Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance.

Graphical abstract

To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection. IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

35Staphylococcus aureus is a leading cause of chronic and recurrent infections of the skin, bones, joints 36 and bloodstream. During infection, S. aureus faces the twin threats of the host immune system and 37 therapeutic antibiotics. Since both host immune cells and antibiotics generate reactive oxygen species, 38we hypothesised that S. aureus may employ a common mechanism to repair damage caused by either 39 threat. We found that staphylococcal DNA is damaged during exposure to both human neutrophils 40 and most bactericidal antibiotics. To understand the nature of this damage and how S. aureus repairs 41 it, we screened a panel of transposon mutants defective for various DNA repair processes. This 42 revealed that loss of the rexBA operon significantly reduced staphylococcal survival in human blood, 43 during incubation with purified neutrophils, in the peritoneal cavity of mice and during exposure to a 44 large panel of bactericidal antibiotics. We then used biochemical assays to demonstrate that RexAB is 45 a member of the AddAB family of ATP-dependent helicase/nucleases that are required for the repair 46 of DNA double strand breaks. Finally, we found that RexAB homologues in Enterococcus faecalis and 47Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that 48 DNA repair constitutes a broadly conserved defence against neutrophils. Together, these data 49 demonstrate that DNA is a target of host immune cells and several antibiotics, leading to double-50 strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of 51Gram-positive pathogens during infection. 52 53 54

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA, leading to induction of the mutagenic SOS response associated with the emergence of drug resistance. However, the type of DNA damage that arises and how this triggers the SOS response is largely unclear. We found that several different classes of antibiotic triggered dose-dependent induction of the SOS response in Staphylococcus aureus , indicative of DNA damage, including some bacteriostatic drugs. The SOS response was heterogenous and varied in magnitude between strains and antibiotics. However, in many cases, full induction of the SOS response was dependent upon the RexAB helicase/nuclease complex, which processes DNA double strand breaks to produce single-stranded DNA and facilitate RecA nucleoprotein filament formation. The importance of RexAB in repair of DNA was confirmed by measuring bacterial survival during antibiotic exposure, with most drugs having significantly greater bactericidal activity against rexB mutants relative to wild type strains. For some, but not all antibiotics there was no difference in bactericidal activity between wild type and rexB mutant under anaerobic conditions, indicative of a role for reactive oxygen species in mediating DNA damage. Taken together, this work confirms previous observations that several classes of antibiotics cause DNA damage in S. aureus and extends them by showing that processing of DNA double strand breaks by RexAB is a major trigger of the mutagenic SOS response and promotes bacterial survival.