Abstract
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, resulting in more than one million deaths each year worldwide. This pathogen generates hydrogen peroxide (H2O2) as part of its normal metabolism, yet it lacks enzymes that remove this oxidant. While the ability of S. pneumoniae and neighbouring cells to cope with H2O2 is of interest, the H2O2 will be predominantly metabolised by host enzymes myeloperoxidase and lactoperoxidase to form the secondary oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). High concentrations of thiocyanate are found in the epithelial lining fluid of the respiratory tract, making HOSCN the major oxidant derived from bacterial H2O2. When quantifying S. pneumoniae oxidant sensitivities to H2O2, HOCl, and HOSCN, I found that S. pneumoniae is considerably more tolerant to HOSCN than another respiratory pathogen, Pseudomonas aeruginosa. Incubation with free myeloperoxidase in chloride-rich buffer was bactericidal, however, the addition of thiocyanate protected S. pneumoniae. Similarly, incubation of S. pneumoniae with lactoperoxidase and thiocyanate did not kill the bacteria.
Tolerance to HOSCN may give S. pneumoniae a survival advantage over other pathogenic bacteria. Understanding the mechanisms by which S. pneumoniae protects itself from HOSCN is likely to reveal novel strategies for limiting the colonisation and pathogenicity of this pathogen. As such, I investigated the role of the low-molecular-weight-thiol glutathione in HOSCN tolerance. S. pneumoniae does not synthesise glutathione, but imports it from the environment via an ABC transporter. Upon treatment of S. pneumoniae with HOSCN, bacterial glutathione was reversibly oxidised in a time- and dose-dependent manner, and intracellular proteins became glutathionylated. Bacterial death was observed when the reduced glutathione pool dropped below 20%. A S. pneumoniae mutant unable to import glutathione was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO, converting bacterial H2O2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidised glutathione. This research highlights the importance of host glutathione in protecting S. pneumoniae from HOSCN. Limiting glutathione utilisation by S. pneumoniae may be a way to limit colonisation and pathogenicity.
To investigate S. pneumoniae HOSCN tolerance further, a S. pneumoniae transposon mutant library containing 37,000 mutants was generated. This library, and a secondary library gifted from collaborators, were exposed to HOSCN selection in an unbiased genome-wide screen. Subsequent investigation identified 37 genes that were involved in the response of S. pneumoniae to HOSCN. This response was multifaceted, with genes from glucose metabolism, capsule and cell wall structural integrity, oxidant detoxification and DNA repair being identified as vital for S. pneumoniae survival in the presence of HOSCN. Targeting the genes identified in this screen holds promise for effectively controlling S. pneumoniae infection by sensitising them to host-generated HOSCN to assist the immune system in controlling this deadly pathogen.