Cloning, expression and purification of pneumococcal proteins = clonagem, expressão e purificação de proteínas de pneumococcus

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2008

Streptococcus pneumoniae is the causative agent of pneumonia, bacteraemia, meningitis and otitis [1]. The first stage of infection by the pneumococcus is nasopharyngeal colonisation and it is a prelude to dissemination to the lower respiratory tract [2]. Protecting the host against the invasion by microorganisms there is mucus coating the apical epithelial surfaces of the nasopharynx and lungs. In contrast, mucus also is a rich source of potential nutrient for colonising microorganisms. Mucin is the main glycoprotein found in mucus and its carbohydrates may be a major source of energy for the pneumococcus. Several glycosidic enzymes such as neuraminidase A and B, β-D-galactosidase, and N-acetyl-β-D-galactosaminidase have been found in pneumococcal cell lysates and it had been demonstrated that S. pneumoniae can utilise mucin as carbon and nitrogen source for growth. Furthermore, the analysis of sequenced strains of the S. pneumoniae genome revealed more ORFs whose products can, potentially, act on the carbohydrate of mucin. The present study carried out an investigation of these pneumococcal glycosidase’s role. The genes encoding these enzymes were mutated and the ability of the pneumococcal mutants to grow in mucin as the sole carbon and nitrogen source was determined. Several of these knock out mutants were impaired in their capacity to utilise mucin, indicating their importance for mucin degradation. One such strain bears a mutation at gene SPR0059, which encodes for a putative beta-galactosidase, and another one with a mutation at the SPR0244 gene, encoding for a putative 6-phospho-beta-glucosidase. The referred mutants had lowered cellular growth in Sicard’s medium supplemented with mucin when compared with the wild type, suggesting that in the absence of those proteins the mucin decomposition was compromised, and so the carbon source provider. These facts lead us to the importance of the SPR0059 and SPR0244 encoded proteins study by its isolation and characterization.