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PageJournal 1 of 36of Agricultural and Food Chemistry

CH2OH H

Multivariate analysis of metabolites from 16 feijoa cultivars

H OH H

H

O O H

H NHCOH3

CH2OH

O H OH H

H

O

H NHCOH3

ACS Paragon Plus Environment 4-cyclopentene1,3-dione

Antifungal activity via chitin synthesis inhibition

Journal of Agricultural and Food Chemistry

1

Bioactivity-guided metabolite profiling of feijoa (Acca sellowiana) cultivars identifies 4-

2

cyclopentene-1,3-dione as a potent antifungal inhibitor of chitin synthesis

Mona Mokhtari1, Michael D. Jackson1, Alistair S. Brown1, David F. Ackerley1,2, Nigel J. Ritson3, Robert A. Keyzers2,4,§ and Andrew B. Munkacsi1,2,§,*

1

School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

2

Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand

3

Foretaste Feijoa Fruit Ltd., Takaka, New Zealand

4

School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington,

New Zealand §

Co-senior author

*

Corresponding author

1 ACS Paragon Plus Environment

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3

ABSTRACT

4

Pathogenic fungi continue to develop resistance against current antifungal drugs. To explore

5

the potential of agricultural waste products as a source of novel antifungal compounds, we

6

obtained an unbiased GC-MS profile of 151 compounds from 16 commercial and

7

experimental cultivars of feijoa peels. Multivariate analysis correlated 93% of the compound

8

profile with antifungal bioactivity. Of 18 compounds that significantly correlated with

9

antifungal activity, five had not previously been described from feijoa. Two novel cultivars

10

were the most bioactive, and 4-cyclopentene-1,3-dione detected in these cultivars was

11

potently antifungal (IC50 = 1-2 µM) against human pathogenic Candida species.

12

Haploinsufficiency and fluorescent microscopy analyses determined that the synthesis of

13

chitin, a fungal cell wall polysaccharide, was the target of 4-cyclopentene-1,3-dione. This

14

fungal-specific mechanism was consistent with 22-70-fold reduction in antibacterial activity.

15

Overall, we identified the agricultural waste product of specific cultivars of feijoa peels as a

16

source of potentially high value antifungal compounds.

17

KEYWORDS: antifungal / chitin synthesis / cyclopentenedione / feijoa / metabolite profiling

18 19 20 21 22 23



24 25

INTRODUCTION

26

Nature has been a rich source for pharmaceutical compounds, underlying 80% of our

27

currently prescribed drugs.1 In particular, many noteworthy drugs in the history of human

28

civilization have been derived from plants (e.g., morphine and aspirin to treat pain and

29

headaches, artemisinin to treat malaria). Just as there is a potential epidemic due to bacteria

30

developing resistance to current antibiotics and delays in the development of new antibiotics,2

31

the same applies to pathogenic fungi. In recent years, the limited nature of our current arsenal

32

of antifungal drugs has been manifest in the resistance of pathogenic fungi to current

33

antifungal drugs, increases in rates of fungal diseases in immunocompromised patients (e.g.,

34

chemotherapy, transplant, and HIV patients), and increases in the reports of new pathogenic

35

species.3 Like antibiotics, most antifungal drugs have historically been based on natural

36

products isolated or derived from cultured microbes. However, given that plants are

37

consistently regulating infections by fungi (i.e., permitting infection by mutualistic fungi and

38

resisting infection by pathogenic fungi), extracts of plants including fruits are also a

39

promising source of novel antifungal compounds.4

40

Feijoa (Acca sellowiana, Myrtacaeae) is native to Southern Brazil, Northern

41

Argentina and Western Paraguay, where it is also known as pineapple guava. It is actively

42

cultivated as an annual multi-million dollar (>$8M USD) agricultural crop in temperate and

43

subtropical climates in the United States of America, South America, Europe, Australia and

44

New Zealand. The pulp of the fruit (Figure 1) is consumed in its entirety or as a juice, as well

45

as in food products such as ice cream, chutney, chocolate, and wine. The fruit pulp has a

46

gritty texture with a distinctive highly perfumed odor and a flavor that is is sweet to mid-sour.

47

Bioactive compounds with anticancer, antioxidant, antiviral, anti-inflammatory, anti-diabetic,

48

probiotic and antibacterial activities have been isolated from the leaves, pulp and peels of


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49

commercial cultivars of the thick-skinned feijoa fruit. However, antifungal compounds in

50

feijoa are dramatically understudied with only four reports to date,12-15 none of which has

51

identified mechanism of action or the bioactive compound.

52

The presence of antifungal compounds in feijoa is particularly compelling given the

53

longstanding observation by feijoa breeders that feijoa fruit is rarely penetrated by fungal

54

pathogens. There is thus great potential to explore feijoa peels for antifungal compounds and

55

turn this waste product into a high value antifungal pharmaceutical. Out of the annual global

56

feijoa crop (105,000 tons), approximately 40% is used in juice production that generates

57

approximately 42,000 tons of fejoa peel waste. This is a significant amount of waste

58

production in the feijoa industry that could potentially be used to generate additional

59

commercial gain. While there has been extensive success in the citrus industry making use of

60

citrus processing byproduct streams (e.g., the production of phytochemicals and

61

nutraceuticals such as citrus molasses, D-limonene and bioflavonoids),16 such efforts have

62

not yet been explored for feijoa.

63

Since bioactivity has been documented without reference to specific feijoa cultivars

64

and antifungal activity has not often been investigated,5-15 here we hypothesize the potential

65

of developing high value antifungal drugs from feijoa peels via compound profiling of 16

66

cultivars of feijoa including commercial and non-commercial cultivars. We correlated 151

67

volatile compounds with antifungal activity, identified two novel cultivars as those with the

68

most antifungal activity, and determined that the compound 4-cyclopentene-1,3-dione,

69

present in both of these cultivars was potently antifungal via the inhibition of chitin synthesis.

70

As the antifungal mechanism of action targets chitin, which does not exist in humans, animals

71

or plants, this antifungal compound with pharmaceutical- and agrichemical-grade potency as

72

well as specificity has the potential to turn feijoa peel waste into a high-value product.

73



74

MATERIALS AND METHODS

75

Plant Material. Fruits of 16 feijoa cultivars were grown and harvested in one location

76

(Takaka, New Zealand): Apollo, FFF5, FFF6, FFF7, FFF8, FFF9, Kakapo, Karamea,

77

Kawatiri, Opal Star, R263, Tagan, Triumph, Unique, Waingaro, and Waitui. Peels were

78

surface-sterilized with ethanol, separated from pulp with a sterile scalpel, homogenized

79

(Breville BLR50S Motiv Blender, Melbourne, Australia) at maximum speed for 5 min,

80

centrifuged at 4000 rpm for 5 min, and the pellet was used for methanolic extraction.

81

Compound Extraction. Compounds were extracted with methanol at a ratio of 700 g

82

of homogenized peel to an equal volume of methanol. The peel:methanol mixture was

83

extracted through shaking at 230 rpm at 30ºC for 24 h, and then centrifuged at 4000 rpm for 5

84

min. The supernatant was filtered through 0.22 µm filters to ensure sterility. Methanol was

85

removed using a cold trap system (Labconco, Kansas City, MO, USA), and the solid

86

methanol extract was resuspended in DMSO (at a ratio of 1 mL DMSO to 1 mL methanol)

87

and stored at -20ºC.

88

Compound Profiling. Volatile and semi-volatile compounds were analyzed on a

89

Shimadzu QP2010-Plus gas chromatograph (Shimadzu, Kyoto, Japan) fitted with a RXI-

90

5SilMS column (30 m x 0.25 mm i.d. x 0.25 µm film thickness) (Restek, Bellefonte, PA,

91

USA) and attached to an electron impact mass spectrometer operating at 70 eV in positive ion

92

mode, scanning from m/z 42-600 every 300 msec. Samples were introduced (1 µL) using an

93

AOC-20i auto-sampler with a split injection (20:1) at an injector temperature of 270°C using

94

helium as the carrier gas at a linear velocity of 43.4 cm/s (1.38 mL/min) at constant flow.

95

Each sample was injected with an initial oven temperature of 50°C for 2 min, followed by a

96

ramp at 10°C/min to 300°C with a final hold for 5 min. Compounds were identified by

97

comparison of fragmentation mass spectra with those contained in the NIST11 MS library

98

with a spectral matching similarity index (SI) >85% in at least two of three experimental


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99

replicates. Compound annotation was performed using Linear Retention Indices (LRIs)

100

compared with freely accessible LRI17 (± 50 LRI units) values using similar column-types

101

while compound identification was achieved by comparison of LRI and mass spectral

102

fragmentation patterns with those of authentic standards based on the proposed minimum

103

reporting standards according to the Chemical Analysis Working Group.18 According to this

104

standard, we report compounds as the following: a, identified compound based on matched

105

mass spectrum and LRI of an authentic standard; b, annotated compound with matched LRI

106

and mass spectrum; c, annotated compound with matched mass spectrum; d, unknown.

107

Antifungal Activity and Haploinsufficiency. All strains of S. cerevisiae were

108

obtained from Open Biosystems (Huntsville, AL, USA) in the haploid BY4741 background

109

and diploid BY4743 background. Pathogenic Candida strains were obtained from American

110

Type Culture Collection (Manassas, VA, USA) and included C. albicans (ATCC 10231), C.

111

glabrata (ATCC 90030), C. parapsilosis (ATCC 90018), and C. tropicalis (ATCC 13803).

112

Fungal growth was measured in a liquid assay as previously described.19 Fungal cultures at 5

113

x 105 cells/mL were grown in Synthetic Complete (SC) media with varying concentrations of

114

feijoa extract or compound, incubated at 30ºC for 14 h, and quantified via optical density at

115

600 nm using a Perkin Elmer Envision plate reader (Perkin Elmer, Waltham, MA, USA).

116

Growth inhibition was calculated using the following formula:

117

Growth inhibition (%) = 100 – [(OD600 of treatment/OD600 of control) x 100]

118

Multivariate Statistical Analysis. Compound profiles and bioactivity were compared

119

using a partial least squares regression (PLSR) analysis incorporated in UNSCRAMBLER

120

(version X10.4, CAMO, Oslo, Norway) as previously described.20 The data were arranged in

121

an X x Y matrix, where the response variable Y (bioactivity) corresponded to growth

122

inhibition of S. cerevisiae of the 16 cultivar extracts and the predictor variable X

123

corresponded to the largest peak area for each compound. All X variables were log



124

transformed, mean centred and scaled to one standard deviation. All Y variables were mean

125

centred. The PLSR analysis used the Non-Linear Iterative Partial Least Squares (NIPALS)

126

algorithm. Compound lists for each cultivar were generated using the weighted regression

127

coefficient and validated using random cross-validation. The Martens Uncertainty Test based

128

on cross-vaidation used factors 1 and 2 to identify compounds statistically significant to the

129

model.

130

Antibacterial Activity. Growth inhibition of Bacillus subtilis (ATCC 6051), a tolC

131

mutant efflux pump-deficient Escherichia coli (W3110), an extended spectrum β-lactamase

132

Klebsiella pneumoniae (NZRM 4387), and methicillin-resistant Staphylococcus aureus

133

(MRSA; ATCC 43300) were measured in liquid media via optical density at 600 nm using an

134

Envision plate reader (Perkin Elmer, Waltham, MA, USA) as previously described.21

135

Percentage growth inhibition was then calculated using the following formula:

136

Growth inhibition (%) = 100 – [(OD600 of treatment/OD600 of control) × 100]

137

Fluorescent Microscopy. Chitin was visualized using the fluorescent stain Calcafluor

138

White (Sigma) as previously described.22 Cells were grown overnight in SC media and then

139

grown to mid-log in SC starting at a common density (OD600 = 0.1). Calcafluor White (1 g/L)

140

was added on the top of the cells and visualized after 5 min with an Evo-Tec OPERA high-

141

throughput confocal microscope (Perkin Elmer) at 60X using the DAPI filter.

142

Standards. 4-Cyclopentene-1,3-dione, β-caryophyllene, and (E)-3-hexenyl butyrate

143

were purchased from Sigma Aldrich. Flavone was isolated from natural sources and provided

144

as a gift (Stephen Bloor, Callaghan Innovation).

145 146

RESULTS

147

Bioactivity of Feijoa Cultivar Extracts. The bioactivity of feijoa extracts has been

148

examined in an undocumented set of commercial cultivars.5-15 To further understand the


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chemical diversity that exists within the genetic diversity of feijoa, here we investigated the

150

bioactivity of ten commercial cultivars (Apollo, Kakapo, Karamea, Kawatiri, Opal Star,

151

Tagan, Triumph, Unique, Waingaro, Waitui) and six novel cultivars (FFF5, FFF6, FFF7,

152

FFF8, FFF9, R263). We quantified the efficacy of methanolic extracts derived from peels of

153

these 16 feijoa cultivars to inhibit the growth of S. cerevisiae (Baker’s yeast), an organism

154

that has extensively been used to examine bioactivity of compounds and extracts.23,24

155

Specifically, we measured the percentage of inhibition of growth of S. cerevisiae over a

156

period of 14 hours, the amount of time that it takes an untreated wild-type strain of yeast to

157

reach the mid-log phase of growth. Based on growth in the presence of a range of extract

158

concentrations, we determined that 0.01% v/v of an extract significantly reduced growth of a

159

wild-type (WT) strain of yeast (BY4741) by 18-85% compared to untreated WT (Figure 2).

160

Methanolic extracts of the commercial cultivars inhibited growth by 18-70% compared to

161

untreated cells, while those from the novel cultivars inhibited growth by 30-85% compared to

162

untreated cells. The most bioactive cultivars were the novel cultivars FFF5 and FFF6, which

163

were each significantly different from the next most bioactive cultivar Waitui (p < 0.0005).

164

These results indicate that there is extensive variation in bioactivity among feijoa cultivars as

165

well as highlighting that the greatest bioactivity is in two novel feijoa cultivars (FFF5 and

166

FFF6).

167

Compound Profiles of Feijoa Cultivars. Antifungal bioactivity has only been

168

reported in a few studies that explored crude acetone extracts and essential oils of feijoa.12-15

169

To further define the compound diversity of feijoa in terms of antifungal properties, we used

170

GC-MS to obtain unbiased metabolomic profiles in the methanolic extracts of peels from 16

171

feijoa cultivars. The identity of the volatile and semi-volatile compounds was tentatively

172

assigned by comparison of mass spectral fragmentation patterns with those of more than

173

212,000 compounds in the NIST11 database, using a cutoff of 85% spectral similarity. 8 ACS Paragon Plus Environment


174

Across the 16 feijoa cultivars, GC-MS analysis distinguished 151 compounds across all

175

cultivars, of which 68 compounds were tentatively annotated on the basis of >85% similarity

176

to a spectrum in the NIST11 database (Figure S1). Additionally, 79 compounds did not match

177

to compounds in the NIST database, and these compounds were thus classified as unknown

178

(Table S1).

179

Multivariate Analysis of Cultivars, Bioactivity and Compounds. To investigate

180

possible relationships between bioactivity and compound profiles of the 16 feijoa cultivars,

181

we conducted a PLSR analysis incorporated in the software UNSCRAMBLER that is

182

regularly used to correlate compounds and different properties.25 We correlated a large

183

number of dependent (151 compounds) and independent (bioactivity) variables for all 16

184

cultivars. The PLSR analysis generated a β-coefficiency value for each compound (ranging

185

from -1 to +1) wherein compounds with higher positive β-coefficiency values were more

186

correlated to bioactivity and negative β-coefficiency values were negatively correlated to

187

bioactivity. The β-coefficiency values were then used to build seven different models

188

(factors) to link bioactivity to the compounds. Notably, the first two factors explained 92.6%

189

of the variance (i.e., X axis is the model factor 1 that explained 73% of the data variance and

190

Y axis is model factor 2 that explained 20% of the data variance) in our data correlating

191

bioactivity and compound identity (Figure 3), a value that is consistent with results in other

192

studies using just the first two factors.26

193

Next, we sought to correlate each cultivar with bioactivity using the first two factors.

194

In agreement with Figure 2, the FFF5 cultivar was most correlated to bioactivity with FFF6

195

as the second closest cultivar, followed by Waitui and R263 (Figure 3A). Based on the β-

196

coefficiency values, these four cultivars were positively correlated with bioactivity in both

197

factor 1 and 2. In contrast, three cultivars (FFF7, Apollo, Kawatiri, FFF9) were positively

198

correlated to factor 1 and negatively correlated to factor 2, while four cultivars (FFF8, Tagan,


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Kakapo, Karamea) were positively correlated to factor 2 and negatively correlated to factor 1.

200

Four cultivars (Unique, Opal Star, Triumph, Waingaro) were negatively correlated to both

201

factors.

202

We then sought to distinguish the compounds that were positively and negatively

203

correlated with bioactivity. The multivariate PLSR analysis determined those compounds to

204

the right side of the mid-line as being positively correlated to factor 1, while those

205

compounds to the top side of the mid-line were positively correlated to factor 2 (Figure 3B).

206

The data analysis illustrated there were 17 compounds negatively correlated to both factors 1

207

and 2, while there were 26 compounds positively correlated to both factors. To define the

208

compounds that were significantly correlated with bioactivity, we conducted further

209

statistical analyses using a Martens uncertainty test based on the first two factors in the PLSR

210

analysis. We detected 22 compounds that were significantly correlated with bioactivity (18

211

positively correlated, four negatively correlated), of which nine matched with >85% SI to

212

compounds in the NIST11 database (Table 1). Using the minimum criterion of 85% similarity

213

to the NIST11 database for identification, we tentatively annotated nine significantly

214

positively correlated compounds based on β-coefficiency: compound 26 (β-phellandrene),

215

compound 141 (flavone), compound 4 (4-cyclopentene-1,3-dione), compound 105 (ledene),

216

compound 113 (unknown), compound 95 (β-caryophyllene), compound 46 (N-methyl-N-

217

nitroso-2-propanamine), compound 20 (2H-pyran-2,6(3H)-dione) and compound 47

218

(unknown). In order of correlation based on β-coefficiency, the four significantly negatively

219

correlated compounds were compounds 52, 61, 131 and 140, none of which could be

220

annotated based on ≥85% similarity with fragmentation patterns in the NIST11 database

221

(Table S1). The results of these statistical correlations with two factors were validated using

222

cross-validation in the PLSR model. The model generates a validation rate for each factor,

223

wherein validation for the model factors was high; for the first factor (r2 = 25.1%) and this



224

was further increased with the second factor (r2 = 35.1%), results that are consistent with

225

previous reports of two factors that reliably correlated biological activity and metabolic

226

profile.26

227

Of the nine significantly positively correlated compounds tentatively identified on the

228

basis of >85% similarity to the NIST database, three were commercially available (flavone,

229

4-cyclopentene-1,3-dione, and β-caryophyllene). We obtained the GC-MS fragmentation

230

patterns and retention times of the authentic standards for these three compounds and

231

compared these profiles with the profiles of the tentatively identified compounds detected in

232

the feijoa cultivars (Table 1A). In each case the fragmentation patterns and retention times

233

were identical, confirming the identity of these three compounds.

234

Unique Compounds in Novel Feijoa Cultivars. To follow up on our identification

235

of the FFF5 and FFF6 cultivars as having the greatest bioactivity (Figure 2; Figure 3A), we

236

investigated compounds with >85% similarity to database matches that were identified only

237

in these cultivars. We therefore examined the GC-MS analyses of these cultivars and detected

238

(E)-3-hexenyl butyrate and two unknown compounds unique to the FFF5 cultivar (Table 1B),

239

while there were no unique compounds detected in FFF6. We then compared the GC-MS

240

fragmentation patterns and retention times of our tentatively identified (E)-3-hexenyl butyrate

241

from the FFF5 cultivar with those of an authentic standards, which were the same as those of

242

the standards, confirming the identity of this compound as being present only in the novel

243

FFF5 feijoa cultivar.

244

Confirmation of Compound Bioactivity. To confirm the prediction of our

245

multivariate analyses that the definitively identified flavone, β-caryophyllene and 4-

246

cyclopentene-1-3-dione were positively correlated with growth inhibition of S. cerevisiae,

247

we measured growth inhibition of S. cerevisiae in the presence and absence of standards.

248

Relative to untreated cells, the IC50 values for the three compounds were 2.3 µM, 35.8 µM


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and 1.3 µM for flavone, ß-caryophyllene, and 4-cyclopentene-1-3-dione, respectively (Table

250

2). The unique compound observed only in the FFF5 cultivar, (E)-3-hexenyl butyrate,

251

conferred growth inhibition via an IC50 of 22.7 µM (Table 2). These results validate our

252

multivariate analysis-based correlation strategy, showing that positively correlated

253

compounds predicted by the PLSR analysis are indeed bioactive in yeast cells.

254

Antifungal Activity of Feijoa Cultivars and Compounds Against Human

255

Pathogens. Candida species commonly cause fungal infections and fatalities in

256

immunocompromised hosts with more than 400,000 fatalities worldwide every year.3 To

257

determine whether the compounds we identified as being inhibitory to growth of the saprobic

258

yeast S. cerevisiae were more widely effective against human pathogenic fungal species, we

259

quantified growth of four Candida species (C. albicans, C. glabrata, C. parapsilosis, and C.

260

tropicalis) in the presence of three compounds that were positively correlated with bioactivity

261

(flavone, β-caryophyllene, 4-cyclopentene-1-3-dione) and one compound that was only

262

identified in the FFF5 cultivar ((E)-3-hexenyl butyrate). Relative to untreated cells, the IC50

263

values for the four compounds were 1.5-2.6 µM for flavone, 1.5-2.3 µM for 4-cyclopentene-

264

1-3-dione, 35.8-75.2 µM for β-caryophyllene, and 16.0-38.2 µM for (E)-3-hexenyl butyrate

265

(Table 2). Overall, the bioactivity of these four compounds against human pathogenic

266

Candida species was consistent with bioactivity against S. cerevisiae (Table 2), providing

267

further validation of our use of S. cerevisiae as a model fungus to study the antifungal

268

bioactivity of feijoa cultivars.

269

As these four compounds are merely a fraction of the 151 compounds that we

270

identified in the 16 feijoa cultivars, we sought to further understand the antifungal potency of

271

the total methanolic extracts derived from these cultivars, testing their abilities to inhibit the

272

growth of C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. At 0.01% v/v, the 16

273

cultivar extracts significantly inhibited growth of the four Candida species by 12-87%



274

compared to untreated cells. Specifically, the FFF5 and FFF6 cultivar extracts inhibited

275

growth of the pathogenic Candida strains by 69-85% compared to untreated cells (Table 3),

276

consistent with these cultivar extracts being the most bioactive against S. cerevisiae (Figure

277

2; Table 3).

278

Antifungal Mechanism of Action of 4-cyclopentene-1-3-dione. Based on potent

279

antifungal activity (IC50 = 1-2 µM), 4-cyclopentene-1,3-dione has the potential to be a lead

280

for developement as a clinical antifungal drug once a mechanism of action is determined.

281

Haploinsufficiency (hypersensitivity) of a heterozygous diploid yeast strain indicates the

282

mutant gene is the drug target based on inhibition of the product of a single gene copy by the

283

drug.24 To determine if the target of 4-cyclopentene-1,3-dione is a known antifungal drug

284

target, we evaluated sensitivity of heterozygous mutants bearing deletions of known

285

antifungal drug targets. We observed that heterozygosity of the chitin synthase-1 and chitin

286

synthase-2 genes (chs1∆/CHS1 and chs2∆/CHS2) conferred significant hypersensitivity to 4-

287

cyclopentene-1,3-dione with 70% and 74% growth reductions, respectively, compared to

288

10% growth reduction seen in the diploid WT strain (BY4743) (Figure 4A). For comparison,

289

other antifungal drug targets were not hypersensitive to 4-cyclopentene-1,3-dione including

290

the cell wall 1,3-β-D-glucan synthase FKS1 gene (target of caspofungin), the ergosterol

291

biosynthesis ERG11 gene (target of ketoconazole), the sphingolipid synthesis LCB1 gene

292

(target of myriocin), ergosterol biosynthesis HMG1 gene (target of atorvastatin), the cytosine

293

deaminase FCY1 gene (resistance to flucytosine) and the RNAse subunits POP1 gene

294

(hypersensitive to nystatin) (Figure 4A). Interestingly, there was significant, albeit slight

295

(21%), inhibition of the hmg1∆/HMG1 strain by 4-cyclopentene-1,3-dione, though not to the

296

same extent as chs1∆/CHS1. The CHS1 and CHS2 genes are the target of polyoxin,27 and the

297

hypersensitivity of the chs1∆/CHS1 and chs2∆/CHS2 strains to 4-cyclopentene-1,3-dione

298

mirrored a 76% and 65% growth reduction in these strains by polyoxin (Figure 4B).


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If a drug target is absent, cells will be resistant to that drug.24 Consistent with CHS1

300

and CHS2 being the target of 4-cyclopentene-1,3-dione, chs1∆ and chs2∆ haploid mutants

301

were resistant to both 4-cyclopentene-1,3-dione and polyoxin, which induced no growth

302

inhibition in chs1∆ and chs2∆ compared to 59% growth inhibition for each compound in the

303

WT strain (Figure 4C). To further characterize chitin synthesis inhibition, we examined the

304

abundance of chitin in the cell wall. Using a fluorescent stain specific for chitin (calcafluor

305

white),28 both 4-cyclopentene-1,3-dione and polyoxin B reduced abundance of chitin in the

306

septum (Figure 4D), a morphology that is consistent with our demonstrated inhibition of the

307

CHS1 and CHS2 gene products, which are the major regulators of chitin distribution in

308

septa.29 Together, these results indicate 4-cyclopentene-1,3-dione and polyoxin share a

309

common mechanism of action by targeting the major chitin synthesis genes CHS1 and CHS2

310

and inhibiting the synthesis of chitin, a major structural component of the fungal cell wall that

311

does not exist in mammalian cells and is critical to growth of all fungi. This specificity for

312

chitin was further supported with an absence of antibacterial activity of 4-cyclopentene-1,3-

313

dione against four bacterial species at 1.3 µM, the concentration that achieved IC50 antifungal

314

activity. Consequently, antibacterial activity of 4-cyclopentene-1,3-dione was reduced 22-70

315

fold relative to antifungal activity with IC50 values of 85±11, 60±1, 33±2 and 105±14 µM

316

against B. subtilis (Gram positive lab strain), E. coli (Gram negative lab strain), S. aureus

317

(pathogenic strain) and K. pneumoniae (pathogenic strain), respectively.

318 319

DISCUSSION

320

In this study, we investigated agronomically diverse feijoa cultivars to identify

321

antifungal compounds with pharmaceutical potential. These compounds were sourced from

322

feijoa peel waste to provide the first explicit attempt to add high value to the peels that are

323

currently discarded by the ton in the feijoa juicing industry. We investigated 16 feijoa



324

cultivars, both commercial and non-commercial within a feijoa breeding program, and in an

325

unbiased fashion correlated antifungal bioactivity with the observed metabolite content (151

326

volatile compounds) rather than in a traditional reductionist fashion of bioassay-guided

327

fractionation of a subset of these compounds. Using established methodology to link

328

metabolomic and bioassay results,25 our multivariate analysis demonstrated strong correlation

329

between antifungal bioactivity and 22 compounds present in methanolic extracts of feijoa

330

fruit peels. One of these compounds, 4-cyclopentene-1,3-dione, was potently antifungal

331

against Candida species that cause the most fungal infections in humans, including C.

332

albicans, via a fungal-specific mechanism targeting chitin in fungal cell walls. As no chitin

333

synthesis inhibitor has been clinically approved for use in humans,29 our results indicate 4-

334

cyclopentene-1,3-dione has such clinical potential, particularly since no cyclopentenedione

335

reported to date has demonstrated inhibition of pathogenic Candida species via a chitin

336

synthesis mechanism.

337

In addition to recovering compounds detected in previous studies such as flavone,30 β-

338

caryophyllene,30 3-octanone,30 trans-calamenene30 and globulol,31 we also detected

339

compounds that have not been reported from feijoa to date, although these are primarily

340

tentative annotations and not formal positive identifications. In one case of a positive

341

identification, we provide the first report of 4-cyclopentene-1,3-dione being present in feijoa.

342

We confirmed the compound identity via GC-MS analysis of an authentic standard, and

343

demonstrated potent antifungal activity via growth inhibition of multiple pathogenic Candida

344

species. The presence of 4-cyclopentene-1,3-dione in a plant species is not specific to feijoa

345

as this compound has previously been identified in walnut oil.32 Interestingly, the most potent

346

FFF5 cultivar contained relatively the most 4-cyclopentene-1,3-dione among the 16 cultivars,

347

raising the possibility that the antifungal potency of FFF5 is largely a consequence of 4-

348

cyclopentene-1,3-dione itself or enhanced 4-cyclopentene-1,3-dione bioactivity due to


Page 16 of 36

Page 17 of 36


349

synergy with other bioactive compounds (e.g., butyrate compounds can increase the

350

bioactivity of other volatile organic compounds33).

351

In regards to the potency of antifungal bioactivity, 4-cyclopentene-1,3-dione is

352

comparable with established antifungal drugs. Clinically used drugs from the polyene group

353

such as simvastatin and atorvastatin exhibit IC50 values of 3 µM and 10 µM, respectively,

354

against Candida albicans,34 while azole drugs used in the clinic such as fluconazole have IC50

355

values of ~3 µM against different Candida species.35 Our study determined that 4-

356

cyclopentene-1,3-dione exhibits IC50 values of 1.5-2.3 µM against four Candida species,

357

indicating that this compound is as potent as antifungal drugs used in the clinic today.

358

Although the antifungal activity of 4-cyclopentene-1,3-dione has not previously been

359

demonstrated, it is consistent with antifungal activity for structurally related coruscanone A.36

360

Coruscanone A and a desmethoxy derivative were each comparable to the clinically approved

361

antifungals fluconazole and amphotericin B with IC50 values of 0.5-3 µM against Candida

362

species . While the antifungal mechanism of coruscanone A is not yet resolved, our results

363

show that 4-cyclopentene-1,3-dione is antifungal via chitin synthesis inhibition.

364

As chitin is a major component of fungal cell walls that is missing in vertebrate

365

organisms, chitin biosynthesis is a specific and promising target to treat fungal infections

366

without inhibiting those same pathways in human hosts.3,29 Nikkomycin and polyoxin are two

367

established chitin synthase inhibitors that were isolated more than 40 years ago from cultures

368

of Streptomyces species.29 While both are pyrimidine nucleoside peptides, neither has been

369

clinically approved as a pharmaceutical drug, albeit polyoxin is an active ingredient in

370

commercial fungicides in the agricultural and horticultural industries.29 Both nikkomycin and

371

polyoxin inhibit CHS1 and CHS2, which is consistent with our results comparing 4-

372

cyclopentene-1,3-dione and polyoxin. Although these three compounds share a mechanism, it

373

is plausible that 4-cyclopentene-1,3-dione will exhibit different pharmacokinetics than



Page 18 of 36

374

nikkomycin and polyoxin given their completely different structural backbones. For example,

375

the more lipophillic cyclopentenedione backbone will exhibit vastly different solubility than

376

pyrimidine nucleoside peptides, and also will be less susceptible to hydrolysis. Given the

377

Michael acceptor

378

cycopentenedione scaffold, covalent inhibition of targets is also plausible. In addition to

379

nikkomycin and polyoxin isolated from bacteria, there have been other structurally diverse

380

natural product chitin synthesis inhibitors with antifungal activity, notably five compounds

381

isolated from four fungal species and 14 compounds isolated from 10 plant species.29 With

382

our results, 4-cyclopentene-1,3-dione can be added to this list of natural chitin synthase

383

inhibitors.

and

acidic

1,3-diketo

functional

groups

within

the

compact

384

Cyclopentenedione compounds are a relatively new group of ~100 secondary

385

metabolites isolated primarily from plants as well as fungi and bacteria.37 It is plausible that

386

other cyclopentenedione compounds, in addition to our demonstration for 4-cyclopentene-

387

1,3-dione, are antifungal by a common mechanism of chitin synthesis inhibition. For

388

example, the cyclopentenediones linderone and methyllinderone exhibited antifungal activity

389

against plant pathogens via chitin synthase inhibition.38 However, these cyclopentenediones

390

did not inhibit CHS1 activity, which was the main reason these compounds did not

391

effectively kill Candida species (IC50 > 700 µM). In contrast, we demonstrated that 4-

392

cyclopentene-1,3-dione inhibited CHS1 and thus inhibited growth of four Candida species

393

with IC50 ~ 1-2 µM. More broadly than specificity for chitin synthase genes, the reports of

394

anti-cancer and anti-inflammatory bioactivities among cyclopentenedione compounds in

395

mammalian cells suggests that chitin synthesis inhibition is not the only bioactivity

396

mechanism in this family of compounds since mammalian cells do not contain chitin.37 A

397

similar phenomenon was evident in this study, with 4-cyclopentene-1,3-dione exhibiting low


Page 19 of 36


398

level antibacterial activity relative to the potent antifungal activity (consistent with bacteria

399

not containing chitin).

400

Recognizing the potential use of cyclopentenedione compounds as novel

401

pharmaceutical drugs, a library of compounds derived from a parental cyclopentenedione

402

compound was generated and chemically characterized but not characterized for any

403

biological activity.39 Our finding of potent antifungal activity of 4-cyclopentene-1,3-dione as

404

a chitin synthesis inhibitor clearly warrants further evaluation of structure-activity

405

relationships. Similar medicinal chemistry approaches have been used to enhance the anti-

406

inflammatory and anti-cancer activities of compounds with a cyclopentenedione backbone.37

407

As the cyclopentenedione backbone is highly reactive, it has a high potential as a lead for

408

commercial synthesis and testing of analogues to probe antifungal structure-activity

409

relationships.

410

In addition to the potential of 4-cyclopentene-1,3-dione as a pharmaceutical

411

antifungal with specificity for a target that does not exist in humans and animals, there is also

412

potential for this compound to be a specific fungicide as well as insecticide (the insect

413

exoskeletion contains chitin) in an agricultural setting. Regarding insects, a number of chitin

414

synthesis inhibitors are currently used worldwide to combat arthropod pests in agriculture and

415

forestry responsible for billions of dollars of damage.40 It will thus be interesting to evaluate

416

chitin-specific insecticide activity of 4-cyclopentene-1,3-dione, particularly since insecticide

417

activity of cylcopentenedione compounds is poorly understood with only one publication and

418

the few cyclopentenedione compounds with insecticide activity were not chitin synthesis

419

inhibitors.41 Specifically, it will be interesting to determine if 4-cyclopentene-1,3-dione

420

behaves in a similar fashion to the chitin synthesis inhibitor polyoxin in only exhibiting

421

insecticide activity when administered orally or injected into the insects.40 Regarding fungal

422

plant pathogens, the cyclopentenedione compounds chrysotrione A and B have exhibited



423

antifungal activity against Fusarium verticilloides, a major pathogen of corn.42 Notably,

424

chrysotrione A and B have not been shown to be antifungal through a chitin synthesis

425

inhibition mechanism such as that seen in this study for 4-cyclopentene-1,3-dione.

426

In conclusion, we conducted the first systematic evaluation of bioactivity across feijoa

427

cultivars and demonstrated there is enormous functional chemical diversity among feijoa

428

cultivars to exploit as a natural source of bioactive compounds from agricultural waste. We

429

used bioactivity-guided metabolite profiling of peels derived from 16 commercial and

430

experimental feijoa cultivars to correlate 151 volatile compounds with antifungal activity.

431

These compounds were identified via unbiased metabolite profiling of both novel, non-

432

commercial and commercially available feijoa cultivars, which collectively indicate that there

433

is extensive chemical diversity within closely related feijoa cultivars. All cultivars were

434

substantially antifungal at 0.01% (v/v) based on growth inhibition of Saccharomyces and

435

Candida species. The two experimental cultivars FFF5 and FFF6 that exhibited the most

436

antifungal activity contained the compound 4-cyclopentene-1,3-dione, which we showed to

437

be potently antifungal against Saccharomyces and Candida species. It will be necessary to

438

measure the abundance of 4-cyclopentene-1,3-dione in each feijoa peel to precisely determine

439

how much of this potent antifungal compound can be isolated from a given amount of feijoa

440

peel waste. Via our identification of the first cyclopentenedione compound to potently kill

441

Candida species via a fungal-specific mechanism of chitin synthesis inhibition, there is

442

potential of a high value antifungal pharmaceutical in a waste product in the feijoa juicing

443

industry, which warrants further investigation of volatile and non-volatile compounds for

444

more activities in addition to antifungal activity.

445

446

ASSOCIATED CONTENT

447

Supporting Information 19 ACS Paragon Plus Environment

Page 20 of 36

Page 21 of 36


448

Figure S1. GC-MS chromatogram of FFF6 feijoa cultivar.

449

Table S1. List of compounds detected in 16 feijoa cultivars, with experimental and literature

450

LRI values and similarity indices to NIST database.

451 452

AUTHOR INFORMATION

453

Corresponding Author

454

(A.B.M.) Phone: +64 4 463 5171. Email: [email protected]

455 456

Funding

457

This work was supported by a Victoria University of Wellington Doctoral Scholarship

458

(M.M.).

459 460

Notes

461

There are no conflicts of interest to report.

462 463

ACKNOWLEDGMENTS

464

We express our gratitude to Jeffrey Sheridan for technical assistance in figure preparation and

465

to all the employees at Foretaste Feijoa Fruit Ltd.

466

467

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601



602

Figure legends

603

604

Figure 1. Representative images of feijoa including feijoa tree (upper left), feijoa flowers

605

(upper right), feijoa fruit (lower left), and cross-section of feijoa fruit (lower right). Images

606

provided by Foretaste Feijoa Fruit Ltd., Takaka, New Zealand.

607

608

Figure 2. Bioactivity of methanolic extracts (0.01% v/v) of 16 feijoa cultivars based on

609

growth inhibition of S. cerevisiae. The growth percentage inhibition was calculated via

610

optical density (OD600) at mid-log in treated cells relative to untreated cells. Data shown as

611

mean ± S.D. **, p < 0.005, Student’s t-test comparing treated and untreated cells. ***, p

Bioactivity-Guided Metabolite Profiling of Feijoa ... - ACS Publications

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