Estudo da regulação transcricional por YgiV e da interação proteica de VisP na sinalização química e montagem do LPS na patogênese de Salmonella enterica sorovar Typhimurium
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Data
2020-11-06
Autores
Silva, Patrick da [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Bactérias patogênicas empregam proteínas para responder a estímulos externos e ativar a patogenicidade. Salmonella enterica serovar Typhimurium apresenta a proteína VisP, a qual possui papel importante na resposta a estresse e na ativação das ilhas de patogenicidade SPI-1 e 2 (Salmonella Patogenicity Island 1 and 2). O gene visP está contido em um operon o qual também apresenta ygiV, que codifica um regulador transcricional da família AraC. O presente estudo avaliou e investigou a regulação transcricional realizada por YgiV e sua correlação com o sistema de dois componentes QseBC no processo patogênico. Realizou-se análise transcriptômica do mutante ΔygiV identificando-se 379 genes diferencialmente expressos comparado com a amostra selvagem. Dentre os genes com aumento de expressão em ΔygiV, 33 pertencem a SPI-1 com função de invasão de células epiteliais. A maioria dos genes com expressão reduzida codificam proteínas de catabolismo em geral, de açúcares, aminoácidos e ácidos nucleicos. Foi realizada uma massiva análise in silico de predição da função de YgiV, assim como determinação de uma sequência consenso de reconhecimento e interação com o fator de transcrição. Determinou-se uma provável interação de YgiV com a molécula 1,2-propanodiol. Ademais, através da identificação da sequência consenso dos elementos cis que sofrem ação reguladora de YgiV, foi predito o provável regulon de YgiV. Corroborando os dados de análise transcriptômica, foram identificados genes de SPI-1 e a via de fermentação de fucose à propionato. Ensaios de análise de expressão gênica sob superexpressão de YgiV confirmaram os dados de predição in silico. Também foi avaliada a expressão gênica de alguns alvos de SPI-1 e do operon pdu, responsável pela degradação de 1,2- propanodiol à propionato. Os resultados mostraram que a atuação natural de YgiV é acentuada em ambiente anaeróbio e que a presença de fucose afeta fortemente o efeito de sua função. Por fim, ensaios de invasão de células HeLa demonstraram que YgiV pode apresentar papel de repressor transcricional, função esta que pode ser intensificada conforme a concentração de 1,2-propanodiol aumenta. Estes resultados demonstraram o papel de YgiV de regulação transcricional no processo patogênico de invasão de células epiteliais mediado por S. Typhimurium e sua relação com açúcares presentes no lúmen intestinal. E que a via de produção de propionato é imprescindível para uma colonização intestinal efetiva por S. Typhimurium, processo que depende da regulação por QseBC e YgiV.
Pathogenic bacteria employ proteins to respond to external stimuli and activate pathogenicity. Salmonella enterica serovar Typhimurium presents the VisP protein, which plays an important role in the response to stress and in the activation of the pathogenicity islands SPI-1 and 2 (Salmonella Patogenicity Island 1 and 2). The visP gene is contained in an operon which also has ygiV, which encodes an AraC family transcriptional regulator. The present study evaluated and investigated the transcriptional regulation carried out by YgiV and its correlation with the two-component QseBC system. A transcriptomic analysis of the ΔygiV mutant was performed, identifying 379 differentially expressed genes compared to the wild-type. Among the genes with increased expression in ΔygiV, 33 belong to SPI-1 with the role of epithelial cell invasion. Most genes with reduced expression code for catabolism associated proteins in general, as sugar, aminoacids and nucleic acids catabolism. A massive in silico analysis was performed to predict YgiV function, as well as the determination of a recognition and transcription factor interaction consensus sequence. A probable YgiV interaction with the 1,2-propanediol molecule was determined. Furthermore, by identifying the consensus sequence of cis elements that undergo YgiV regulatory action, the likely YgiV regulon was predicted. Confirming the transcriptomic analysis data, SPI-1 genes and the fucose fermentation pathway to propionate were identified. Gene expression analysis assays under YgiV overexpression confirmed the in silico prediction data. The gene expression of some SPI-1 targets and the pdu operon, responsible for the degradation of 1,2-propanediol to propionate, was also evaluated. The results showed that the natural performance of YgiV is accentuated in an anaerobic environment and that the presence of fucose strongly affects the effect of its function. Finally, HeLa cell invasion assays have shown that YgiV can play the role of transcriptional repressor, a function that can be enhanced as the 1,2-propanediol concentration increases. These results demonstrated the transcriptional regulation role of YgiV in in the pathogenic process of epithelial cell invasion mediated by S. Typhimurium and its relationship with sugars present in the intestinal lumen. As well as the propionate production pathway being essential for an effective intestinal colonization by S. Typhimurium, a process that depends on regulation by QseBC and YgiV.
Pathogenic bacteria employ proteins to respond to external stimuli and activate pathogenicity. Salmonella enterica serovar Typhimurium presents the VisP protein, which plays an important role in the response to stress and in the activation of the pathogenicity islands SPI-1 and 2 (Salmonella Patogenicity Island 1 and 2). The visP gene is contained in an operon which also has ygiV, which encodes an AraC family transcriptional regulator. The present study evaluated and investigated the transcriptional regulation carried out by YgiV and its correlation with the two-component QseBC system. A transcriptomic analysis of the ΔygiV mutant was performed, identifying 379 differentially expressed genes compared to the wild-type. Among the genes with increased expression in ΔygiV, 33 belong to SPI-1 with the role of epithelial cell invasion. Most genes with reduced expression code for catabolism associated proteins in general, as sugar, aminoacids and nucleic acids catabolism. A massive in silico analysis was performed to predict YgiV function, as well as the determination of a recognition and transcription factor interaction consensus sequence. A probable YgiV interaction with the 1,2-propanediol molecule was determined. Furthermore, by identifying the consensus sequence of cis elements that undergo YgiV regulatory action, the likely YgiV regulon was predicted. Confirming the transcriptomic analysis data, SPI-1 genes and the fucose fermentation pathway to propionate were identified. Gene expression analysis assays under YgiV overexpression confirmed the in silico prediction data. The gene expression of some SPI-1 targets and the pdu operon, responsible for the degradation of 1,2-propanediol to propionate, was also evaluated. The results showed that the natural performance of YgiV is accentuated in an anaerobic environment and that the presence of fucose strongly affects the effect of its function. Finally, HeLa cell invasion assays have shown that YgiV can play the role of transcriptional repressor, a function that can be enhanced as the 1,2-propanediol concentration increases. These results demonstrated the transcriptional regulation role of YgiV in in the pathogenic process of epithelial cell invasion mediated by S. Typhimurium and its relationship with sugars present in the intestinal lumen. As well as the propionate production pathway being essential for an effective intestinal colonization by S. Typhimurium, a process that depends on regulation by QseBC and YgiV.
Descrição
Palavras-chave
YgiV, Salmonella enterica, Transcrição bacteriana, Regulação metabólica, Fucose, Propanodiol