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

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 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 to tolerate HOSCN. We identified 37 genes associated with HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes , , and were generated and their ability to survive HOSCN was assessed. With the exception of Δ, 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 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 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. IMPORTANCE is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.