Resumo

Pneumococci is an asymptomatic colonizer of the human nasopharynx and accounts for most cases of community-acquired pneumonia and can cause non-invasive and invasive diseases. The currently available vaccines are serotype specific with low immunogenicity activity and high production cost. To overcome these problems, several research groups have been investigating proteins that would be associated with virulence as promising targets to compose vaccines. In this work, we sequenced and analyzed S. pneumoniae serotype 5 genome and also produced a database containing pneumococcal proteins. We selected two genes from this database that codify to surface-exposed proteins. The cellular localization prediction was performed by in silico analysis. To confirm the expression of proteins on the cell surface were used FACS and IEF. We also evaluated the ability of these proteins to interact with extracellular matrix proteins, as well as the presence and degree of identity of these genes in clinical samples by PCR and sequencing. The genes were amplified by PCR, cloned into expression vectors and the soluble proteins were purified by “IMAC”. Antisera were produced by immunization of mouse with recombinant proteins adsorbed in Alhydrogel. Recombinant proteins were shown to react by immublotting with these antisera. The antisera were used to confirm the extracellular localization of the proteins in 10 serotypes of pneumococci. The CbpE protein was able to interact with fibronectin, laminin, fibrinogen and collagen IV, while PiuA protein showed no binding to any of these matrices. We confirm by PCR that both genes are present in 51 pneumococcal clinical isolates of different serotype. The degree of nucleotide and protein identity showed more than 95%, by sequencing. Furthermore, both proteins were expressed on the bacterial surface and conserved among clinical isolates, only CbpE showed interaction with extracellular matrix proteins. Data together suggest that this protein could be considered a good candidate for the development protein based – vaccine. Protection studies in animal models using both proteins are in progress in order to better evaluate the potential of these vaccine antigens. Financing institution: Bio-Manguinhos/FIOCRUZ and PAPES VI