O papel da sinalização química via sensor histidina quinase QseC na virulência de Escherichia coli enteroagregativa Stx+
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Data
2021-11-30
Autores
Machado Ribeiro, Tamara Renata
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Escherichia coli enteroagregativa (EAEC) está envolvida em casos de diarreia aguda e persistente (≥14 dias) em crianças e adultos de países desenvolvidos e em desenvolvimento. Possui um padrão de adesão exclusivo em cultivo de células epiteliais, denominado adesão agregativa (AA). São descritas adesinas e toxinas nos mecanismos de patogenicidade de EAEC. A formação de um espesso biofilme, mediada principalmente por fímbrias de adesão agregativa (AAF), é característica marcante desse patótipo. A EAEC O104:H4 Stx+ foi responsável por um grande surto de colite hemorrágica e síndrome hemolítica urêmica (SHU) em 2011, na Alemanha. A cepa foi considerada um híbrido EAEC/EHEC, pois havia sido lisogenizada com um fago que codifica para a toxina de Shiga, comumente encontrada em EHEC (Escherichia coli enterohemorrágica). Foi evidenciada uma combinação altamente patogênica a humanos, devido ao seu elevado potencial de causar SHU. As bactérias utilizam sinalização química mediada por sistemas de 2 componentes para se comunicarem com o hospedeiro e sua respectiva microbiota, de modo a regularem seus mecanismos de sobrevivência. O sistema QseBC encontra-se diretamente relacionado, em enterobactérias, com a regulação da expressão de fatores de virulência em importantes patógenos humanos. Sua sinalização ocorre via Autoindutor-3 (AI-3), produzido por bactérias, Epinefrina (Epi) e Norepinefrina (NE), hormônios adrenérgicos do hospedeiro. Trabalhos anteriores evidenciaram que o bloqueio da via de sinalização do sistema QseBC por LED209 diminui a expressão de genes de virulência em bactérias Gram-negativas. Essa molécula atua especificamente em QseC, não interfere no crescimento dos patógenos e não apresenta toxicidade para modelos animais. O objetivo deste estudo foi investigar a sinalização química na virulência de EAEC O104:H4 Stx+ e Stx- via sensor histidina quinase QseC in vitro e in vivo. Foram realizados nocautes gênicos via vetor suicida pJP5603 para caracterização fenotípica e de expressão gênica, bem como ensaios in vivo para avaliar a cinética da infecção na ausência e presença de LED209. A interrupção dessa via mediada por QseC resultou na redução da formação de biofilme e de adesão em células Caco-2, na alteração de expressão de importantes fatores de virulência, bem como na menor eficiência na colonização in vivo para a cepa C227-11 (Stx+). Por outro lado, as mesmas diferenças não foram observadas para a cepa BA3826 (Stx-). Além disso, foi evidenciado um importante papel de QseC para a cepa C227-11 (Stx+) na diminuição da captação de vesículas de membrana externa por células intestinais, assim como na modulação da microbiota intestinal humana. Desta forma, conclui-se que QseC é um excelente alvo para o desenvolvimento de terapias, assim como o uso de LED209, para a cepa C227-11 (Stx+).
Enteroaggregative Escherichia coli (EAEC) is involved in cases of acute and persistent diarrhea (≥14 days) in children and adults from developed and developing countries. It has a unique adhesion pattern in epithelial cell cultures, called aggregative adhesion (AA). There are several adhesins and toxins in the pathogenicity mechanisms of EAEC. The formation of a thick biofilm, mediated mainly by aggregative adhesion fimbriae (AAF), is a hallmark of this pathotype. EAEC O104:H4 Stx+ was responsible for a major outbreak of hemorrhagic colitis and hemolytic uremic syndrome (HUS) in 2011 in Germany. The strain was considered an EAEC/EHEC hybrid, as it was lysogenized with a phage that codes for a Shiga toxin, commonly found in EHEC (enterohemorrhagic Escherichia coli). A highly pathogenic combination to humans has been shown, due to its high potential to cause HUS. These bacteria employ chemical signaling mediated by 2-component systems to communicate with the host and its microbiota, in order to regulate their survival mechanisms. The QseBC system in enterobacteria is directly related with the regulation of the expression of virulence factors in important human pathogens. The signaling occurs via Autoinducer-3 (AI-3), produced by bacteria, Epinephrine (Epi) and Norepinephrine (NE), host adrenergic hormones. Previous studies have shown that blocking the signaling pathway of the QseBC system by LED209 results in the lower expression of virulence genes in Gram-negative bacteria. This molecule acts precisely on QseC sensor kinase, it does not interfere with the growth of pathogens and has no toxicity for animal models. The aim of the study was to investigate chemical signaling in the virulence of EAEC O104:H4 Stx + and Stx- via histidine kinase QseC sensor in vitro and in vivo. Gene knockouts were generated via pJP5603 suicide vector, and it was used to perform phenotypic and gene expression assays, as well as in vivo assays to assess the infection kinetics in the absence and presence of LED209. Disruption of this QseC-mediated pathway resulted in reduced biofilm formation, altered expression of important virulence factors, and reduced in vivo colonization efficiency for the C227-11 (Stx +) strain. On the other hand, these differences were not observed for the BA3826 (Stx-) strain. Furthermore, QseC showed to have a role in the uptake of outer membrane vesicles by intestinal cells and modulation of the human intestinal microbiota. Therefore, the QseC sensor kinase is an excellent target for the development of novel therapies, as well as the use of LED209 in the C227-11 (Stx +) outbreak strain.
Enteroaggregative Escherichia coli (EAEC) is involved in cases of acute and persistent diarrhea (≥14 days) in children and adults from developed and developing countries. It has a unique adhesion pattern in epithelial cell cultures, called aggregative adhesion (AA). There are several adhesins and toxins in the pathogenicity mechanisms of EAEC. The formation of a thick biofilm, mediated mainly by aggregative adhesion fimbriae (AAF), is a hallmark of this pathotype. EAEC O104:H4 Stx+ was responsible for a major outbreak of hemorrhagic colitis and hemolytic uremic syndrome (HUS) in 2011 in Germany. The strain was considered an EAEC/EHEC hybrid, as it was lysogenized with a phage that codes for a Shiga toxin, commonly found in EHEC (enterohemorrhagic Escherichia coli). A highly pathogenic combination to humans has been shown, due to its high potential to cause HUS. These bacteria employ chemical signaling mediated by 2-component systems to communicate with the host and its microbiota, in order to regulate their survival mechanisms. The QseBC system in enterobacteria is directly related with the regulation of the expression of virulence factors in important human pathogens. The signaling occurs via Autoinducer-3 (AI-3), produced by bacteria, Epinephrine (Epi) and Norepinephrine (NE), host adrenergic hormones. Previous studies have shown that blocking the signaling pathway of the QseBC system by LED209 results in the lower expression of virulence genes in Gram-negative bacteria. This molecule acts precisely on QseC sensor kinase, it does not interfere with the growth of pathogens and has no toxicity for animal models. The aim of the study was to investigate chemical signaling in the virulence of EAEC O104:H4 Stx + and Stx- via histidine kinase QseC sensor in vitro and in vivo. Gene knockouts were generated via pJP5603 suicide vector, and it was used to perform phenotypic and gene expression assays, as well as in vivo assays to assess the infection kinetics in the absence and presence of LED209. Disruption of this QseC-mediated pathway resulted in reduced biofilm formation, altered expression of important virulence factors, and reduced in vivo colonization efficiency for the C227-11 (Stx +) strain. On the other hand, these differences were not observed for the BA3826 (Stx-) strain. Furthermore, QseC showed to have a role in the uptake of outer membrane vesicles by intestinal cells and modulation of the human intestinal microbiota. Therefore, the QseC sensor kinase is an excellent target for the development of novel therapies, as well as the use of LED209 in the C227-11 (Stx +) outbreak strain.
Descrição
Palavras-chave
Escherichia coli enteroagregativa, Sinalização química, Virulência, Enteroaggregative Escherichia coli, Chemical signaling, Virulence