Abstract
Streptococcus pneumoniae is a leading cause of death from infectious disease. The bacterium generates large amounts of hydrogen peroxide during its normal metabolism. In the presence of thiocyanate (SCN-) in the respiratory tract, this hydrogen peroxide is converted to hypothiocyanous acid (HOSCN) by the host enzymes lactoperoxidase and myeloperoxidase. We have shown that S. pneumoniae has a high tolerance to HOSCN. The aim of our research is to understand how S. pneumoniae copes with this oxidant and to develop strategies to neutralize its HOSCN defences. We identified two essential components of the pneumococcal HOSCN defence: 1) a flavoprotein disulfide reductase that directly breaks down HOSCN, named hypothiocyanous acid reductase (Har), and 2) a membrane transporter that enables the bacteria to import glutathione from their host environment. We showed that genetic deletion of both systems renders the bacteria completely incapable to grow in the presence of HOSCN. Importantly, loss of either Har or the glutathione importer results in an attenuated phenotype in mouse models of pneumococcal infection, with a lower bacterial burden observed in the lungs and blood. We generated recombinant Har and showed that it reduces HOSCN with near diffusion-limited catalytic efficiency. We identified a potent inhibitor of Har by screening a pool of known flavoprotein disulfide reductase inhibitors. Encouragingly, this Har inhibitor was able to increase S. pneumoniae susceptibility to HOSCN. We also purified the glutathione-scavenging component of the pneumococcal glutathione importer and investigated its interaction with glutathione using NMR chemical shift perturbation assays and differential scanning fluorimetry. Our results indicate that electrostatic interactions between lysine and arginine residues in the protein’s ligand binding site and the carboxylate groups of glutathione are critical for binding. Collectively, our research lays the foundation for inhibiting the HOSCN reductase enzyme and disrupting glutathione import in S. pneumoniae.