Article pubs.acs.org/JAFC
A Mini HIP HOP Assay Uncovers a Central Role for Copper and Zinc in the Antifungal Mode of Action of Allicin Thomas A. K. Prescott*,† and Barry Panaretou‡ †
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom King’s College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom
‡
S Supporting Information *
ABSTRACT: Garlic contains the organosulfur compound allicin which exhibits potent antifungal activity. Here we demonstrate the use of a highly simplified yeast chemical genetic screen to characterize its mode of action. By screening 24 validated yeast gene deletion “signature” strains for which hypersensitivity is characteristic for common antifungal modes of action, yeast lacking the high affinity Cu2+ transporter Ctr1 was found to be hypersensitive to allicin. Focusing on transition metal related genes identified two more hypersensitive strains lacking the Cu2+ and Zn2+ transcription factors Mac1 and Zap1. Hypersensitivity in these strains was reversed by the addition of Cu2+ and Zn2+ ions, respectively. The results suggest the antifungal activity of allicin is mediated through restricted Cu2+ and Zn2+ uptake or inhibition of Cu2+ and Zn2+ metalloproteins. As certain antimicrobial modes of action are much more common than others, the approach taken here provides a useful way to identify them early on. KEYWORDS: garlic, copper, CTR1, yeast deletion, allicin, chelation, mode-of-action
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INTRODUCTION Garlic exhibits biocidal activity toward a diverse range of microorganisms; its in vitro activity against pathogenic yeasts is particularly impressive, with fungicidal activity comparable to fluconazole.1−3 When garlic cloves are crushed the enzyme alliinase converts the organosulfur precursor compound alliin into the active antimicrobial compound allicin.4,5 In a mouse model of systemic candidiasis, treatment with 5 mg kg−1 day−1 allicin increased survival times from 8.4 to 15.8 days.3 Furthermore, garlic extract and allicin have shown some promise as topical agents for treating vaginal thrush in new born infants and chronic oral candidiasis in adults.6,7 Various modes of action have been proposed to explain the antifungal activity of allicin. As far back as 1956 E. D Willis demonstrated that allicin acted as an inhibitor of a variety of different thiol containing enzymes and that the inhibition was reversed by the addition of glutathione. This gave rise to the suggestion that allicin acts through a nonspecific mode of action by covalently attaching to enzyme thiol groups.8 Modern revaluation of this mode of action has reconfirmed the interaction of allicin with thiol containing enzymes and demonstrated the rapid diffusion of allicin across lipid bilayers.9,10 In addition to its interaction with protein thiol groups several other modes of action have been proposed. The antibacterial mode of action of allicin was studied in Salmonella typhimurium, and RNA, protein, and DNA syntheses rates were measured using radiolabeled precursors. Although all three processes were inhibited, inhibition of RNA synthesis was much more rapid.11 Disruption of the cells electrochemical potential has also been proposed as a possible mode of action; a redox sensitive GFP reporter in yeast demonstrated allicin induced changes in cellular electrochemical potential followed by caspase activation and apoptosis.12 More recently, global gene expression profiling in S. cerevisiae has been carried out; © XXXX American Chemical Society
major responses were associated with the proteasome, iron ion transporter activity, and amino acid control.13 A similar approach in Candida albicans identified nearly 3000 genes that were differentially expressed covering diverse biological processes. Subsequent analysis with 2D-DIGE identified four upregulated proteins; cytoplasmic adenylate kinase, pyruvate decarboxylase, hexokinase and the heat shock protein Ssc1.14 Previous work has demonstrated fungicidal synergy between allicin and Cu2+ although these effects occur at concentrations of Cu2+ considerably higher than that of the trace amounts present in culture medium suggesting this effect is not connected to the mode of action of allicin.15,16 In the present study we use a simplified screen comprising a subset of yeast gene deletion “signature” strains to probe the mode of action of allicin. Yeast chemical genetic HIP HOP screens carried out with ∼6000 S. cerevisiae gene deletion strains have proved to be a powerful technique in the elucidation of the mode of action of small molecules that act to inhibit the growth of eukaryotic cells.17,18 They have been shown to correctly identify the mode of action of known drugs as well as uncovering the mode of action of novel compounds.19,20 The premise behind HIP HOP profiling is that rather than attempt to assay isolated proteins with the inhibitor compound (a process that would be prohibitively time-consuming); instead collections of yeast gene deletion strains can be used to measure the sensitivity of whole cells to the compound. In the case of a highly targeted compound that acts though a single receptor protein, the yeast strain that expresses reduced levels of that receptor will exhibit hyperReceived: Revised: Accepted: Published: A
January 18, 2017 March 9, 2017 April 19, 2017 April 19, 2017 DOI: 10.1021/acs.jafc.7b00250 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Table 1. Yeast Strains Used in the Mini HIP HOP Screen Comprising Signature Strains That Exhibit Hypersensitivity to Compounds That Act through Common Modes of Action Where a Specific Protein Target Is Lackinga
a
gene
systematic name
acc. no.
HIP/HOP
genetic notation
likely mode of action
NMD3 REI1 LSG1 SSL2 NEO1 TIM54 PIK1 CMD1 TDA10 LEM3 FEN1 LRO1 MIA40 TOM40 AFT1 FTR1 CTR1 FET3 IRE1 HAC1 MRPL19 RIM101 SEC13 SEC31
YHR170W YBR267W YGL099W YIL143C YIL048W YJL054W YNL267W YBR109C YGR205W YNL323W YCR034W YNR008W YKL195W YMR203W YGL071W YER145C YPR124W YMR058W YHR079C YFL031W YNL185C YHL027W YLR208W YDL195W
Y26418 Y23407 Y24466 Y22302 Y21441 Y21369 Y26958 Y23248 Y24835 Y31121 Y35763 Y35383 Y27033 Y20789 Y34438 Y36142 Y35539 Y36192 Y31907 Y35650 Y22027 Y30936 Y24157 Y23893
HIP HIP HIP HIP HIP HIP HIP HIP HIP HOP HOP HOP HIP HIP HOP HOP HOP HOP HOP HOP HIP HOP HIP HIP
nmd3Δ/NMD3 rei1Δ/REI1 lsg1Δ/LSG1 ssl2Δ/SSL2 neo1Δ/NEO1 tim54Δ/TIM54 pik1Δ/PIK1 cmd1Δ/CMD1 tda10Δ/TDA10 lem3Δ/lem3Δ fen1Δ/fen1Δ lro1Δ/lro1Δ mia40Δ/MIA40 tom40Δ/TOM40 af t1Δ/aft1Δ f tr1Δ/f tr1Δ ctr1Δ/ctr1Δ fet3Δ/fet3Δ ire1Δ/ire1Δ hac1Δ/hac1Δ mrpl19Δ/MRPL19 rim101Δ/rim101 Δ sec13Δ/SEC13 sec31Δ/SEC31
DNA intercalation DNA intercalation DNA intercalation DNA intercalation membrane perturbation membrane perturbation membrane perturbation membrane perturbation membrane perturbation membrane perturbation membrane perturbation/lipids fatty acid disruption mitochondrial membrane perturbation mitochondrial membrane perturbation cation chelation cation chelation cation chelation cation chelation ER stress ER stress uncoupling pH stress Lipid stress interfering with trafficking Lipid stress interfering with trafficking
As determined by large scale genome wide screening of yeast inhibitory small molecules.20 Strains supplied by EUROSCARF.
the full range of ∼6000 yeast gene deletion strains that are used in genome wide profiling. In the present study a subset of just 24 signature genes (a mini HIP HOP screen) are used to further refine the antifungal mode of action of garlic. This highly simplified approach provides a means to eliminate common modes of action early on and makes yeast chemical genetic screening accessible for laboratories that lack the means to screen large collection of yeast gene deletion strains.
sensitivity to the test compound. The combination of reduced levels of functional receptor created through gene deletion and chemical inhibition of the receptor by the inhibitor compound results in decreased growth. Conversely strains that express reduced levels of proteins that are unrelated to the receptor protein do not exhibit hypersensitivity. Thus, by measuring the growth rate of individual gene deletion strains in the presence of a sublethal concentration of an inhibitory test compound, a measurable link is provided between protein levels in whole cells (including the drug target itself) and the relative fitness of the cells.21 In the HIP assay (HaploInsufficiency Profiling) a library of diploid yeast strains each deleted for one copy of a different gene and therefore expressing approximately half the normal level of a different protein are screened against the test compound.18 Haploinsufficiency profiling allows essential genes to be included as protein function is reduced rather than completely eliminated. In the HOP assay (HOmozygous Profiling), yeast deleted for both copies of a gene are screened enabling complete elimination of proteins from the cell although essential genes cannot be included.18 The genetically tractable nature of yeast and the genetic conservation of essential drug targets makes it an ideal system to understand the mode of action of diverse drug candidates from chemotherapy agents to antifungal compounds.18 Previous work using high throughput HIP HOP screening of several thousand yeast inhibitory small molecules has demonstrated that certain nonspecific modes of action where a specific protein receptor is lacking such as membrane perturbation, DNA binding, or metal ion chelation are considerably more common than targeted and specific ligandprotein interactions.20 So called signature genes that are associated with these common modes of action could provide revealing insights into compound mode action without testing
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EXPERIMENTAL SECTION
Preparation of Garlic Extract and Activity-Guided Fractionation. Allium sativum L. was obtained from a commercial supplier (Tesco Supermarket, Kew, London, U.K.). Freshly peeled garlic bulbs (40g) were crushed to a mulch in a mortar and pestle to provide a homogeneous paste which was then extracted with 40 mL deionized water to produce a highly concentrated slurry. Solid matter and fine particles were removed by repeated centrifugation at 13 400×g followed by filtration through a 0.22 μ PTFE syringe filter. The extract was then stored in single use aliquots at −80 °C. Fractionation of the aqueous garlic extract was carried out on a Waters HPLC fitted with a Phenomenex 5 μ C-18 4.6 × 250 mm2 column and diode array detector, using a gradient of 10 to 100% MeCN over 30 min. Fractions were collected every 20 s into a 96 well plate, aliquots of which were assayed for yeast growth inhibition. Isolation of Allicin. Fresh garlic (370 g) was crushed and extracted twice with 50 mL chloroform. The chloroform was removed by rotary evaporation and the resulting yellow solution was fractionated by HPLC using a 5 μ Phenomenex 10 × 150 mm C-18 column and an isocratic 60% MeOH elution (flow rate 5 mL/min). The active fraction comprising a well resolved HPLC peak was collected through multiple runs which were pooled and extracted with chloroform. Rotary evaporation followed by concentration under dry nitrogen yielded a pale yellow liquid with the characteristic odor of garlic. Liquid chromatography−mass spectrometry (LC−MS) of the purified sample was performed on a Thermo Scientific LTQ-orbitrap XL′ mass B
DOI: 10.1021/acs.jafc.7b00250 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry spectrometer fitted with an electrospray source (system hardware and parameters as described previously).21 Yeast Strains Used in This Study. All strains were purchased from the EUROpean Saccharomyces cerevisiae ARchive for Functional analysis (EUROSCARF). Heterozygous diploids were obtained for essential signature genes: nmd3Δ/NMD3, rei1Δ/REI1, lsg1Δ/LSG1, ssl2Δ/SSL2, neo1Δ/NEO1, tim54Δ/TIM54, pik1Δ/PIK1, cmd1Δ/ CMD1, tda10Δ/TDA10, mia40Δ/MIA40, tom40Δ/TOM40, mrpl19Δ/MRPL19, sec13Δ/SEC13, and sec31Δ/SEC31. Homozygous diploids were obtained for nonessential signature genes: lem3Δ/lem3Δ, fen1Δ/fen1Δ, lro1Δ/lro1Δ, af t1Δ/af t1Δ, f tr1Δ/f tr1Δ, ctr1Δ/ctr1Δ, fet3Δ/fet3Δ, ire1Δ/ire1Δ, hac1Δ/hac1Δ, rim101Δ/rim10 Δ, mac1Δ/mac1Δ, cup2Δ/cup2Δ, zap1Δ/zap1Δ, smf1Δ/smf1Δ, and ctr2Δ/ctr2Δ. The diploids have the following isogenic background: MATa/α; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; met15Δ0/MET15; LYS2/lys2Δ0; and ura3Δ0/ura3Δ0. In all cases the genes were deleted with KANMX4. Yeast Growth Curves. The growth curves of yeast deletion strains were measured in a 384 well microplate. Each well contained 50 μL liquid YPD medium in which the yeast culture in question had been diluted to an optical density of OD600 0.005 using a 1 cm path length spectrophotometer. Test substances were diluted in the same 50 μL volume, with serial dilution and other liquid handling performed with an Integra Viaflo 16 channel pipet. The edge wells of the plate were used to form an evaporation barrier of water. The microplate with lid was incubated at 30 °C in a Tecan Infinite M200 plate reader recording OD600 readings in 9 separate locations per well every 20 min for 23 h. Growth curves were then plotted using plate reader OD600 raw data over time.
Figure 1. Growth curves of isogenic control yeast strain BY4743 with increasing concentrations of aqueous garlic extract. Black filled circles no extract, filled gray squares 0.0125% v/v extract, filled gray circles 0.025% v/v extract, open squares 0.05% v/v extract, black open circles 0.1% v/v extract, and gray open circles 0.2% v/v extract. Each data point is an average of readings taken from 22 replicate wells.
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RESULTS AND DISCUSSION Growth Curves of 24 Yeast Deletion Signature Strains in the Presence of Aqueous Garlic Extract Reveals Hypersensitivity of Yeast Lacking the Cu2+ Transporter Ctr1. Previous high-throughput genome-wide screening of yeast inhibitory small molecules has shown that targeted protein−ligand inhibition type modes of action are relatively rare; a more common explanation for the yeast inhibitory activity of a small molecule is likely to be found in modes of action where a single protein target is absent.20 Table 1 lists 24 gene deletion signature strains that exhibit hypersensitivity to compounds acting through common nonprotein type modes of action such as plasma membrane perturbation, DNA binding and metal ion chelation.20 Growth curves of the BY4743 isogenic control strain were measured with titrations of garlic extract to determine the optimal screening dose (Figure 1). Growth curves for each of the 24 strains were then determined in the presence of a subinhibitory dose of 0.01% v/v aqueous extract and used to determine relative growth inhibition for each strain (Figure 2). Out of the 24 signature strains only ctr1Δ/ctr1Δ shows hypersensitivity to the garlic extract, relative to the BY4743 control strain. Yeast CTR1 codes for a membrane protein that has been demonstrated to act as a high affinity Cu2+ transporter under low Cu2+ conditions.22 Other common modes of action such as membrane damage, DNA intercalation, and so forth could be discounted as they are unlikely to be responsible for the antifungal activity of garlic. Allicin is Responsible for the Yeast Inhibitory Effects of Aqueous Garlic Extract. Although allicin is well-known to be the active antimicrobial principle of garlic, activity-guided fractionation of aqueous garlic extract was carried out to demonstrate that allicin was responsible for all of the yeast inhibitory activity of the extract used in this study. Assay of HPLC fractions assigned the activity of the extract to a single well resolved peak eluting at 13 min. Subsequent collection,
Figure 2. The ctr1Δ/ctr1Δ strain exhibits hypersensitivity to aqueous garlic extract relative to the BY4743 isogenic control. Relative growth inhibition of 24 yeast gene deletion signature strains (see Table 1.) were recorded in the presence of aqueous garlic extract. Growth curves were recorded for each strain with and without 0.01% v/v aqueous garlic extract and growth inhibition between 10 and 20 h determined relative to that of each untreated control strain. Error bars are SEM, n = 3 from three independent experiments.
concentration, and assay of the active fraction confirmed that its minimal inhibitory concentration was the same (0.2% v/v) as the equivalent prefractionation 5 μL HPLC injection sample. Thus, all of the yeast inhibitory activity of the extract was attributed to a single HPLC peak. Subsequent isolation of the active fraction by semipreparative HPLC yielded an oil with MS−MS spectra consistent with those reported previously for allicin.23 Hypersensitivity of the CTR1 Deletion Strain to Allicin Is Reversed by Cu2+. Yeast lacking Ctr1 exhibited hypersensitivity to allicin relative to the isogenic BY4743 control strain (Figure 3A,B). We reasoned that if a lack of intracellular Cu2+ increased the activity of allicin then adding back Cu2+ would reduce the activity of allicin. As expected, addition of Cu2+ reverses allicin-ctr1Δ/ctr1Δ hypersensitivity in a dose dependent-manner (Figure 4A,B). Treating the ctr1Δ/ctr1Δ no allicin control with increasing Cu2+ did not alter growth to the same degree (Figure 4C). These results suggest that Cu2+ homeostasis is integral and closely connected to the yeast inhibitory activity of allicin. C
DOI: 10.1021/acs.jafc.7b00250 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. ctr1Δ/ctr1Δ strain displays hypersensitivity to allicin (B) relative to the isogenic BY4743 control strain (A). Black filled circles no allicin, gray filled circles 3.85 μM allicin, gray triangles 7.7 μM allicin, black open circles 15.4 μM allicin, and gray open circles 30.8 μM allicin. Each data point is an average of 5 replicate wells.
Figure 4. Addition of Cu2+ reverses the activity of allicin in ctr1Δ/ctr1Δ yeast. ctr1Δ/ctr1Δ yeast was treated with 3.85 μM allicin (A) and 7.7 μM allicin (B), and no allicin (control) (C) with increasing concentrations of Cu2+ as follows. Gray open circles no Cu, black open circles 1 μM Cu2+, gray triangles 2 μM Cu2+, gray filled circles 4 μM Cu2+, and black filled circles 32 μM Cu2+. Each data point is an average of 5 replicate wells.
Results Suggest Allicin Acts to Restrict Cu2+ and Zn2+ Uptake. Hypersensitivity of ctr1Δ/ctr1Δ yeast is considered indicative of a mode of action mediated via Cu2+ chelation.20 Compounds that act as metal ion chelators such as curcumin are known to induce hypersensitivity with ctr1Δ/ctr1Δ yeast, however curcumin also induces hypersensitivity with yeast lacking the iron transporter Ftr1 which was not observed in the present study.20,28 Interestingly Mac1 which regulates Ctr1 expression under low Cu2+ conditions contains dual cysteine rich C-terminal Cu+ binding motifs while Zap1 also contains a cysteine rich N-terminal region.25,29 Thus, the antimicrobial effects of allicin which is known to conjugate to thiol groups could be mediated though inhibition of Cu2+ and Zn2+ uptake by inactivating thiol residues on Cu2+ and Zn2+sensing transcription factors. Another possibility would be direct chelation of Cu2+ or Zn2+ by allicin, a relatively common antifungal mode of action. This is exemplified by compounds such as disulfiram, a strong metal ion chelator which is yeast inhibitory and whose HIP HOP profile is characterized by hypersensitivity with ctr1Δ/ctr1Δ and mac1Δ/mac1Δ yeast strains.30 It is also possible that allicin acts on one or more Cu2+ and Zn2+ metalloproteins a mode of action whose activity would be enhanced by deletion of Cu2+ and Zn2+ import proteins. Further work is needed to delineate these modes of
Focusing on Transition Metal Related Genes Identifies Mac1Δ and Zap1Δ Yeast as Hypersensitive. Next we widened our screen to include genes related to transition metal import and homeostasis. Six strains mac1Δ/mac1Δ, cup2Δ/ cup2Δ, zap1Δ/zap1Δ, smf1Δ/smf1Δ, and ctr2Δ/ctr2Δ were screened for hypersensitivity to allicin; mac1Δ/mac1Δ and zap1Δ/zap1Δ both exhibited hypersensitivity (Figure 5). Mac1 is a Cu2+ sensing transcription factor that controls Cu2+ uptake under low Cu2+ conditions. It controls the expression of a variety of Cu2+ import proteins including Ctr1; Zap1 is a Zn2+ sensing transcription factor that controls Zn2+ homeostasis and the response to low zinc conditions.24−26 Two other copper homeostasis gene deletion strains cup2Δ/cup2Δ and ctr2Δ/ ctr2Δ did not display hypersensitivity to allicin. Unlike Ctr1, Cup2, and Ctr2 are not required for low Cu2+ conditions suggesting that allicin acts to induce Cu2+ starvation. Smf1 is metal ion transporter with diverse substrates including manganese and iron, smf1Δ/smf1Δ did not exhibit hypersensitivity.27 As Zap1 controls zinc uptake, we reasoned that addition of Zn2+ might reverse the allicin hypersensitivity of the strain lacking Zap1 in a similar manner to that observed with Ctr1 and Cu2+ ions. This is indeed the case (Figure 6) although the effect is not as prominent as with Ctr1. D
DOI: 10.1021/acs.jafc.7b00250 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Focusing on transition metal related genes identifies mac1Δ/mac1Δ and zap1Δ/zap1Δ yeast as hypersensitive. Gray circles no allicin, gray triangles 3.85 μM allicin, and black circles 7.7 μM allicin. Each data point is an average of 5 replicate wells.
Figure 6. Addition of Zn2+ antagonizes allicin growth inhibition of zap1Δ/zap1Δ yeast. zap1Δ/zap1Δ with no allicin (A), zap1Δ/zap1Δ with 7.7 μM allicin (B). Gray circles no added Zn2+, gray triangles 80 μM Zn2+, and black circles 160 μM Zn2+. Each data point is an average of 5 replicate wells.
action. Lastly, the utility of the mini HOP HOP screen lies not just in its ability to uncover areas of biology connected to the mode of action of the test compound but also in its ability to eliminate common modes of action early on. If a compound produces no hits in the mini HIP HOP screen, then the compound may act through a specific protein receptor which would justify a full HIP HOP screen or a genome wide variomics mutation based screen.31
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Figure S5 ion trap MS/MS of precursor ion m/z 163 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 208 332 5393; fax: +44 208 332 5310; e-mail: t.
[email protected] (T.A.K.P.). ORCID
ASSOCIATED CONTENT
Thomas A. K. Prescott: 0000-0002-3039-7067
S Supporting Information *
Notes
The authors declare no competing financial interest.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00250. Figures S1 and S2 growth curves of yeast signature strains grown in the presence of a sublethal dose of garlic extract. Figure S3, HPLC-UV profile of aqueous garlic extract. Figure S4 positive ion HRESIMS of allicin.
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ACKNOWLEDGMENTS We would like to thank Dominic Hoepfner (Novartis Institutes for BioMedical Research) for his ideas and general support and Geoffrey Kite (RBG Kew) for assistance with LC−MS. E
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