Development of a Streptococcus pneumoniae system to control exposure of immature peptidoglycan

• Autor(a) principal: Pinheiro, Sara Filipa dos Santos
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/113709
• topic: Streptococcus pneumoniae; LytA; IPTG-inducible promoter; immature peptidoglycan; immunofluorescence; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Streptococcus pneumoniae is a Gram-positive bacterial pathogen capable of causing from mild infections to more severe conditions such as pneumonia and meningitis. Pneumococcal virulence is dependent on the production of different virulence factors, such as particular peptidoglycan-associated surface proteins or the production of a capsular polysaccharide structure surrounding the bacteria. Peptidoglycan, a major cell wall component, offers structural stability to the cell and is dynamically tailored by hydrolases, such as LytA, during physiological mechanisms like cell elongation, division and septation. Capsule production, which is strictly regulated, requires expression of several genes present in the cps locus, including genes with a regulatory role such as wzd and wze. The interaction between proteins Wzd and Wze, in the presence of ATP, is thought to play a central role in the regulation of capsule synthesis. The aim of this dissertation was to develop a S. pneumoniae system capable of controlling the exposure of immature peptidoglycan to enzymes present in the surrounding medium. To achieve this goal, pneumococcal strains were constructed where the Escherichia coli LacI repressor (encoded by the lacI gene) is constitutively expressed and the capsule regulatory genes pneumococcal wzd and wze are placed under the control of an IPTG-inducible promoter (Plac). This was performed in the background of S. pneumoniae wzd and wze deletion mutants, where immature peptidoglycan is exposed at the surface of the septal region due to absence of capsule at the division septum. One of the constructed strains, 6314Δwze-lacI-Plac-wze, was analyzed by immunofluorescence microscopy to assess if septal capsule expression was restored at the division septum upon expression of IPTG-inducible wze. This analysis showed that the constructed mutant, in the presence of IPTG, presented a seemingly intermediate phenotype between absence of capsule in the division septum and full encapsulation.