Pneumococcal gene expression during its interaction with human basal epithelial...

• Autor(es): Nunes, Ana Margarida Lameira
• Data: 2008
• Identificador Persistente: http://hdl.handle.net/10400.1/846
• Origem: Sapientia - Universidade do Algarve
• Assunto(s): Teses; Células epiteliais; Genética; Virologia; Streptococcus pneumonial
• Tipo de Documento: Dissertação de Mestrado
• Idioma: Inglês
• Orientador(es): Andrew, Peter; Faleiro, Maria Leonor

Descrição

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2008 The aim of this research was to analyze pneumococcal gene expression during interactions with human nasal epithelial cells and comparison with other different infection models. As the number one cause of bacterial pneumonia and one of the major causes of mortality and morbidity, S. pneumoniae has been a target of several studies in past years. Oggioni and colleagues (2006) studied pneumococcal gene expression during infection in two different scenarios, bacteria in the blood and bacteria in the tissue (as brain and lung). In this current study the target genes analyzed were nanA, ply, comX, nox, sodA, since these were reported as up regulated in the lung (Oggioni et al., 2006), except for ply, that was chosen because of its importance in pneumococcal infection. It has been reported that induction of competence system by the quorum sensing peptide stimulates the bacteria to grow in biofilm and also increases S. pneumoniae virulence. Based on these results, we attempted to induce biofilm formation prior to infection with the aim to increase pneumococci virulence before adherence to nasal epithelial cells. However, unlike Oggioni et al., (2006), our method was unsuccessful in producing biofilm and therefore it was impossible to proceed. A further idea was to analyze pneumococcal gene expression in apical fluid that is released and present on the surface of human epithelial cells. It was thought that this could elucidate mechanisms of how pneumococci overcome host defense. However, antibiotics that were used for cell culture were detected in the apical fluid and therefore, it was not possible to continue with this experiment since all the bacteria died within 30 minutes of exposure. For these reasons the main aim of this study was to explore the mechanisms of pneumococcal adherence to basal cells. Gene expression analysis was performed using three reference conditions, S. pneumoniae in BEBM, in TSB and the non adhered pneumococci. BEBM proved to be the best reference condition and using this we have shown that all the genes we targeted were up regulated within 2h of exposure to basal cells, for adhered and non adhered bacteria, except comX and sodA. NanA (neuraminidase gene) showed the highest increase in expression levels compared to the other genes, 25,47±2,21 for the adhered pneumococcus to patient sp282 basal cells and 23,10±0,47 for the non adhered bacteria to patient 455 basal cells. After 6h, all genes were up regulated except ply.