Combating Black Fungus: Using Allicin as a Potent Antifungal Agent against Mucorales.

Invasive fungal (IF) diseases are a leading global cause of mortality, particularly among immunocompromised individuals. The SARS-CoV-2 pandemic further exacerbated this scenario, intensifying comorbid IF infections such as mucormycoses of the nasopharynx. In the work reported here, it is shown that zygomycetes, significant contributors to mycoses, are sensitive to the natural product allicin. Inhibition of Mucorales fungi by allicin in solution and by allicin vapor was demonstrated. Mathematical modeling showed that the efficacy of allicin vapor is comparable to direct contact with the commercially available antifungal agent amphotericin B (ampB). Furthermore, the study revealed a synergistic interaction between allicin and the non-volatile ampB. The toxicity of allicin solution to human cell lines was evaluated and it was found that the half maximal effective concentration (EC50) of allicin was 25-72 times higher in the cell lines as compared to the fungal spores. Fungal allicin sensitivity depends on the spore concentration, as demonstrated in a drop test. This study shows the potential of allicin, a sulfur-containing defense compound from garlic, to combat zygomycete fungi. The findings underscore allicin's promise for applications in infections of the nasopharynx via inhalation, suggesting a novel therapeutic avenue against challenging fungal infections.

To Cut the Mustard: Antimicrobial Activity of Selenocyanates on the Plate and in the Gas Phase.

Organic selenocyanates (RSeCN) are among the most reactive and biologically active Se species, often exhibiting a pronounced cytotoxic activity against mammalian cells and microorganisms. Various aromatic selenocyanates have been synthesized and, similar to some of the most Reactive Sulfur Species (RSS), such as allicin, found to be active against a range of bacteria, including Escherichia coli, Pseudomonas syringae and Micrococcus luteus, and fungi, including Verticillium dahlia, Verticillium longisporum, Alternaria brassicicola, and Botrytis cinerea, even via the gas phase. The highest antimicrobial activity has been observed for benzyl selenocyanate, which inhibited the growth of all bacteria considerably, even at the lowest tested concentration of 50 µM. Notably, neither the analogues thiocyanate (BTC) nor isothiocyanate (BITC) show any of these activities, rendering this selenium motif rather special in activity and mode of action. Eventually, these findings advocate a range of potential applications of organic selenocyanates in medicine and agriculture.

Allicin as a Volatile or Nebulisable Antimycotic for the Treatment of Pulmonary Mycoses: In Vitro Studies Using a Lung Flow Test Rig.

Fungal infections of the lung are an increasing problem worldwide and the search for novel therapeutic agents is a current challenge due to emerging resistance to current antimycotics. The volatile defence substance allicin is formed naturally by freshly injured garlic plants and exhibits broad antimicrobial potency. Chemically synthesised allicin was active against selected fungi upon direct contact and via the gas phase at comparable concentrations to the pharmaceutically used antimycotic amphotericin B. We investigated the suppression of fungal growth by allicin vapour and aerosols in vitro in a test rig at air flow conditions mimicking the human lung. The effect of allicin via the gas phase was enhanced by ethanol. Our results suggest that allicin is a potential candidate for development for use in antifungal therapy for lung and upper respiratory tract infections.

Allicin, the Odor of Freshly Crushed Garlic: A Review of Recent Progress in Understanding Allicin's Effects on Cells.

The volatile organic sulfur compound allicin (diallyl thiosulfinate) is produced as a defense substance when garlic (Allium sativum) tissues are damaged, for example by the activities of pathogens or pests. Allicin gives crushed garlic its characteristic odor, is membrane permeable and readily taken up by exposed cells. It is a reactive thiol-trapping sulfur compound that S-thioallylates accessible cysteine residues in proteins and low molecular weight thiols including the cellular redox buffer glutathione (GSH) in eukaryotes and Gram-negative bacteria, as well as bacillithiol (BSH) in Gram-positive firmicutes. Allicin shows dose-dependent antimicrobial activity. At higher doses in eukaryotes allicin can induce apoptosis or necrosis, whereas lower, biocompatible amounts can modulate the activity of redox-sensitive proteins and affect cellular signaling. This review summarizes our current knowledge of how bacterial and eukaryotic cells are specifically affected by, and respond to, allicin.

The Sulfilimine Analogue of Allicin, S-Allyl-S-(S-allyl)-N-Cyanosulfilimine, Is Antimicrobial and Reacts with Glutathione.

When cells of garlic (Allium sativum) are disrupted by wounding, they produce the defense substance allicin (diallylthiosulfinate). Allicin is an efficient thiol trap and readily passes through cell membranes into the cytosol, where it behaves as a redox toxin by oxidizing the cellular glutathione (GSH) pool and producing S-allylmercaptoglutathione (GSSA). An N-cyanosulfilimine analogue of allicin (CSA), which was predicted to have similar reactivity towards thiol groups but be more stable in storage, was synthesized and its properties investigated. Similarly to allicin, CSA was shown to inhibit the growth of various bacteria, a fungus (baker's yeast), and Arabidopsis roots. A chemogenetic screen showed that yeast mutants with compromised GSH levels and metabolism were hypersensitive to CSA. GSH reacted with CSA to produce allyltrisulfanylglutathione (GS3A), which was a white solid virtually insoluble in water. Yeast Δgsh1 mutants are unable to synthesize GSH because they lack the γ-glutamylcysteine synthetase (GSH1) gene, and they are unable to grow without GSH supplementation in the medium. GS3A in the growth medium supported the auxotrophic requirement for GSH in Δgsh1 mutants. This result suggests that GS3A is being reduced to GSH in vivo, possibly by the enzyme glutathione reductase (GR), which has been shown to accept GSSA as a substrate. The results suggest that CSA has a mode of action similar to allicin and is effective at similar concentrations.

Allicin, a Potent New Ornithine Decarboxylase Inhibitor in Neuroblastoma Cells.

The natural product allicin is a reactive sulfur species (RSS) from garlic (Allium sativum L.). Neuroblastoma (NB) is an early childhood cancer arising from the developing peripheral nervous system. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are oncometabolites that contribute to cell proliferation in NB and other c-MYC/MYCN-driven cancers. Both c-MYC and MYCN directly transactivate the E-box gene ODC1, a validated anticancer drug target. We identified allicin as a potent ODC inhibitor in a specific radioactive in vitro assay using purified human ODC. Allicin was ∼23 000-fold more potent (IC50 = 11 nM) than DFMO (IC50 = 252 µM), under identical in vitro assay conditions. ODC is a homodimer with 12 cysteines per monomer, and allicin reversibly S-thioallylates cysteines. In actively proliferating human NB cells allicin inhibited ODC enzyme activity, reduced cellular polyamine levels, inhibited cell proliferation (IC50 9-19 µM), and induced apoptosis. The natural product allicin is a new ODC inhibitor and could be developed for use in conjunction with other anticancer treatments, the latter perhaps at a lower than usual dosage, to achieve drug synergism with good prognosis and reduced adverse effects.

Investigation of the deposition behaviour and antibacterial effectivity of allicin aerosols and vapour using a lung model.

Allicin is a natural antibiotic produced by garlic as a defence against pathogens and pests. Due to the worldwide increase in antibiotic resistance, new antibiotics are desperately required. Allicin is such a candidate and is active against several multidrug-resistant (MDR) strains of human pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). When administered orally, allicin is titrated out by glutathione in the cells and blood, and effective therapeutic concentrations are difficult to achieve at the site of an infection. However, in the case of lung infections, allicin can be delivered directly to pathogens via the pulmonary route. In this study, we designed and constructed an in vitro lung test rig, which allowed us to model accurately the exposure of lung air-passage surfaces to allicin and gentamicin, in order to examine the feasibility of combating lung infections by direct inhalation. A prototype test rig of lung bronchi with three bifurcations was constructed, which could be coated internally with a thin layer of bacteria-seeded agar medium. The deposition of antimicrobial aerosols on the modelled bronchial surfaces was followed in preliminary tests without the need for animal experiments. The differential sensitivity of the test bacteria to different antibiotics and the dose-dependency of inhibition was shown using the model. Furthermore, a synergistic effect of allicin vapour and ethanol in inhibiting bacterial growth was demonstrated. The modelling of the axial velocity air-flow distribution correlated with the regions indicating the inhibition of bacterial growth, demonstrating that the model has predictive value and can reduce the requirement for animal sacrifice in pre-clinical trials of novel antibiotics.

The human allicin-proteome: S-thioallylation of proteins by the garlic defence substance allicin and its biological effects.

A single clove of edible garlic (Allium sativum L.) of about 10 g produces up to 5 mg of allicin (diallylthiosulfinate), a thiol-reactive sulfur-containing defence substance that gives injured garlic tissue its characteristic smell. Allicin induces apoptosis or necrosis in a dose-dependent manner but biocompatible doses influence cellular metabolism and signalling cascades. Oxidation of protein thiols and depletion of the glutathione pool are thought to be responsible for allicin's physiological effects. Here, we studied the effect of allicin on post-translational thiol-modification in human Jurkat T-cells using shotgun LC-MS/MS analyses. We identified 332 proteins that were modified by S-thioallylation in the Jurkat cell proteome which causes a mass shift of 72 Da on cysteines. Many S-thioallylated proteins are highly abundant proteins, including cytoskeletal proteins tubulin, actin, cofilin, filamin and plastin-2, the heat shock chaperones HSP90 and HSPA4, the glycolytic enzymes GAPDH, ALDOA, PKM as well the protein translation factor EEF2. Allicin disrupted the actin cytoskeleton in murine L929 fibroblasts. Allicin stimulated the immune response by causing Zn2+ release from proteins and increasing the Zn2+-dependent IL-1-triggered production of IL-2 in murine EL-4 T-cells. Furthermore, allicin caused inhibition of enolase activity, an enzyme considered a cancer therapy target. In conclusion, our study revealed the widespread extent of S-thioallylation in the human Jurkat cell proteome and showed effects of allicin exposure on essential cellular functions of selected targets, many of which are targets for cancer therapy.

S-allylmercaptoglutathione Is a Substrate for Glutathione Reductase (E.C. 1.8.1.7) from Yeast (Saccharomyces cerevisiae).

Allicin (diallylthiosulfinate) is a potent thiol reagent and natural defense substance produced by garlic (Allium sativum) tissues when damaged. Allicin acts as a redox toxin and oxidizes the cellular glutathione (GSH) pool producing S-allylmercaptoglutathione (GSSA). The cellular enzyme glutathione reductase (GR) uses NADPH to reduce glutathione disulfide (GSSG) back to GSH and replenishes the GSH pool. It was not known whether GR could accept GSSA as a substrate. Here, we report that GR from yeast (Saccharomyces cerevisiae) shows Michaelis⁻Menten kinetics with GSSA as substrate in vitro (Km = 0.50 mM), but that GSSA is not as good a substrate as GSSG (Km = 0.07 mM). Furthermore, cells unable to synthesize GSH because the γ-glutamylcysteine synthetase (GSH1) gene is deleted, cannot grow without GSH supplementation and we show that the auxotrophic requirement for GSH in Δgsh1 mutants can be met by GSSA in the growth medium, suggesting that GSSA can be reduced to GSH in vivo.

A Comparison of the Antibacterial and Antifungal Activities of Thiosulfinate Analogues of Allicin.

Allicin (diallylthiosulfinate) is a defence molecule from garlic (Allium sativum L.) with broad antimicrobial activities in the low µM range against Gram-positive and -negative bacteria, including antibiotic resistant strains, and fungi. Allicin reacts with thiol groups and can inactivate essential enzymes. However, allicin is unstable at room temperature and antimicrobial activity is lost within minutes upon heating to >80 °C. Allicin's antimicrobial activity is due to the thiosulfinate group, so we synthesized a series of allicin analogues and tested their antimicrobial properties and thermal stability. Dimethyl-, diethyl-, diallyl-, dipropyl- and dibenzyl-thiosulfinates were synthesized and tested in vitro against bacteria and the model fungus Saccharomyces cerevisiae, human and plant cells in culture and Arabidopsis root growth. The more volatile compounds showed significant antimicrobial properties via the gas phase. A chemogenetic screen with selected yeast mutants showed that the mode of action of the analogues was similar to that of allicin and that the glutathione pool and glutathione metabolism were of central importance for resistance against them. Thiosulfinates differed in their effectivity against specific organisms and some were thermally more stable than allicin. These analogues could be suitable for applications in medicine and agriculture either singly or in combination with other antimicrobials.

The Chemistry of Alliums.

Physiologically active sulfur-containing compounds produced by Allium spp. have long fascinated chemists, biochemists, and biologists.[...].

Yap1p, the central regulator of the S. cerevisiae oxidative stress response, is activated by allicin, a natural oxidant and defence substance of garlic.

Allicin is a thiol-reactive sulfur-containing natural product from garlic with a broad range of antimicrobial effects against prokaryotes and eukaryotes. Previous work showed that the S. cerevisiae OSI1 gene is highly induced by allicin and other thiol-reactive compounds, and in silico analysis revealed multiple Yap1p binding motifs in the OSI1 promoter sequence. An OSI1-promoter::luciferase reporter construct expressed in Wt and Δyap1 cells showed absolute Yap1p-dependence for allicin-induced OSI1-expression. A GFP: Yap1p fusion protein accumulated in the nucleus within 10min of allicin treatment and a Δyap1 mutant was highly sensitive to allicin. Yap1p regulates glutathione (GSH) metabolism genes, and Δgsh1, Δgsh2 and Δglr1 mutants showed increased sensitivity to allicin. Allicin activated the OSI1-promoter::luciferase reporter construct in Δgpx3 and Δybp1 cells, indicating that allicin activates Yap1p directly rather than via H2O2 production. A systematic series of C-to-A Yap1p exchange mutants showed that the C-term C598 and C620 residues were necessary for allicin activation. These data suggest that Yap1p is an important transcriptional regulator for the resistance of yeast cells to allicin, and that activation occurs by direct modification of C-term cysteines as shown for other electrophiles.

The Effects of Allicin, a Reactive Sulfur Species from Garlic, on a Selection of Mammalian Cell Lines.

Garlic (Allium sativum L.) has been used as a spice and medicinal plant since ancient times. Garlic produces the thiol-reactive defence substance, allicin, upon wounding. The effects of allicin on human lung epithelium carcinoma (A549), mouse fibroblast (3T3), human umbilical vein endothelial cell (HUVEC), human colon carcinoma (HT29) and human breast cancer (MCF7) cell lines were tested. To estimate toxic effects of allicin, we used a standard MTT-test (methylthiazoltetrazolium) for cell viability and ³H-thymidine incorporation for cell proliferation. The glutathione pool was measured using monobromobimane and the formation of reactive species was identified using 2',7'-dichlorofluoresceine-diacetate. The YO-PRO-1 iodide staining procedure was used to estimate apoptosis. Allicin reduced cell viability and cell proliferation in a concentration dependent manner. In the bimane test, it was observed that cells treated with allicin showed reduced fluorescence, suggesting glutathione oxidation. The cell lines tested differed in sensitivity to allicin in regard to viability, cell proliferation and glutathione oxidation. The 3T3 and MCF-7 cells showed a higher proportion of apoptosis compared to the other cell types. These data show that mammalian cell lines differ in their sensitivity and responses to allicin.

Effect of silver nanoparticles on the standard soil arthropod Folsomia candida (Collembola) and the eukaryote model organism Saccharomyces cerevisiae.

BACKGROUND: Because of their antimicrobial properties, silver nanoparticles (AgNPs) have been widely used and have come into contact with the environment. In the present work, an effect of AgNPs on a standard soil organism, Folsomia candida, was studied (in comparison to silver nitrate) focusing on molecular and cellular alterations as ecotoxicological endpoints. RESULTS: At the molecular level, an up-regulation of metallothionein-containing protein (MTC) mRNA in AgNP-treated groups indicated toxic heavy metal stress effects caused by the release of silver ions from AgNPs, which is similar to animal groups treated with silver nitrate. Alteration of the steady-state level of glutathione S-transferase (GST) mRNA was detected in animal treated with AgNPs and AgNO3. At the cellular level, the relation between GST activity and the size of the glutathione (GSH) was examined. Change of GST activity from different animal groups was not significant, whereas the GSH pool (reduced and oxidized forms) decreased with increasing concentration of AgNPs. In order to obtain direct evidence whether AgNPs cause oxidative stress, treated animals were incubated with the non-fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). A fluorescence signal was observed in both AgNPs- and AgNO3-treated groups pointing to the production of reactive species (RS). Since RS formation in F.candida is difficult to quantify, yeast strain BY4742 (wild-type) and mutants lacking of oxidative stress-related protective enzymes were exploited as a further eukaryote model organism. AgNPs and AgNO3 were found to also affect growth of yeast and induced oxidative stress. CONCLUSIONS: An effect of AgNPs on Collembola and yeast strains is similar to the one from AgNO3. However, AgNPs is less toxic due to the slow release of silver ions. In summary, the toxic effect of AgNPs on F. candida is caused by the combination of the release of silver ions from AgNPs and the formation of reactive species.

The defense substance allicin from garlic permeabilizes membranes of Beta vulgaris, Rhoeo discolor, Chara corallina and artificial lipid bilayers.

BACKGROUND: Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers. METHODS: Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped. RESULTS: Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers. CONCLUSIONS: Allicin increases the permeability of membranes. GENERAL SIGNIFICANCE: Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicin's ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.

Allicin: chemistry and biological properties.

Allicin (diallylthiosulfinate) is a defence molecule from garlic (Allium sativum L.) with a broad range of biological activities. Allicin is produced upon tissue damage from the non-proteinogenic amino acid alliin (S-allylcysteine sulfoxide) in a reaction that is catalyzed by the enzyme alliinase. Current understanding of the allicin biosynthetic pathway will be presented in this review. Being a thiosulfinate, allicin is a reactive sulfur species (RSS) and undergoes a redox-reaction with thiol groups in glutathione and proteins that is thought to be essential for its biological activity. Allicin is physiologically active in microbial, plant and mammalian cells. In a dose-dependent manner allicin can inhibit the proliferation of both bacteria and fungi or kill cells outright, including antibiotic-resistant strains like methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, in mammalian cell lines, including cancer cells, allicin induces cell-death and inhibits cell proliferation. In plants allicin inhibits seed germination and attenuates root-development. The majority of allicin's effects are believed to be mediated via redox-dependent mechanisms. In sub-lethal concentrations, allicin has a variety of health-promoting properties, for example cholesterol- and blood pressure-lowering effects that are advantageous for the cardio-vascular system. Clearly, allicin has wide-ranging and interesting applications in medicine and (green) agriculture, hence the detailed discussion of its enormous potential in this review. Taken together, allicin is a fascinating biologically active compound whose properties are a direct consequence of the molecule's chemistry.

Sulfur and sulfur compounds in plant defence.

The multiplicity of chemical structures of sulfur containing compounds, influenced in part by the element's several oxidation states, directly results in diverse modes of action for sulfur-containing natural products synthesized as secondary metabolites in plants. Sulfur-containing natural products constitute a formidable wall of defence against a wide range of pathogens and pests. Steady progress in the development of new technologies have advanced research in this area, helping to uncover the role of such important plant defence molecules like endogenously-released elemental sulphur, but also deepening current understanding of other better-studied compounds like the glucosinolates. As studies continue in this area, it is becoming increasingly evident that sulfur and sulfur compounds play far more important roles in plant defence than perhaps previously suspected.

The biology of reactive sulfur species (RSS).

Sulfur is an essential and quantitatively important element for living organisms. Plants contain on average approximately 1 g S kg⁻¹ dry weight (for comparison plants contain approximately 15 g N kg⁻¹ dry weight). Sulfur is a constituent of many organic molecules, for example amino acids such as cysteine and methionine and the small tripeptide glutathione, but sulfur is also essential in the form of Fe-S clusters for the activity of many enzymes, particularly those involved in redox reactions. Sulfur chemistry is therefore important. In particular, sulfur in the form of thiol groups is central to manifold aspects of metabolism. Because thiol groups are oxidized and reduced easily and reversibly, the redox control of cellular metabolism has become an increasing focus of research. In the same way that oxygen and nitrogen have reactive species (ROS and RNS), sulfur too can form reactive molecular species (RSS), for example when a -SH group is oxidized. Indeed, several redox reactions occur via RSS intermediates. Several naturally occurring S-containing molecules are themselves RSS and because they are physiologically active they make up part of the intrinsic plant defence repertoire against herbivore and pathogen attack. Furthermore, RSS can also be used as redox-active pharmacological tools to study cell metabolism. The aim of this review is to familiarize the general reader with some of the chemical concepts, terminology and biology of selected RSS.

Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast.

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes.