Impact of S.pneumoniae type-I pilus and its subunits on bacterial adherence to human epithelial cells

S. pneumoniae is a human pathogen able to cause both invasive and non-invasive diseases as well as to colonize the nasopharyngeal tract of children and adults. Despite pneumococcus is a leading cause of morbidity and mortality worldwide, the pathogenic mechanisms exploited by S. pneumoniae are not yet clear. A critical step is colonization of the nasopharynx and the initial interaction of pneumococci with host cells. Recently, pili were discovered in gram-positive bacteria, mediating critical host-bacterial interactions, such as adherence to the epithelium, biofilm formation and translocation of host mucosal barriers. Thirty to 50% S. pneumoniae strains harbor pilus islet-1 (PI-1) coding for a surface exposed pilus reported to be important both for colonization and virulence. In particular it has been clearly demonstrated that pilus component RrgA is the major determinant for adhesion to epithelial cells in vitro whereas RrgB is important to allow the formation of the polymeric structure into which the adhesin is incorporated. The data reported in this work contribute to better define the involvement of S. pneumoniae pili in adhesion to human epithelial cells and providing evidence that anti-pilus antibodies are able to prevent bacterial adherence. In order to test the adhesive attitude of S.pneumoniae strains, we selected a panel of epithelial cell lines differing for their anatomical origin and the capacity to constitute polarized monolayers. Me180 cervical epithelial cells, characterized to form weak adherence junctions and thus expose basolateral surface, result the best model to study pneumococcal adhesiveness. This evidence led us to hypothesize that the pilus ligands may reside in some of the extracellular matrix components rather than in cell associated receptors. Once identified the most appropriate cellular model we started the characterization of pilus dependent adhesion by using TIGR4 mutants lacking pilus components or subpopulations derived from the wild type strain and differing only in pilus expression (highly or poorly piliated). The results reveal that piliated bacteria were drastically more adherent to cells compared to non piliated strains. However, both the lack of the pilus adhesion moiety (RrgA) or of the pilus backbone (RrgB) determined a dramatic reduction of adhesion, suggesting that both the presence of the adhesin and its correct spacing from the bacterial cell were equally important. In addiction we tested the pilus mediated adhesiveness of different pneumococcal strains that could be successfully divided into sub-populations highly (H) or poorly (L) expressing pili. By comparing the H sub-populations we noticed striking difference in their capacity to adhere to host cells. Conventional and immune electron microscopy studies reveal that the capsule thickness is inversely related with the bacterial ability to adhere to host cells likely due to the different exposure of pili on the bacterial surface. Moreover the deletion of the capsular locus in the poorly adherent strain 19FTaiwan14 results in a considerable increase in the adhesion to epithelial cells, at levels comparable to that of poorly capsulated TIGR4 strain. Additionally, in this study, we demonstrate that anti-pilus antibodies were able to significantly impair pneumoccal adhesion to human epithelial cells in strains prominently extruding pili from the capsule. Of interest, we found a monoclonal antibody against RrgA that was able to compete with adhesion in a similar manner with respect to the polyclonal serum against the whole protein. Epitope mapping experiments resulted in the identification of the possible binding region on the RrgA molecule, located in the c-terminal domain. At the moment we are trying to confirm this result by producing point-mutated forms of RrgA with the scope to select mutants that are not recognized by the functional monoclonal antibody and finally complement rrga ko strain with these sequences to confirm the importance of the epitope in the adhesion to human epithelial cells