Garlic plants (Allium sativum L.) produce antimicrobial compounds, such as diallyl thiosulfinate (allicin) and diallyl polysulfanes. Here, we investigated the transcriptome and protein S-thioallylomes under allicin and diallyl tetrasulfane (DAS4) exposure in the Gram-positive bacterium Bacillus subtilis. Allicin and DAS4 caused a similar thiol-specific oxidative stress response, protein and DNA damage as revealed by the induction of the OhrR, PerR, Spx, YodB, CatR, HypR, AdhR, HxlR, LexA, CymR, CtsR, and HrcA regulons in the transcriptome. At the proteome level, we identified, in total, 108 S-thioallylated proteins under allicin and/or DAS4 stress. The S-thioallylome includes enzymes involved in the biosynthesis of surfactin (SrfAA, SrfAB), amino acids (SerA, MetE, YxjG, YitJ, CysJ, GlnA, YwaA), nucleotides (PurB, PurC, PyrAB, GuaB), translation factors (EF-Tu, EF-Ts, EF-G), antioxidant enzymes (AhpC, MsrB), as well as redox-sensitive MarR/OhrR and DUF24-family regulators (OhrR, HypR, YodB, CatR). Growth phenotype analysis revealed that the low molecular weight thiol bacillithiol, as well as the OhrR, Spx, and HypR regulons, confer protection against allicin and DAS4 stress. Altogether, we show here that allicin and DAS4 cause a strong oxidative, disulfide and sulfur stress response in the transcriptome and widespread S-thioallylation of redox-sensitive proteins in B. subtilis. The results further reveal that allicin and polysulfanes have similar modes of actions and thiol-reactivities and modify a similar set of redox-sensitive proteins by S-thioallylation.

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 0 mutant was highly susceptible to HOCl stress during the stationary phase. Transcriptome analysis of the (p)ppGpp 0 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)ppGpp 0 mutant showed a slight oxidative shift in the bacillithiol (BSH) redox potential ( E BSH ) and an impaired H 2 O 2 detoxification due to higher endogenous ROS levels. The increased ROS levels in the (p)ppGpp 0 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)ppGpp 0 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)ppGpp 0 mutant, while Fur-regulated uptake systems for iron, heme or siderophores ( efeOBU , isdABCDEFG , sirABC and sstADBCD ) were repressed. Finally, the susceptibility of the (p)ppGpp 0 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 .

Staphylococcus aureus is a major human pathogen, which encounters reactive oxygen, nitrogen, chlorine, electrophile and sulfur species (ROS, RNS, RCS, RES and RSS) by the host immune system, during cellular metabolism or antibiotics treatments. To defend against redox active species and antibiotics, S. aureus is equipped with redox sensing regulators that often use thiol switches to control the expression of specific detoxification pathways. In addition, the maintenance of the redox balance is crucial for survival of S. aureus under redox stress during infections, which is accomplished by the low molecular weight (LMW) thiol bacillithiol (BSH) and the associated bacilliredoxin (Brx)/BSH/bacillithiol disulfide reductase (YpdA)/NADPH pathway. Here, we present an overview of thiol-based redox sensors, its associated enzymatic detoxification systems and BSH-related regulatory mechanisms in S. aureus, which are important for the defense under redox stress conditions. Application of the novel Brx-roGFP2 biosensor provides new insights on the impact of these systems on the BSH redox potential. These thiol switches of S. aureus function in protection against redox active desinfectants and antimicrobials, including HOCl, the AGXX® antimicrobial surface coating, allicin from garlic and the naphthoquinone lapachol. Thus, thiol switches could be novel drug targets for the development of alternative redox-based therapies to combat multi-drug resistant S. aureus isolates.

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Allicin (diallyl thiosulfinate) is the major thiol-reactive organosulfur compound produced by garlic plants (Allium sativum) upon tissue damage. Allicin exerts its strong antimicrobial activity against bacteria and fungi via S-thioallylation of protein thiols and low molecular weight thiols. Here, we investigated the effect of allicin on SARS-CoV-2 infected Vero E6 and Calu-3 cells. Toxicity tests revealed that Calu-3 cells showed greater allicin tolerance, probably due to >4-fold higher GSH levels compared to the very sensitive Vero E6 cells. Exposure of infected Vero E6 and Calu-3 cells to biocompatible allicin doses led to a ∼60–70% decrease of viral RNA and infectious viral particles. Label-free quantitative proteomics was used to investigate the changes in the Calu-3 proteome after SARS-CoV-2 infection and the effect of allicin on the host-virus proteome. SARS-CoV-2 infection of Calu-3 cells caused a strong induction of the antiviral interferon-stimulated gene (ISG) signature, including several antiviral effectors, such as cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, OAS, and ISG15, pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTCL1, and ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin treatment of infected Calu-3 cells reduced the expression of IFN signaling pathways and ISG effectors and reverted several host pathways to levels of uninfected cells. Allicin further reduced the abundance of the structural viral proteins N, M, S and ORF3 in the host-virus proteome. In conclusion, our data demonstrate the antiviral and immunomodulatory activity of biocompatible doses of allicin in SARS-CoV-2-infected cell cultures. Future drug research should be directed to exploit the thiol-reactivity of allicin derivatives with increased stability and lower human cell toxicity as antiviral lead compounds.