Allicin, a natural antimicrobial defence substance from garlic, inhibits DNA gyrase activity in bacteria.

Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests. Allicin is a reactive sulfur species (RSS) that oxidizes accessible cysteines in glutathione and proteins. We used a differential isotopic labelling method (OxICAT) to identify allicin targets in the bacterial proteome. We compared the proteomes of allicin-susceptible Pseudomonas fluorescens Pf0-1 and allicin-tolerant PfAR-1 after a sublethal allicin exposure. Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation. Protein oxidation increased after exposure to allicin, and only 50% of proteins from allicin-susceptible Pf0-1, but 65% from allicin-tolerant PfAR-1, remained less than 20% oxidised. DNA gyrase was identified as an allicin target. Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria. After allicin treatment the degree of Cys433 oxidation increased to 55% in susceptible Pf0-1 but only to 10% in tolerant PfAR-1. Allicin inhibited E. coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid. Purified PfAR-1 DNA gyrase was inhibited to greater extent by allicin in vitro than the Pf0-1 enzyme. Substituting PfAR-1 GyrA into Pf0-1 rendered the exchange mutants more susceptible to allicin than the Pf0-1 wild type. Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-tolerant PfAR-1 background, rather than the PfAR-1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0-1 GyrA subunit. DNA gyrase is a target for medicinally important antibiotics; thus, allicin and its analogues may have potential to be developed as gyrase inhibitors, either alone or in conjunction with other therapeutics.

Antimicrobial properties of ternary eutectic aluminum alloys.

Several Escherichia coli deletion mutants of the Keio collection were selected for analysis to better understand which genes may play a key role in copper or silver homeostasis. Each of the selected E. coli mutants had a deletion of a single gene predicted to encode proteins for homologous recombination or contained functions directly linked to copper or silver transport or transformation. The survival of these strains on pure copper surfaces, stainless steel, and alloys of aluminum, copper and/or silver was investigated. When exposed to pure copper surfaces, E. coli ΔcueO was the most sensitive, whereas E. coli ΔcopA was the most resistant amongst the different strains tested. However, we observed a different trend in sensitivities in E. coli strains upon exposure to alloys of the system Al-Ag-Cu. While minor antimicrobial effects were detected after exposure of E. coli ΔcopA and E. coli ΔrecA to Al-Ag alloys, no effect was detected after exposure to Al-Cu alloys. The release of copper ions and cell-associated copper ion concentrations were determined for E. coli ΔcopA and the wild-type E. coli after exposure to pure copper surfaces. Altogether, compared to binary alloys, ternary eutectic alloys (Al-Ag-Cu) had the highest antimicrobial effect and thus, warrant further investigation.

Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines.

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.

Extracting iron and manganese from bacteria with ionophores - a mechanism against competitors characterized by increased potency in environments low in micronutrients.

To maintain their metal ion homeostasis, bacteria critically depend on membrane integrity and controlled ion translocation. Terrestrial Streptomyces species undermine the function of the cytoplasmic membrane as diffusion barrier for metal cations in competitors using ionophores. Although the properties of the divalent cation ionophores calcimycin and ionomycin have been characterized to some extent in vitro, their effects on bacterial ion homeostasis, the factors leading to bacterial cell death, and their ecological role are poorly understood. To gain insight into their antibacterial mechanism, we determined the metal ion composition of the soil bacterium Bacillus subtilis after treatment with calcimycin and ionomycin. Within 15 min the cells lost approximately half of their cellular iron and manganese content whereas calcium levels increased. The proteomic response of B. subtilis provided evidence that disturbance of metal cation homeostasis is accompanied by intracellular oxidative stress, which was confirmed with a ROS-specific fluorescent probe. B. subtilis showed enhanced sensitivity to the ionophores in medium lacking iron or manganese. Furthermore, in the presence of ionophores bacteria were sensitive to high calcium levels. These findings suggest that divalent cation ionophores are particularly effective against competing microorganisms in soils rich in available calcium and low in available iron and manganese.

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

Reactive chlorine species (RCS), such as hypochlorous acid (i.e., bleach), are antimicrobial oxidants produced by the innate immune system. Like many redox-regulated transcription factors, the Escherichia coli repressor NemR responds to RCS by using the reversible oxidation of highly conserved cysteines to alter its DNA-binding affinity. However, earlier work showed that RCS response in NemR does not depend on any commonly known oxidative cysteine modifications. We have now determined the crystal structure of NemR, showing that the regulatory cysteine, Cys106, is in close proximity to a highly conserved lysine (Lys175). We used crystallographic, biochemical, and mass spectrometric analyses to analyze the role of this lysine residue in RCS sensing. Based on our results, we hypothesize that RCS treatment of NemR results in the formation of a reversible Cys106-Lys175 sulfenamide bond. This is, to our knowledge, the first description of a protein whose function is regulated by a cysteine-lysine sulfenamide thiol switch, constituting a novel addition to the biological repertoire of functional redox switches.

Activation of RidA chaperone function by N-chlorination.

Escherichia coli RidA is a member of a structurally conserved, yet functionally highly diverse protein family involved in translation inhibition (human), Hsp90-like chaperone activity (fruit fly) and enamine/imine deamination (Salmonella enterica). Here, we show that E. coli RidA modified with HOCl acts as a highly effective chaperone. Although activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system and glutathione, it is independent of cysteine modification. Instead, treatment with HOCl or chloramines decreases the amino group content of RidA by reversibly N-chlorinating positively charged residues. N-chlorination increases hydrophobicity of RidA and promotes binding to a wide spectrum of unfolded cytosolic proteins. Deletion of ridA results in an HOCl-sensitive phenotype. HOCl-mediated N-chlorination thus is a cysteine-independent post-translational modification that reversibly turns RidA into an effective chaperone holdase, which plays a crucial role in the protection of cytosolic proteins during oxidative stress.

Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins.

Reactive nitrogen species (RNS) function as powerful antimicrobials in host defence, but so far little is known about their bacterial targets. In this study, we set out to identify Escherichia coli proteins with RNS-sensitive cysteines. We found that only a very select set of proteins contain cysteines that undergo reversible thiol modifications upon nitric oxide (NO) treatment in vivo. Of the 10 proteins that we identified, six (AtpA, AceF, FabB, GapA, IlvC, TufA) have been shown to harbour functionally important thiol groups and are encoded by genes that are considered essential under our growth conditions. Media supplementation studies suggested that inactivation of AceF and IlvC is, in part, responsible for the observed NO-induced growth inhibition, indicating that RNS-mediated modifications play important physiological roles. Interestingly, the majority of RNS-sensitive E. coli proteins differ from E. coli proteins that harbour H2O2-sensitive thiol groups, implying that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. We confirmed this specificity by analysing the activity of one of our target proteins, the small subunit of glutamate synthase. In vivo and in vitro activity studies confirmed that glutamate synthase rapidly inactivates upon NO treatment but is resistant towards other oxidative stressors.

Global characterization of disulfide stress in Bacillus subtilis.

We used DNA macroarray and proteome analysis to analyze the regulatory networks in Bacillus subtilis that are affected by disulfide stress. To induce disulfide stress, we used the specific thiol oxidant diamide. After addition of 1 mM diamide to an exponentially growing culture, cell growth stopped until the medium was cleared of diamide. Global analysis of the mRNA expression pattern during growth arrest revealed 350 genes that were induced by disulfide stress by greater than threefold. Strongly induced genes included known oxidative stress genes that are under the control of the global repressor PerR and heat shock genes controlled by the global repressor CtsR. Other genes that were strongly induced encode putative regulators of gene expression and proteins protecting against toxic elements and heavy metals. Many genes were substantially repressed by disulfide stress, among them most of the genes belonging to the negative stringent response. Two-dimensional gels of radioactively labeled protein extracts allowed us to visualize and quantitate the massive changes in the protein expression pattern that occurred in response to disulfide stress. The observed dramatic alteration in the protein pattern reflected the changes found in the transcriptome experiments. The response to disulfide stress seems to be a complex combination of different regulatory networks, indicating that redox-sensing cysteines play a key role in different signaling pathways sensing oxidative stress, heat stress, toxic element stress, and growth inhibition.

Proteomic approach to understanding antibiotic action.

We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.