Publicação
The impact of the phytopathogen Pectobacterium carotovorum in Drosophila melanogaster development
| Resumo: | Cell-to-cell communication or quorum sensing is an important bacterial process of intercellular communication that enables bacteria to determine their numbers by producing, secreting and detecting the accumulation of signalling molecules, called autoinducers. This process allows bacterial populations to synchronize gene expression and coordinate, at the group level, complex behaviours such as antibiotic production, biofilm formation or virulence. Pectobacterium carotovorum are Gram-negative bacteria which inhabit the soil and infect vegetables, causing large economic problems. Production of virulence factors in this specie is tightly regulated by two quorum sensing mechanisms: ExpI/ExpR system which uses the N-acyl homoserine lactone signal; and the GacSA/Rsm system in which the signal is unknown. Ecc15 is a strain of P. carotovorum that is able to persist in the gut, when oral ingested, by Drosophila melanogaster larvae. This persistence ability was attributed to the acquisition of a gene denominated Erwinia virulence factor (evf). Although the host immune response to this bacterium was extensively studied, the effect at the host development level upon bacterial infection was never addressed. Additionally, the genetic network controlling evf expression and the Evf specific mechanism of action in the gut remains poorly understood. In this work we studied the effect of the exposure to Ecc15 in the host development by following the time to pupation of larvae exposed to bacteria. We also investigated the Evf specific mechanism of action in the context of the host-microbe interaction by testing the effect of bacterial free supernatants and purified Evf protein. Moreover, we investigated the genetic network regulating evf transcription by monitoring the expression of the promoter regions of the known and putative regulators of evf (hor and evr, respectively). Finally we studied the role of quorum sensing and evf possible regulators in this specific interaction by exposing larvae to mutants in these genetic systems. In conclusion we observed that larvae exposed to wild-type Ecc15 have a development delay of approximately one day and that this effect is evf-mediated. We concluded the evf-effect is caused by interruption of feeding behaviour and that the product of the evf gene is not extracellularly toxic. In addition, we optimized a method to construct mutants in Ecc15 that allowed us to elucidate part of the genetic network involved in regulation of evf. Lastly we showed the role of the quorum sensing in the evf-mediated effect of Ecc15 in Drosophila development in vivo |
|---|---|
| Autores principais: | Vieira, Filipe José Dias, 1991- |
| Assunto: | Expressão génica Drosophila melanogaster Recombinação genética Teses de mestrado - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Cell-to-cell communication or quorum sensing is an important bacterial process of intercellular communication that enables bacteria to determine their numbers by producing, secreting and detecting the accumulation of signalling molecules, called autoinducers. This process allows bacterial populations to synchronize gene expression and coordinate, at the group level, complex behaviours such as antibiotic production, biofilm formation or virulence. Pectobacterium carotovorum are Gram-negative bacteria which inhabit the soil and infect vegetables, causing large economic problems. Production of virulence factors in this specie is tightly regulated by two quorum sensing mechanisms: ExpI/ExpR system which uses the N-acyl homoserine lactone signal; and the GacSA/Rsm system in which the signal is unknown. Ecc15 is a strain of P. carotovorum that is able to persist in the gut, when oral ingested, by Drosophila melanogaster larvae. This persistence ability was attributed to the acquisition of a gene denominated Erwinia virulence factor (evf). Although the host immune response to this bacterium was extensively studied, the effect at the host development level upon bacterial infection was never addressed. Additionally, the genetic network controlling evf expression and the Evf specific mechanism of action in the gut remains poorly understood. In this work we studied the effect of the exposure to Ecc15 in the host development by following the time to pupation of larvae exposed to bacteria. We also investigated the Evf specific mechanism of action in the context of the host-microbe interaction by testing the effect of bacterial free supernatants and purified Evf protein. Moreover, we investigated the genetic network regulating evf transcription by monitoring the expression of the promoter regions of the known and putative regulators of evf (hor and evr, respectively). Finally we studied the role of quorum sensing and evf possible regulators in this specific interaction by exposing larvae to mutants in these genetic systems. In conclusion we observed that larvae exposed to wild-type Ecc15 have a development delay of approximately one day and that this effect is evf-mediated. We concluded the evf-effect is caused by interruption of feeding behaviour and that the product of the evf gene is not extracellularly toxic. In addition, we optimized a method to construct mutants in Ecc15 that allowed us to elucidate part of the genetic network involved in regulation of evf. Lastly we showed the role of the quorum sensing in the evf-mediated effect of Ecc15 in Drosophila development in vivo |
|---|
Atividades financiadas
Não foram encontrados projetos financiados.