Systemic overview of key factors for oxidative stress by CRISPRi-seq in Streptococcus pneumoniae

The unique ability of Streptococcus pneumoniae to produce and withstand high levels of hydrogen peroxide (H2O2) during growth has been a topic of interest. One on hand, the H2O2 promotes pneumococcal colonization by killing its competitors at nasopharynx and pathogenesis by induction of apoptosis in lung cell. One the other hand, high H2O2 levels also cause strong oxidative stress to itself. We found that S. pneumoniae produced much higher levels of H2O2 when cultured under ambient air conditions (~1 mM) compared to the 5% CO2 condition (~50 uM). To explore the mechanism of H2O2 tolerance in S. pneumoniae, we utilized a genome-wide CRISPRi-seq to identify the key factors by comparing gene fitness between air and 5% CO2 conditions, or air incubation with or without catalase which eliminates H2O2. The results of the two comparisons were highly consistent, indicating the self-produced high level H2O2 is a major stress of S. pneumoniae grown in air conditions. Genes involved in DNA repair and homologous recombination were identified to be conditionally essential in high level H2O2 production condition, confirmed that DNA damage is the major consequence of oxidative stress. Other important genes include sufCDSE2 in iron-sulfur cluster biosynthesis, the two-component system ciaRH, mreC and uppP in peptidoglycan biosynthesis. Notably, these genes were also essential for pneumococcal colonization in a murine model, making them potential targets for anti-colonization efforts. Our findings shed light on the pneumococcal unique strategy to master the dual powers of H2O2.