The alarmone (p)ppGpp confers tolerance to oxidative stress during the stationary phase by maintenance of redox and iron homeostasis in Staphylococcus aureus

Verena Nadin Fritsch,Vu Van Loi,Tobias Busche,Quach Ngoc Tung,Roland Lill,Petra Horvatek,Christiane Wolz,Jörn Kalinowski,Haike Antelmann

doi:10.1016/j.freeradbiomed.2020.10.322

Abstract

Slow growing stationary phase bacteria are often tolerant to multiple stressors and antimicrobials. Here, we show that the pathogen Staphylococcus aureus develops a non-specific tolerance towards oxidative stress during the stationary phase, which is mediated by the nucleotide second messenger (p)ppGpp. The (p)ppGpp0 mutant was highly susceptible to HOCl stress during the stationary phase. Transcriptome analysis of the (p)ppGpp0 mutant revealed an increased expression of the PerR, SigB, QsrR, CtsR and HrcA regulons during the stationary phase, indicating an oxidative stress response. The (p)ppGpp0 mutant showed a slight oxidative shift in the bacillithiol (BSH) redox potential (EBSH) and an impaired H2O2 detoxification due to higher endogenous ROS levels. The increased ROS levels in the (p)ppGpp0 mutant were shown to be caused by higher respiratory chain activity and elevated total and free iron levels. Consistent with these results, N-acetyl cysteine and the iron-chelator dipyridyl improved the growth and survival of the (p)ppGpp0 mutant under oxidative stress. Elevated free iron levels caused 8 to 31-fold increased transcription of Fe-storage proteins ferritin (ftnA) and miniferritin (dps) in the (p)ppGpp0 mutant, while Fur-regulated uptake systems for iron, heme or siderophores (efeOBU, isdABCDEFG, sirABC and sstADBCD) were repressed. Finally, the susceptibility of the (p)ppGpp0 mutant towards the bactericidal action of the antibiotics ciprofloxacin and tetracycline was abrogated with N-acetyl cysteine and dipyridyl. Taken together, (p)ppGpp confers tolerance to ROS and antibiotics by down-regulation of respiratory chain activity and free iron levels, lowering ROS formation to ensure redox homeostasis in S. aureus.

Highlights

Staphylococcus aureus is an opportunistic pathogen, which colonizes the nose and the skin of one quarter of the human population, but can cause severe life-threatening infections [1,2,3,4,5]
We were interested if S. aureus is able to develop non-specific tolerance to hypochlorous acid (HOCl) and MHQ during the stationary phase
While growth of the (p)ppGpp0 and relsyn mutants could be fully restored with N-acetyl cysteine, the survival could not be fully restored to wild types (WT) level (Fig. 9A–E). These results indicate that the HOCl susceptibility of the (p) ppGpp0 and relsyn mutants is caused by ROS formation, which can be limited by ROS scavengers

Summary

Introduction

Staphylococcus aureus is an opportunistic pathogen, which colonizes the nose and the skin of one quarter of the human population, but can cause severe life-threatening infections [1,2,3,4,5]. The success of S. aureus as major human pathogen is further caused by the increasing prevalence of multiple antibiotic-resistant strains with limited treatment options, such as methicillin-resistant S. aureus isolates (MRSA) [6,7]. Activated macro phages and neutrophils produce large amounts of reactive oxygen and chlorine species (ROS, RCS), such as H2O2 and HOCl as the first line defense to kill invading pathogens [8,9,10,11]. S. aureus has to adapt to antimicrobial compounds and reactive electrophilic species (RES), such as quinones during host-pathogen interactions. It is of utmost importance to study the defense and resistance mechanisms of S. aureus under ROS, RCS, RES and antibiotics for identification of new drug targets and development of alternative therapy strategies to com bat infections with multi-resistant S. aureus isolates [12]

Methods

Results

Conclusion