The characterization of the interaction between Streptococcus pneumoniae PspC and Homo sapiens pIgR

Streptococcus pneumoniae is a commensal bacterium in the human nasopharyngeal tract and is known to cause severe respiratory related diseases in humans. The pneumococcal surface protein C (PspC) has been demonstrated to interact with human polymeric immunoglobulin receptor (pIgR) and its free form secretory component (SC). S. pneumoniae utilizes the pIgR recycling mechanism to bypass the nasopharyngeal epithelial barrier of the host through the PspC-pIgR interaction. Studies have shown the YPT motifs in PspC R domains to be involved in this interaction (Hammerschmidt et al., 2000). The exact amino acid sequences in pIgR/SC have not yet been elucidated but Elm et al., (2004), Lu et al., (2003) and Venables et al., (2013) have demonstrated that Ig-like domains 3 and 4 of SC are mutually critical in this interaction. Mutagenesis studies were conducted to give insight into regions of these domains responsible for this interaction. In order to select possible candidates for mutagenesis, homology modelling, protein-protein docking and multiple sequence alignments were performed. PspC-SC interaction is human specific (Hammerschmidt et al., 2000) and the conservation between known species was used to select amino acids located on highly variable loop regions of the SC molecule. Amino acids Ser257, Asp312 and Gln373 were suggested by previous studies and investigated in this study. Protein-protein docking was performed with Cluspro 2.0 and Haddock 2.2 webservers. The docking results, coupled with conservation information resulted in Arg304 also being selected for investigation. Additional candidates were identified for future studies. Point mutations of the selected amino acids were introduced with overlap PCR and confirmed by DNA sequencing. The SC-D3D4 proteins were expressed in vitro and refolded by an on-column method developed by Venables et al., (2013). The Biorad ProteOn™ XPR36 protein interaction array system at Rhodes University was utilized for measuring the effects of the SC-D3D4 mutations on affinity for PspC-R1R2. The affinities of expressed SC-D3D4 mutants for PspC-R1R2 were compared to the wild type (WT) SC-D3D4 in concentration dependent kinetic analyses. The KD values for WT SC-D3D4 and Ser257, Arg304, Asp312 and Gln373 mutations were 1.4, 2.9, 4.1, 2.0, and 2.1 μM, respectively. It was concluded that these mutations had no impact on the affinity of SC-D3D4 with PspC-R1R2 and therefore probably excludes these four residues as interaction motifs for PspC