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

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 (E_BSH) 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.

SUBMITTER: Fritsch VN

PROVIDER: S-EPMC7754856 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Fritsch Verena Nadin VN Loi Vu Van VV Busche Tobias T Tung Quach Ngoc QN Lill Roland R Horvatek Petra P Wolz Christiane C Kalinowski Jörn J Antelmann Haike H

Free radical biology & medicine 20201101

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)ppGpp<sup>0</sup> mutant was highly susceptible to HOCl stress during the stationary phase. Transcriptome analysis of the (p)ppGpp<sup>0</sup> mutant revealed an increased expression of th ...[more]

PMID: 33144262

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Project description:During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically modulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.

Dataset Information

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

Publications

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

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