Publicação
Control of Streptococcus pneumoniae virulence by manipulation of the genetic sequence of virulence factor pneumolysin
| Resumo: | Streptococcus pneumoniae is one of the most important pathogens of humans. It produces a toxin, pneumolysin (PLY), able to form extraordinarily large pores in the membrane of attacked cells, for which they are dependent on the presence of membrane cholesterol. Murine models of acute pneumonia have shown that PLY is essential for the survival of pneumococcus in both the upper and lower respiratory tracts, as well as it is required for bacterial spread from the lungs to the bloodstream. Besides colonization and invasive disease, PLY has a role in immunogenic protection response. It has been proposed as a strong serotype-independent vaccine candidate. In this thesis, we demonstrate that manipulation of 5’ mRNA folding free energy (ΔG) by introduction of synonymous (silent) mutations allows for projected changes in PLY translation by pneumococcus without the need for chemical inducers or heterologous promoters. Hence, we created a panel of isogenic strains differing only in a few silent mutations at the 5’ end of the ply mRNA that are predicted to alter ΔG by a wide range. In vitro, such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. We demonstrate that production of PLY increases due to faster translation initiation with unchanged mRNA stability and decay. In vivo virulence was assessed using a mouse model in which we show differential outcome of pneumococcal pneumonia according to the 5’ mRNA ΔG. Inflammation features on lung histology and proinflammatory cytokine profile both positively and significantly correlate to PLY expression. This strategy of rheostat control of virulence factors is useful and provides proof-ofprinciple as a new tool for understanding the role of specific factors upon host-bacterial interactions. Further research on mRNA ΔG manipulation is a resourceful tool in the study of bacterial pathogenesis. |
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| Autores principais: | Amaral, Fábio André Eleutério |
| Assunto: | Ciências Médicas::Medicina Básica |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Streptococcus pneumoniae is one of the most important pathogens of humans. It produces a toxin, pneumolysin (PLY), able to form extraordinarily large pores in the membrane of attacked cells, for which they are dependent on the presence of membrane cholesterol. Murine models of acute pneumonia have shown that PLY is essential for the survival of pneumococcus in both the upper and lower respiratory tracts, as well as it is required for bacterial spread from the lungs to the bloodstream. Besides colonization and invasive disease, PLY has a role in immunogenic protection response. It has been proposed as a strong serotype-independent vaccine candidate. In this thesis, we demonstrate that manipulation of 5’ mRNA folding free energy (ΔG) by introduction of synonymous (silent) mutations allows for projected changes in PLY translation by pneumococcus without the need for chemical inducers or heterologous promoters. Hence, we created a panel of isogenic strains differing only in a few silent mutations at the 5’ end of the ply mRNA that are predicted to alter ΔG by a wide range. In vitro, such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. We demonstrate that production of PLY increases due to faster translation initiation with unchanged mRNA stability and decay. In vivo virulence was assessed using a mouse model in which we show differential outcome of pneumococcal pneumonia according to the 5’ mRNA ΔG. Inflammation features on lung histology and proinflammatory cytokine profile both positively and significantly correlate to PLY expression. This strategy of rheostat control of virulence factors is useful and provides proof-ofprinciple as a new tool for understanding the role of specific factors upon host-bacterial interactions. Further research on mRNA ΔG manipulation is a resourceful tool in the study of bacterial pathogenesis. |
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