eae) and T3SS translocon (escN) showed a decrease in the number of cell-associated bacteria (P<0.05), as compared to the wild-type strain. In trans complementation restored the ability of the eae and escN mutants to adhere efficiently to HeLa cells. The screening for polymorphisms in functional domains of the SST3 demonstrated not to interfere with the adherence of the isolate chosen for this study. In conclusion, it was demonstrated that the establishment of the hybrid LA/DA adhesion phenotype in the aEPEC 282/14 isolate is dependent exclusively on the Intimin-Tir interaction.
The main virulence mechanism of enteropathogenic Escherichia coli (EPEC) is the capacity to cause a histopathological lesion on the intestinal mucosa, termed Attaching and Effacing (AE); characterized by intimate bacterial adherence, microvillus destruction and formation of F-actin rich pedestal-like structures, in infected enterocytes. Genes of the locus of enterocyte effacement (LEE region) encode all proteins necessary for AE lesion formation. EPEC are divided in typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in the former group. From a collection of seven aEPEC O2:H16, obtained from outbreak and sporadic cases of diarrhea, we showed that five of them produced a hybrid localized/diffuse adherence (LA/DA) in HeLa cells. In this study, we selected one aEPEC isolate of this serotype (282/14) that produced the LA/DA pattern, to investigate the bacterial structures involved in its adhesive phenotype. For this purpose, aEPEC 282/14 was mutagenized using the EZ::TN < R6Kyori/KAN-2 > Tnp transposome kit, generating a library of Tn5 insertions. These Tn5 insertion mutants were screened for non-adherent or less adherent mutants, in assays performed in 6 h of incubation with HeLa cells. Among the 320 clones screened, nine were considered deficient in their ability to interact with epithelial cells, and four of them presented the Tn5 insertion in genes within the LEE region, such as tir, escV, and grlR. In order to confirm the role of Intimin-Tir interaction in the establishment of this hybrid phenotype, we constructed isogenic mutants in the eae and escN genes, which encode the adhesin Intimin and the ATPase necessary for the T3SS (type III secretion system) assembly, respectively. The isogenic mutants generated in the Intimin (eae) and T3SS translocon (escN) showed a decrease in the number of cell-associated bacteria (P<0.05), as compared to the wild-type strain. In trans complementation restored the ability of the eae and escN mutants to adhere efficiently to HeLa cells. The screening for polymorphisms in functional domains of the SST3 demonstrated not to interfere with the adherence of the isolate chosen for this study. In conclusion, it was demonstrated that the establishment of the hybrid LA/DA adhesion phenotype in the aEPEC 282/14 isolate is dependent exclusively on the Intimin-Tir interaction.
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Palavras-chave
aEPEC, Escherichia coli enteropatogênica atípica, Sistema de secreção do tipo III, Adesão, Adhesion, Type III secretion system (T3SS)