Structural and biophysical studies of pneumococcal capsular surface proteins

Abstract

Streptococcus pneumoniae (pneumococcus) is a major human pathogen and a leading cause of morbidity and mortality worldwide, especially in children and the elderly. The casualties due to respiratory infections are estimated to be over 4 million per year, where pneumococcus is the predominant species. Moreover, the increasing number of antibiotic-resistant strains and the suboptimal clinical efficacy of available vaccines confines control of this pathogen. In view of this situation, substantial attention has focused on novel virulence-related pneumococcal proteins as potential targets for future drug targets. One such target is the pneumococcal serine-rich repeat protein (PsrP), an important virulence factor present in a majority of the strains capable of causing invasive pneumococcal disease (IPD).

The functional binding region (BR) of this protein binds to keratin-10 (KRT10) and also promotes biofilm formation through self-oligomerization. The crystal structure of the KRT10-binding region of PsrP (BR187-385) reveals an extended β-sheet on one side of a compressed two-sided β-barrel presents a basic groove that could accommodate the acidic helical rod domain of KRT10. Well-ordered loop regions distort the other side of the barrel and form a papercliplike sub-structure for more specific interaction with KRT10. In vitro alanine substitution of residues localized within this paperclip structure efficiently disrupted BR187-385/KRT10 complex formation.

Within the work of this thesis we also found that BR187-385, which lacks the putative oligomerization region, forms stable oligomers in vitro. Small angle X-ray scattering and circular dichroism experiments revealed a non-globular and possibly disordered structure of the N-terminal region. A comparative analysis of the long (BR120-395) and short (BR187-385) domain constructs even suggested an inhibitory role for the N-terminal BR122-166 domain in oligomerization. Indeed, we could show that the N-terminal region is released by cleavage through the human furin protease that specifically recognizes a sequence localized between the globular BR187-385 domain and the disordered N-terminal part. The crystal structure of the dimer of the KRT10-binding domain of PsrP reveals a domain swap mechanism for dimerization, this process, although energetically costly, is probable when PsrP is involved in biofilm formation.

The minor ancillary pilus protein RrgC is believed to anchor the pneumococcal pilus to the cell wall, and until recently very little was known about its structure. In this work we have evaluated the structure of the protein in solution with SAXS, and shown that the protein is a multidomain protein with flexible linkers and adopts extended conformations in solution.

Through this work we have proposed a more specific interaction between PsrP and KRT10 than previously reported, as well as shown that oligomerization is possible despite the removal of the N-terminal region. These findings are very important for a deeper understanding of the details in PsrP’s role in pneumococcal invasion. The structural studies of RrgC provide a platform for future studies. The results presented within this thesis will hopefully aid in the future development of novel drugs and vaccines.