Protein glycosylation is an important post-translational event that can impact protein presentation on the surface of cells, thereby affecting host-cell adhesion and bacteria to bacteria interactions. psrP-secY2A2 is a pneumococcal pathogenicity island which encodes the adhesin pneumococcal serine-rich repeat protein (PsrP), 10 putative glycosyltransferases, and 7 accessory components of an alternate secretion (Sec) system. Based on the homology of these genes to those found in other serine-rich repeat protein (SRRPs) encoding loci, these accessory proteins putatively glycosylate PsrP, and are responsible for the transport of PsrP to the bacterial surface. Our preliminary data show that PsrP is glycosylated and that deletion of the accessory genes results in an unsoluble PsrP protein. To investigate and compare the composition of the glycoconjugates present on PsrP, glycosylated and unglycosylated purified protein was analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection. This analysis determined that glycosylated PsrP contains GlcNAc, galactose, glucose and mannose. Additionally, deletion of each glycosyltransferase in psrP-secY2A2 resulted in decreased, increase, or no change in adhesion to lung cells. Finally, deletion of individual genes specific for transport of PsrP attenuated adhesion to lung cells similarly to the PsrP deficient mutant;suggest that the accessory transport system is required for normal PsrP function. Our findings indicate that the glycosyltransferases are important for protein stability, and adhesion. And, the accessory transport genes are important for normal PsrP function. The purpose of this application is to further investigate the role of PsrP glycosylation as an adhesin. We will address this by focusing on two aims: 1) To determine the glycosylation patterns and quantity of monosaccharides on wild type PsrP and individual glycosyltransferase deletion mutants correlation with effects on adhesion in vitro and transport to the bacterial surface. 2) To define the effect of the seven accessory secretion system proteins in S. pneumoniae on PsrP surface transport. The proposed studies are significant because PsrP is involved bacterial pathogenesis in a manner that is distinct from other SRRPs. The application is innovative because this is the first attempt to look at how glycosylation of PsrP is important for pathogenesis as a specific lung adhesin to cause pneumonia. Thus, the proposed studies will improve our understanding of PsrP glycosylation and will have impact on studies to identify new therapeutic targets for this important virulence determinant. These studies will also provide support for my continued research training and career development, knowledge and skills towards my goal to become an independent investigator.
The Pneumococcal serine-rich repeat protein (PsrP) is a virulence factor present in a variety of invasive S. pneumoniae serotypes that mediates attachment to host lung cells to cause pneumonia. As a member of the serine-rich repeat family, PsrP is glycosylated. Our studies will increase our understanding how PsrP glycosylation impacts pathogenesis, and may provide future directions to identify new therapeutic targets.