Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of Delta ahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen.

Automated summary

By using saturation transposon mutagenesis and deep sequencing, this study identified 37 genes associated with hypothiocyanous acid (HOSCN) tolerance in Streptococcus pneumoniae. These genes are involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Validation experiments showed that most of the single-gene deletion mutants exhibited increased sensitivity to HOSCN, and some mutants showed enhanced activity of antioxidant defense systems. The double deletion of glutathione reductase and sodA significantly sensitized the bacteria. The HOSCN defense systems identified in this study may serve as viable targets for novel therapeutics against S. pneumoniae.