Estudo do sensor quinase QseC de Salmonella Typhimurium com análise transcriptômica e sequenciamento de inserção de transposon (TraDIS)
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Data
2021-12-08
Autores
Manieri, Fernanda Zani
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Universidade Estadual Paulista (Unesp)
Resumo
Sinalização química em bactérias patogênicas é um mecanismo empregado para interagir com o hospedeiro e sua respectiva microbiota. Através desta interação patógeno-hospedeiro ocorre a regulação da virulência em bactérias. Salmonella Typhimurium é uma bactéria de grande importância clínica muito utilizada no estudo da interação patógeno-hospedeiro. O estudo da sinalização química via QseBC e demais sistemas de dois componentes é essencial na compreensão das vias regulatórias que compõem o processo patogênico. O objetivo do trabalho é investigar o papel do sensor quinase QseC e demais mecanismos moleculares na regulação da virulência de S. Typhimurium, e elucidar o conjunto de genes essenciais para a sobrevivência de Salmonella ao estresse ácido. Desta forma, análises transcriptômica (RNA-seq) e computacionais foram direcionadas para análise da expressão de genes alvos via qRT-PCR, mutagênese via Lambda Red, ensaio de estresse ácido, sequenciamento via inserção de transposon dirigida (TraDIS), ensaio de motilidade, ensaio de resistência a peptídeos antimicrobianos e análise da eficácia do composto LED209 em camundongos. O RNA-seq evidenciou a alteração na expressão de 1371 genes após a deleção de qseC (cerca de 29% do genoma), sendo que 766 genes tiveram sua expressão reduzida e 605 genes tiveram expressão aumentada. Entre os principais resultados estavam processos de regulação de genes de virulência, metabolismo anaeróbico, modificações de membrana, montagem flagelar e secreção das flagelinas, motilidade, resistência a polimixina B, além de evidências de sua interação com o sistema de dois componentes PmrAB. A motilidade swarming apresentou redução na cepa mutante, apesar do aumento da secreção de flagelina detectada via Western blot. Também foi detectada a interação da proteína QseB com sua própria região regulatória e do gene ygiV. A análise via TraDIS revelou um conjunto de genes essenciais de resistência ao estresse ácido de Salmonella distinto dos descritos previamente para Escherichia coli, com destaque para genes que participam da resposta ao estímulo de temperatura. Desse modo, é notável a participação do sistema QseBC na regulação do metabolismo e virulência em S. Typhimurium, bem como a utilização de análises robustas têm sido promissoras para investigação de novos alvos e terapias anti-virulência.
Chemical signaling in pathogenic bacteria is a mechanism employed for communication with the host and microbiota. The bacterial virulence regulation occurs through host-pathogen interaction. Salmonella Typhimurium is a bacterium of clinical relevance, considered a model in host-pathogen interaction studies. The study of chemical signaling via QseBC, and other two-component systems is essential to understand the regulatory pathways that are part of the pathogenic process. The aim of the study was to investigate the role of QseC kinase sensor and molecular mechanisms in S. Typhimurium virulence regulation and elucidate the genes that are essential for Salmonella acid survival. Thus, transcriptomic (RNA-seq) and computational analysis were done, followed by gene expression via qRT-PCR, Lambda Red mutagenesis, acid stress resistance, transposon-directed insertion site sequencing (TraDIS), motility assays, antimicrobial peptides resistance assays and efficacy analysis of the compound LED209 in mice. The RNA-seq have shown changes in gene expression after the deletion of qseC, (around 29% of the genome), in which 766 genes were downregulated and 605 were upregulated. The main processes involved were virulence, anaerobic metabolism, membrane modification, flagella assembly and flagellin secretion, motility, polymyxin B resistance and evidence of interaction with PmrAB system. The swarming motility was reduced in the mutant stran, even with the increase in flagellin secretion detected by Western blot. The QseB interaction with both ygiV regulatory region and its own regulatory region was detected. The TraDIS analysis revealed a group of essential genes of Salmonella acid resistome that is different from the previous described in Escherichia coli, with a highlight in genes involved with temperature stimuli response. Thus, it is notable the QseBC participation in S. Typhimurium metabolism and virulence regulation, as well as the use of robust analyses have been promising for the investigation of new targets and antivirulence therapies.
Chemical signaling in pathogenic bacteria is a mechanism employed for communication with the host and microbiota. The bacterial virulence regulation occurs through host-pathogen interaction. Salmonella Typhimurium is a bacterium of clinical relevance, considered a model in host-pathogen interaction studies. The study of chemical signaling via QseBC, and other two-component systems is essential to understand the regulatory pathways that are part of the pathogenic process. The aim of the study was to investigate the role of QseC kinase sensor and molecular mechanisms in S. Typhimurium virulence regulation and elucidate the genes that are essential for Salmonella acid survival. Thus, transcriptomic (RNA-seq) and computational analysis were done, followed by gene expression via qRT-PCR, Lambda Red mutagenesis, acid stress resistance, transposon-directed insertion site sequencing (TraDIS), motility assays, antimicrobial peptides resistance assays and efficacy analysis of the compound LED209 in mice. The RNA-seq have shown changes in gene expression after the deletion of qseC, (around 29% of the genome), in which 766 genes were downregulated and 605 were upregulated. The main processes involved were virulence, anaerobic metabolism, membrane modification, flagella assembly and flagellin secretion, motility, polymyxin B resistance and evidence of interaction with PmrAB system. The swarming motility was reduced in the mutant stran, even with the increase in flagellin secretion detected by Western blot. The QseB interaction with both ygiV regulatory region and its own regulatory region was detected. The TraDIS analysis revealed a group of essential genes of Salmonella acid resistome that is different from the previous described in Escherichia coli, with a highlight in genes involved with temperature stimuli response. Thus, it is notable the QseBC participation in S. Typhimurium metabolism and virulence regulation, as well as the use of robust analyses have been promising for the investigation of new targets and antivirulence therapies.
Descrição
Palavras-chave
Sinalização química, Virulência, Patogenicidade, Sistema de dois componentes, Chemical signaling, Virulence, Pathogenicity, Two-component system