Staphylococcal DNA Repair Is Required for Infection

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.

RexAB Promotes the Survival of Staphylococcus aureus Exposed to Multiple Classes of Antibiotics

“…As expected, we found that cotrimoxazole, ciprofloxacin, nitrofurantoin, and oxacillin triggered SOS induction in both the SH1000 and JE2 strains, albeit to various degrees and with different temporal dynamics ( Fig. 1A to D ) ( 30 , 34 – 36 , 45 ). In all cases, however, there was evidence of dose-dependent induction of the SOS response (Table S1).…”

“…DNA damage in most bacteria, including S. aureus , triggers activation of the SOS response, which leads to the transcription of genes whose products contribute to DNA repair ( 30 – 34 ). These genes include recA , which encodes the RecA protein required for homologous recombination and, together with LexA, is a key regulator of the SOS response ( 30 – 36 ).…”

“…To determine whether antibiotics caused DNA damage in S. aureus , we used a well-characterized P recA-gfp reporter system in two distinct genetic backgrounds: SH1000, a methicillin-sensitive S. aureus (MSSA) strain, and JE2, a community-associated methicillin-resistant S. aureus (CA-MRSA) strain of the USA300 lineage ( 30 , 34 , 42 , 43 ). This system has been shown to produce a dose-dependent fluorescent response to DNA damage caused by the ROS generator paraquat, mitomycin C, and cotrimoxazole ( 30 , 34 ).…”

“…For example, we have shown previously that the combination antibiotic cotrimoxazole (trimethoprim plus sulfamethoxazole) caused DNA double-strand breaks (DSB) and that processing of these by the RexAB nuclease/helicase complex was required for induction of the SOS DNA repair response ( 30 ). RexAB is a member of the AddAB family of ATP-dependent helicase/nucleases that process DNA DSBs to produce a 3′ single strand of DNA ( 34 , 39 – 41 ). RecA binds to the single-stranded DNA, resulting in a nucleoprotein filament that triggers autocleavage of the LexA transcriptional repressor and induction of the SOS response ( 39 , 41 ).…”

Microbial Musings – November 2020

“…aureus that enable it to survive the oxidative burst produced when they are engulfed by neutrophils in blood. They identified RexAB as being required for repair of DNA damaged by reactive oxygen species (ROS) produced by this host response [9]. In this issue Nisha Rangananthan (@drNJ_RJ) and colleagues from Edward’s group have studied the role of the general stress response in the same process in S.…”