RexAB Is Essential for the Mutagenic Repair of Staphylococcus aureus DNA Damage Caused by Co-trimoxazole

Clarke, Rebecca S.; Bruderer, Maya S.; Ha, Kam Pou; Edwards, Andrew M.

doi:10.1128/aac.00944-19

Cited by 22 publications

(54 citation statements)

References 61 publications

(103 reference statements)

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“…Therefore, we tested whether ROS produced by the respiratory burst triggers the SOS response and whether this was dependent upon RexAB. To do this, we used a P recA-gfp reporter construct and validated it by showing dose-dependent activity with mitomycin C, a well-established trigger of the SOS response ( 38 ) ( Fig. S6 ).…”

Section: Resultsmentioning

confidence: 99%

“…In addition to providing protection from oxidative damage caused by neutrophils, we have previously shown that RexAB provides S. aureus with tolerance to the combination antibiotic co-trimoxazole ( 38 ). That work showed that DNA damage was partly due to endogenous oxidative stress that occurred during exposure to the combination antibiotic, in addition to direct damage to DNA caused by thymidine limitation ( 38 ). The data presented here show that the loss of RexAB also sensitizes S. aureus to ciprofloxacin, even though the JE2 strain is resistant.…”

Section: Discussionmentioning

confidence: 99%

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Staphylococcal DNA Repair Is Required for Infection

Clarke

Kim

et al. 2020

mBio

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Staphylococcal DNA Repair Is Required for Infection

Clarke

Kim

et al. 2020

mBio

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“…Therefore, we tested whether the neutrophil oxidative burst triggers the SOS response and whether this was dependent upon RexAB. To do this we used a P recA-gfp reporter construct and validated it by showing dose-dependent activity with mitomycin C, a well-established trigger of the SOS response 38 (Supplementary Fig. S6).…”

Section: Resultsmentioning

confidence: 99%

“…In addition to providing protection from oxidative damage caused by neutrophils, we have previously shown that RexAB provides tolerance to the combination antibiotic co-trimoxazole 38 . That work showed that DNA damage was partly due to endogenous oxidative stress that occurred during exposure to the combination antibiotic, in addition to direct damage to DNA caused by thymidine limitation 38 . The data presented here show that loss of RexAB also sensitises S .…”

Section: Discussionmentioning

confidence: 99%

“…As detailed previously 38 , promoter-reporter gene constructs in the JE2 background was used to directly assess expression of recA . Antibiotic two-fold dilutions were made in flat-bottomed black-walled 96-well plates containing TSB and kanamycin (90 µg ml -1 ) and inoculated with 1/10 dilution of a stationary phase culture of the reporter strains.…”

Section: Methodsmentioning

confidence: 99%

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Staphylococcal DNA repair is required for infection

Clarke

Kim

et al. 2020

Preprint

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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

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Defects in DNA double‐strand break repair resensitize antibiotic‐resistant Escherichia coli to multiple bactericidal antibiotics

et al. 2022

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Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance‐breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double‐strand break repair as a promising target for the development of resistance‐breaking co‐therapies. We examined genetic variants of Escherichia coli that combined antibiotic‐resistance determinants with DNA repair defects. We observed that defects in the double‐strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double‐strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break‐repair inhibitors can be developed they could resensitize antibiotic‐resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic‐resistance mutations.

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RexAB Is Essential for the Mutagenic Repair of Staphylococcus aureus DNA Damage Caused by Co-trimoxazole

Cited by 22 publications

References 61 publications

Staphylococcal DNA Repair Is Required for Infection

Staphylococcal DNA Repair Is Required for Infection

Staphylococcal DNA repair is required for infection

Defects in DNA double‐strand break repair resensitize antibiotic‐resistant Escherichia coli to multiple bactericidal antibiotics

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