Publication
Design of strategies to prevent synthesis of S. pneumoniae capsular polysaccharide at the bacteria division septum
| Summary: | Streptococcus pneumoniae is a common respiratory bacterial pathogen and a frequent cause of community-acquired pneumonia in developed countries. The genes encoding for the capsule polysaccharide (CPS), one of the most important virulence factors of these bacteria, are organized in an operon and in almost all the serotypes the two conserved Wzd and Wze proteins are expressed. Previous results suggest that if these two proteins cannot interact, forming a Wzd/Wze protein complex, pneumococcal bacteria will be prevented from producing capsule at the septum, which was shown to abolish the ability of these bacteria to cause bacteremia in mice after intranasal challenge. In this work, we aimed to find a method capable of screening and identifying small inhibitory (SI) peptides that prevent the interaction between Wzd and Wze. This could, consequently, represent a breakthrough in the development of strategies to replace vaccines against this important clinical pathogen. Initially, a derivative of the Escherichia coli bacterial two-hybrid assay was used. Here, T25- and T18-tagged proteins Wzd and Wze were expressed in the presence of a protein that should compete and interfere with their interaction. However, expression of this control protein, untagged and fully functional Wze, did not prevent the interaction between T25-Wzd and T18-Wze when expressed in a different plasmid. Afterwards we decided to screen for SI peptides directly in S. pneumoniae. For that purpose, we constructed a mutant strain that encodes in the chromosome both proteins, Wzd and Wze, functional and fused to different fluorescent proteins. Accordingly, we observed that Wzd and Wze were localized at the division septum of bacteria and that this localization was lost when a competitor was expressed from a replicative plasmid. We will now screen for SI peptides that can cause delocalization of Wzd and/or Wze and determine their effect on the synthesis of pneumococcal CPS. |
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| Main Authors: | Figueiredo, Joana Silva |
| Subject: | Streptococcus pneumoniae Virulência Pneumonia Microbiologia molecular Teses de mestrado - 2012 |
| Year: | 2012 |
| Country: | Portugal |
| Document type: | master thesis |
| Access type: | open access |
| Associated institution: | Universidade de Lisboa |
| Language: | English |
| Origin: | Repositório da Universidade de Lisboa |
| Summary: | Streptococcus pneumoniae is a common respiratory bacterial pathogen and a frequent cause of community-acquired pneumonia in developed countries. The genes encoding for the capsule polysaccharide (CPS), one of the most important virulence factors of these bacteria, are organized in an operon and in almost all the serotypes the two conserved Wzd and Wze proteins are expressed. Previous results suggest that if these two proteins cannot interact, forming a Wzd/Wze protein complex, pneumococcal bacteria will be prevented from producing capsule at the septum, which was shown to abolish the ability of these bacteria to cause bacteremia in mice after intranasal challenge. In this work, we aimed to find a method capable of screening and identifying small inhibitory (SI) peptides that prevent the interaction between Wzd and Wze. This could, consequently, represent a breakthrough in the development of strategies to replace vaccines against this important clinical pathogen. Initially, a derivative of the Escherichia coli bacterial two-hybrid assay was used. Here, T25- and T18-tagged proteins Wzd and Wze were expressed in the presence of a protein that should compete and interfere with their interaction. However, expression of this control protein, untagged and fully functional Wze, did not prevent the interaction between T25-Wzd and T18-Wze when expressed in a different plasmid. Afterwards we decided to screen for SI peptides directly in S. pneumoniae. For that purpose, we constructed a mutant strain that encodes in the chromosome both proteins, Wzd and Wze, functional and fused to different fluorescent proteins. Accordingly, we observed that Wzd and Wze were localized at the division septum of bacteria and that this localization was lost when a competitor was expressed from a replicative plasmid. We will now screen for SI peptides that can cause delocalization of Wzd and/or Wze and determine their effect on the synthesis of pneumococcal CPS. |
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