Combination of isotope labeling and molecular networking of tandem

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Combination of isotope labeling and molecular networking of tandem mass spectrometry data to reveal 69 unknown metabolites produced by Penicillium nordicum Thaïs Hautbergue, Emilien L Jamin, Robin Costantino, Souria Tadrist, Lauriane Meneghetti, Jean-Claude Tabet, Laurent Debrauwer, Isabelle P. Oswald, and Olivier Puel Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01634 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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Analytical Chemistry

1

Combination of isotope labeling and molecular networking of tandem mass

2

spectrometry data to reveal 69 unknown metabolites produced by Penicillium nordicum

3 4

Thaïs Hautberguea,b, Emilien L. Jamina,b,*, Robin Costantinoa,b, Souria Tadrista, Lauriane

5

Meneghettia,b, Jean-Claude Tabetc,d, Laurent Debrauwera,b, Isabelle P. Oswalda, Olivier Puela

6 7

a Toxalim

8

Purpan, UPS, F-31027 Toulouse, France

9

b

(Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-

Axiom platform, MetaToul-MetaboHUB, National Infrastructure for Metabolomics and

10

Fluxomics, F-31027 Toulouse, France

11

c

12

des Médicaments, CEA, INRA, Université Paris Saclay, MetaboHUB, F-91191 Gif-sur-Yvette,

13

France

14

d

15

Cedex 05, France

16

* Corresponding author: [email protected]

17

Abstract

Service de Pharmacologie et d’Immunoanalyse (SPI), Laboratoire d’Etude du Métabolisme

Sorbonne Universités, Campus Pierre et Marie Curie, IPCM, 4 place Jussieu, 75252 Paris

18

The secondary metabolome of Penicillium nordicum is poorly documented despite its

19

frequent detection on contaminated food, and its capacity to produce toxic metabolites such as

20

ochratoxin A. To characterize metabolites produced by this fungi, we combined a full stable

21

isotopes labeling with the dereplication of tandem mass spectrometry (MS/MS) data by

22

molecular networking. Firstly, the untargeted metabolomic analysis by high-resolution mass

23

spectrometry of a double stable isotope labeling of P. nordicum enabled the specific detection

24

of its metabolites and the unambiguous determination of their elemental composition. Analyses

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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showed that infection of substrate by P. nordicum lead to the production of at least 92

26

metabolites, and that 69 of them were completely unknown. Then, curated molecular networks

27

of MS/MS data were generated with GNPS and MetGem, specifically on the features of interest,

28

which allowed highlighting 13 fungisporin-related metabolites that had not previously been

29

identified in this fungus and 8 that had never been observed in any fungus. The structures of

30

the unknown compounds, namely, a native fungisporin and seven linear peptides, were

31

characterized by tandem mass spectrometry experiments. The analysis of P. nordicum growing

32

on its natural substrates, i.e. pork ham, turkey ham and cheese, demonstrated that 10 of the

33

known fungisporin-related metabolites and three of the new metabolites were also synthesized.

34

Thus, the curation of data for molecular networking using a specific detection of metabolites of

35

interest with stable isotopes labeling, allowed the discovery of new metabolites produced by

36

the food contaminant P. nordicum.

37

Keywords: molecular network, mass spectrometry, stable isotope labeling, Penicillium

38

nordicum, metabolomics, fungisporin

39 40

Food and feed contamination by fungal species is a major agronomic issue, particularly in

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the contexts of the increasing volume of food required and climate change1. Globally,

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approximately 10% of crops are infested, which each year, leads to the destruction of food that

43

could feed 600 million people, i.e., 8.5% of the world population1. Fungal toxicity and the

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spreading of fungi into food stocks are caused by secondary metabolites. It is estimated that the

45

11,250 fungal metabolites currently listed represent less than 20% of all the secondary 2


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metabolites that fungi are able to synthesize, highlighting the vast number of compounds still

47

to be discovered2. The characterization metabolites produced by fungi is therefore crucial to

48

investigate the processes involved in food contamination and to control food contamination by

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mycotoxins.

50

Among food pathogenic fungi, ochratoxin A-producing species such as Penicillium

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nordicum are responsible for annual economic losses exceeding, e.g., in Canada, 260 million

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Canadian dollars3 due to the level of ochratoxin A being above the maximum permitted level.

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Ochratoxin A is one of the eight mycotoxins that is internationally regulated4 since it is

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considered the most potent renal carcinogen of natural origin5. In temperate regions, P.

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nordicum is one of the main ochratoxin A-producing species6, and it regularly contaminates

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dry-cured meat products, cheese and occasionally wheat supplies. Aside from the production

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of ochratoxin A, metabolites produced by of P. nordicum is poorly documented. The toxicity

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of this fungus may also result from the production of other individual mycotoxins or through

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synergistic effects of several mycotoxins7.

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Metabolites produced by fungi can be investigated by untargeted metabolomics methods

61

based on high-resolution mass spectrometry8. Complex samples such as fungal extracts can

62

contain several isomers that can be differentiated by coupling mass spectrometry with liquid

63

chromatography. However, metabolite identification remains particularly challenging in such

64

experiments9. Additionally, bioinformatics tools could be used to facilitate not only the

65

assignment of known metabolites but also the identification of new compounds of interest and

66

the elucidation of their structures. GNPS Molecular Networking10 and MetGem11 are two tools

67

for organizing hundreds of product ion spectra according to their similarity based on the

68

assumption that similar product ion spectra may come from similar molecules. Used alone these

69

tools is not able to detect isomers, which can be prevented using MZmine 212. However, they

3



70

could also generate complex graphs with many non-specific nodes coming from the chemical

71

noise, in-source fragment ions or adducts.

72

To obtain curated molecular networks allowing a good characterization of unknown

73

metabolites produced by P. nordicum, targeted MS/MS experiments were achieved specifically

74

on the signals of interest. For that purpose, metabolites were annotated with a strategy based on

75

double isotope labeling and untargeted metabolomic approach13. Fungal metabolites were

76

labeled with both carbon (13C) and nitrogen (15N) isotopes by growing the fungus on three

77

different substrates: (i) a nonlabelled substrate (containing almost 100% 12C), (ii) a fully labeled

78

substrate (with almost 100% 13C) and (iii) a doubly labeled substrate (with 100% 15N and 50%

79

13C).

80

natural substrate, thanks to their characteristic isotopic patterns and the unambiguous

81

determination of their elemental compositions by high-resolution mass spectrometry.

82



This method enabled the specific detection of metabolites produced by a fungus on its

MATERIALS AND METHODS

83

Fungus. The fungal strain NRRL 6062 was used, and it was confirmed to be P. nordicum14

84

using both morphological criteria15, DNA sequencing of ITS (ITS 4 and 5) and β-tub (a and b)

85

with previously published methods and primers16,17.

86

Chemicals and reagents. Formic acid and analytical solvents (acetonitrile, ethyl acetate

87

and methanol) were purchased from Fisher Scientific (Thermo Fisher Scientific, Illkirch,

88

France). The solvents used for sample preparation were HPLC grade. Acetonitrile was MS

89

grade. Ultra-pure water was generated from a Milli-Q system (Millipore, Saint-Quentin en

90

Yvelines, France). Ochratoxin A was purchased from Sigma (Sigma-Aldrich, Saint-Quentin

91

Fallavier, France), and ochratoxin B was purchased from Fermentek (Fermentek Ltd,

92

Jerusalem, Israel). The peptides were ordered from GeneCust (GeneCust, Dudelange,

93

Luxembourg). All standards were dissolved in methanol for qualitative analysis by HPLC-MS.

4


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Fungal cultures on unlabeled and labeled media. P. nordicum cultures on natural and

95

stable isotopes enriched wheat grains17 were performed according to previously described

96

method12. All experimental details are available in supplementary information.

97

Extraction of the metabolites. The fungal cultures and the control were separately

98

extracted with ethyl acetate by shaking on a horizontal shaker table (IKA Labor technik HS501,

99

IKA, Staufen, Germany) at 180 rpm for 36 h. Then, the samples were filtered to remove the

100

mycelium and the substrate. The crude extracts were concentrated, and the resulting residues

101

were resuspended in 400 µL of methanol. Before analysis, the four samples were filtered

102

through 0.45-μm Whatman filters (Whatman, GE Healthcare, Kent, U.K.).

103

Liquid Chromatography (HPLC), High-Resolution Mass Spectrometry (HRMS)

104

and Ultra-Violet (UV) spectroscopy. The samples were analyzed by coupling HPLC with

105

HRMS. The chromatographic separation was performed with an Ultimate 3000 system

106

(Thermo Scientific, Les Ulis, France). The mobile phases were A: 0.1% formic acid in water

107

and B: 100% acetonitrile. The two solvents were eluted at a flow rate of 0.2 mL/min according

108

to the following gradient: 0 min 20% B, 30 min 50% B, from 35 to 45 min 90% B, and from 50

109

to 60 min 20% B. The injection volume was 10 μL, and the samples were separated on a Luna

110

C18(2) column (150 mm × 2.0 mm, 5 μm) (Phenomenex, Torrance, CA, U.S.A.). HRMS

111

analyses were performed on an LTQ Orbitrap XL hybrid high-resolution mass spectrometer

112

(Thermo Scientific, Les Ulis, France) using both positive and negative electrospray ionization

113

(ESI) modes. The ionization parameters were set as follows: for the positive mode, spray

114

voltage, +4.5 kV; capillary temperature, 350 °C; sheath gas (N2) flow rate, 40 a.u. (arbitrary

115

units); and auxiliary gas (N2) flow rate, 6 a.u. For the negative mode, spray voltage, −3.7 kV;

116

capillary temperature, 350 °C; sheath gas flow rate, 30 a.u. (N2); and auxiliary gas flow rate, 10

117

a.u. (N2). High-resolution mass spectra were acquired at a resolution of 60,000 (at m/z 400)

118

from m/z 50 to m/z 800. The high-resolution mass analyzer was calibrated in each ionization

5



119

mode based on the supplier’s protocol and calibration mixtures (Thermo Scientific, Les Ulis,

120

France).

121

Targeted high-resolution MS/MS experiments were performed in both resonant and non-

122

resonant modes on each metabolite. In this article, CID refers to MS fragmentations in the

123

resonant excitation mode, and HCD refers to fragmentation in the non-resonant mode according

124

to the nomenclature used by Thermo Scientific. The HCD mode allows the mass range to be

125

expanded (absence of a low mass cut-off) and the observation of consecutive decompositions.

126

The generated results can provide structural information complementary to the data obtained in

127

the CID mode, which favors competitive processes. The parameters for the CID mode were set

128

as follow: resolution 7500, maximum injection time 200 ms, isolation width 1.5 u, activation

129

qz 0.250, and activation time 30 ms. The parameters for the HCD mode were set as follow:

130

resolution 7500, maximum injection time 200 ms, isolation width 1.5 u, and activation time 30

131

ms. HCD and CID normalized collision energies (NCE) were adjusted to obtain appropriate

132

fragmentation patterns (at least 10% relative to the abundance of the precursor ion) and

133

maximum structural information.

134

HPLC coupled with diode array detection (Ultimate 3000 system, Thermo Scientific, Les

135

Ulis, France) at 3D and different wavelengths was used to identify some metabolites. The

136

chromatographic system and parameters were the same as those used for HPLC-HRMS

137

analyses.

138

Identification of metabolites and molecular networking. An in-house software was

139

used to compare the results from the natural and labeled wheats13. The elemental composition

140

of each secondary metabolite was determined with a mass measurement accuracy of 5 ppm and

141

included the following elements: C, H, O, N, S, P, Cl, K and Na.

142

Metabolite identifications were classified according to the Metabolomics Standard

143

Initiative18. Metabolites identified at level 1 displayed the same retention time, elemental

6


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composition and product ion spectrum as the standard compound analyzed under the same

145

conditions. The metabolites identified at level 2 displayed product ion spectra and/or UV

146

spectra identical to those referenced in the literature. Some metabolites were identified at level

147

3, which corresponds to displaying product ion spectra that were in agreement with their

148

putative structures. In silico fragmentations were performed using CFM-ID and compared with

149

experimental product ion spectra to support the assignments of the metabolites identified at

150

level 319.

151

Molecular networks were generated with GNPS and MetGem from the targeted high-

152

resolution CID spectra of all metabolites produced by P. nordicum detected in this study

153

together with metabolites detected in P. verrucosum in our previous study20. MS/MS spectra

154

were extracted using MZmine 2 using the following parameters: mass detection noise level 10;

155

ADAP chromatogram builder group intensity threshold 1E3, min highest intensity 2E3, m/z

156

tolerance 0.005 m/z or 20 ppm; chromatogram deconvolution wavelets, S/N threshold 3, min

157

feature height 1,000, peak duration range 0.05-0.7, RT wavelet range 0.01-0.7, m/z range for

158

MS2 scan paring 0.05, RT range for MS2 scan paring 0.2. Molecular networks parameters

159

(MetGem and GNPS) were set as follows: precursor ion mass tolerance 0.2 Da; fragment ion

160

mass tolerance 0.02 Da; min pairs cosine score 0.4; minimum matched fragment ions 4;

161

minimum peak intensity 1000. The molecular network resulting from GNPS was visualized in

162

Cytoscape 2.8.2.

163

Analysis of cultures of P. nordicum on meat and cheese. The strain NRRL 6062 was

164

cultured on several of the preferential substrates of P. nordicum, i.e., cooked pork ham, turkey

165

ham and Raclette cheese purchased in a local supermarket. The substrate slices (0.5-cm thick)

166

were cut to the diameter of the Petri dishes. For each substrate, four slices were immersed in a

167

1% NaOCl solution for 1 min, rinsed twice with sterile water and placed in separate Petri dishes.

168

Three slices were inoculated with 10 µL of P. nordicum spore solution (at 106 spore/mL). One

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169

slice was not inoculated to serve as a control sample. The Petri dishes were stored at 25 °C in

170

the dark for one week. Produced metabolites were extracted, and the samples were prepared as

171

described above for the wheat cultures. Using an injection volume of 10 μL, separations were

172

performed on an Accela 600 HPLC system (Thermo Scientific, Les Ulis, France) using the

173

same chromatographic parameters as described above. Targeted analyses were performed by

174

HPLC-MS using a TSQ Vantage triple-stage quadrupole mass spectrometer (Thermo

175

Scientific) using positive ESI mode based on two MRM transitions for each targeted metabolite.

176

In a second step, the diffusion of metabolites in the substrate was investigated. For this, the

177

strain NRRL 6062 was grown on pork ham, turkey ham and cheese as previously described,

178

but a cellophane sheet was placed on the slice before inoculation. After one week of culture,

179

the mycelium (on the cellophane sheet) was extracted separately from the substrate slice.

180

Metabolites produced by the fungus that can spread within the substrate were found in the slice,

181

and the secondary metabolites that remained in the mycelium were found only in the extract of

182

the cellophane/fungus part. RESULTS AND DISCUSSION

183



184

Metabolites produced by P. nordicum was characterized according to a protocol previously

185

developed in our laboratory13. The fungus was grown on natural wheat grains, 13C grains and

186

13C/15N

187

subjected to the same conditions. To avoid overlapping signals due to the complex isotopic

188

composition of the labeled molecules, the resulting secondary metabolomes were extracted and

189

separately analyzed by HPLC-ESI-HRMS. Then, data mining of the three resulting fungal

190

metabolomes was carried out manually. Chromatograms and mass spectra obtained from the

191

infected 13C/15N labeled grains were analyzed first to take advantage of the specific isotopic

192

pattern of the compounds labeled with 50%

193

variables were specifically detected in the mass spectra generated after positive and negative

grains. A control sample corresponding to non-infected natural wheat grains was

13C

8

and 100%


15N.

Thus, 99 and 105 labeled

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194

electrospray ionization, respectively. Analysis of the chromatograms showed that 15 (in the

195

positive mode) and 25 (in the negative mode) of these specific signals were generated by adduct

196

ions or product ions generated in the instrument. Thereafter, only protonated ions were

197

considered21. Several sodium adduct ions were used when the corresponding protonated ions

198

were not found, which led to a list of 84 signals in the positive mode and 78 in the negative

199

mode. The data mining was then conducted by analyzing the 13C sample and the 12C sample.

200

Finally, comparison of the 12C grains infected by P. nordicum with the control sample enabled

201

the exclusion of metabolites not related to fungal infection (19 for the positive mode and 26 for

202

the negative mode).

203

By knowing the accurate mass of a given molecule under the three different labeling

204

conditions, it was possible to unambiguously determine its elemental composition. The number

205

of carbon atoms contained in each metabolite was determined by subtracting the m/z ratio of

206

the 12C signal from the corresponding 13C signal. Comparison of the accurate masses of ions

207

labeled with

208

Considering these two restrictions and considering the seven golden rules22, only one elemental

209

composition remained common among the three accurate masses. In that way, the formulas of

210

the 92 metabolites produced by P. nordicum were unambiguously determined after accounting

211

for the 25 redundant signals between the positive and the negative ionization modes (Table 1).

212

To share the data for further Penicillium studies and to ensure the utility of all these data,

213

chemical information about each detected metabolite is presented in the Supporting Information

214

(Table S1). Fragmentation spectra are presented as a list of product ions together with their

215

relative abundance in HCD and/or CID modes.

216

Table 1: Produced metabolites detected in P. nordicum infecting wheat grains. Numbers in

217

brackets refer to the number attached to each metabolite throughout the article. proposed identification

12C, 13C

molecular formula

and

Delta ppm

13C/15N

12C

m/z

allowed the number of nitrogen atoms to be calculated.

parent ion

RT (min)

proposed identification

9


molecular formula

Delta ppm

12C

m/z

parent ion

RT (min)


C5H9NO

-1.9

100.0755

[M+H]+

2.9

C6H10O4

-1.9

147.0649

[M+H]+

3.1

C10H13NO2

-1.7

180.1016

[M+H]+

3.9

C11H16O4

-0.6

213.1120

[M+H]+

15.6

C11H16O5

0.2

229.1071

[M+H]+

5.5

C11H16O5

0.2

229.1071

[M+H]+

6.1

LL-P880α (7)

C11H18O4

-0.4

215.1277

[M+H]+

13.9

LL-P880β (8)

C11H18O5

-0.4

231.1226

[M+H]+

5.7

C12H9N3O2

-0.8

226.0620

[M-H]-

15.2

C12H9N3O2

-0.8

226.0620

[M-H]-

12.7

C12H9N3O2

-0.8

226.0620

[M-H]-

18.0

C13H17NO

-0.7

202.1236

[M-H]-

12.8

275.1849

[M+H]+

18.1 17.2

Verrucolone (6)

PC-2 (10) LL-P880γ (9)

C14H26O5 C15H24O2

-0.9

237.1847

[M+H]+

C15H24O2

-0.9

237.1847

[M+H]+

22.1

C15H24O2

-0.9

237.1847

[M+H]+

23.1

C15H24O3

-0.5

253.1797

[M+H]+

9.7

-0.5

253.1797

[M+H]+

-1.1

251.1650

[M-H]-

-1.1

251.1650

[M-H]-

-0.5

253.1797

[M+H]+

C15H24O3 C15H24O3 C15H24O3

Sclerotigenin (5)

-1.5

23.2 251.1650

[M-H]-

C15H26O2

-1.3

237.1857

[M-H]-

40.4

C16H11N3O2

-0.7

278.0922

[M+H]+

9.9

C16H11N3O2

-0.7

278.0922

[M+H]+

15.1

C16H14O3

-3.2

253.0862

[M-H]-

11.5

-2.0

295.0595

[M+H]+

293.0450

[M-H]-

293.0923

[M-H]-

11.3 38.6

-1.9

C17H14N2O3

Anacine (3)

35.5

-1.1

C17H10O5

Aurantiomide C (4)

22.1

-2.9

Hydrolyzed Cyclo(VIVF) VIVF (25)

Cyclo(R1VVF) (24) R1 side chain=C7H7O2

Page 10 of 32

C25H40N4O5

-2.4

477.3060

[M+H]+

16.9

C26H42O7

-4.9

467.2980

[M+H]+

30.5

-4.9

525.2682

[M+H]+

-3.6

523.2543

[M-H]-

-1.8

511.2906

[M+H]+

0.1

509.2770

[M-H]-

-1.8

511.2906

[M+H]+

0.1

509.2770

[M-H]-

-2.7

527.2850

[M+H]+

-0.1

525.2718

[M-H]-

C28H42O5

-4.0

481.2905

[M+Na]+

43.8

C28H42O5

-4.0

481.2905

[M+Na]+

44.2

0.5

537.3050

[M+Na]+

3.4

535.2920

[M-2H+Na]-

0.5

537.3050

[M+Na]+

3.4

535.2920

[M-2H+Na]-

0.5

537.3050

[M+Na]+

3.4

535.2920

[M-2H+Na]-

-0.8

539.2860

[M+H]+

-3.8

537.2698

[M-H]-

C29H40N4O5

-4.8

525.3046

[M+H]+

19.5

C29H40N4O5

-4.8

525.3046

[M+H]+

20.2

3.4

541.3039

[M+H]+

-2.8

539.2860

[M-H]-

3.4

541.3039

[M+H]+

-2.8

539.2860

[M-H]-

4.5

497.3736

[M+Na]+

43.6

446.3994

[M+H]+

38.6

-3.2

532.2901

[M+H]+

-4.3

530.2750

[M-H]-

-4.1

548.2845

[M+H]+

-2.7

546.2707

[M-H]-

C28H36N4O6

Hydrolyzed Fungisporin A C28H38N4O5 VFFV (15) Hydrolyzed Cyclo(VFVF) VFVF (21)

C28H38N4O5

Hydrolyzed Fungisporin B C28H38N4O6 VYFV (11)

(29)

C28H42N4O5

(28)

C28H42N4O5

Hydrolyzed Cyclo(VR2FV) VR2FV (27) R2 side chain=C7H11

C28H42N4O5

Hydrolyzed Cyclo(R1FFV) R1FFV (30) R1 side chain=C4H7O

C29H38N4O6

Hydrolyzed Cyclo(FFVI) IFFV (13) Hydrolyzed Cyclo(FFIV) VFFI (12) Hydrolyzed Cyclo(YFVI) IYFV (14)

C29H40N4O6

Hydrolyzed Cyclo(YFIV) VYFI (23)

C29H40N4O6 C29H50N2O3

11.2

C29H51NO2 Fungisporin D (20)

C30H37N5O4

Cyclo(YWVV) (22)

C30H37N5O5

0.3

C17H26O4

-2.1

315.1571

[M-2H+Na]-

C17H34O5

-4.2

317.2320

[M-H]-

39.1

C17H34O5

-4.2

317.2320

[M-H]-

39.5

C17H34O5

-4.2

317.2320

[M-H]-

41.2

C30H37N5O6

-3.9

562.2649

[M-H]-

C17H34O5

-4.2

317.2320

[M-H]-

-1.6

550.3015

[M+H]+

C17H34O5

-4.2

317.2320

[M-H]-

38.1 Hydrolyzed Fungisporin D C30H39N5O5 VFWV (19) 41.0

-0.6

548.2875

[M-H]-

C18H20N4O3

-0.6

341.1606

[M+H]+

20.1

-1.6

550.3015

[M+H]+

-0.6

341.1606

[M+H]+

-0.6

548.2875

[M-H]-

-1.4

339.1458

[M-H]-

-2.5

566.2959

[M+H]+

C18H22N4O3

-1.1

343.1761

[M+H]+

12.7

-0.6

564.2824

[M-H]-

C18H22N4O3

-1.1

343.1761

[M+H]+

17.8

-2.5

566.2959

[M+H]+

C18H22N4O4

-3.2

381.1521

[M+Na]+

15.3

-0.6

564.2824

[M-H]-

C18H24N2O2

-3.5

301.1900

[M+H]+

37.7

1.5

576.3165

[M+Na]+

312.2896

[M+H]+

3.3

574.3030

[M-2H+Na]-

C18H20N4O3

C19H37NO2

-0.3

15.2

Hydrolyzed Fungisporin D C30H39N5O5 WVVF (17) Hydrolyzed Cyclo(YWVV) VYWV (18)

C30H39N5O6

Hydrolyzed Cyclo(YWVV) WVVY (16)

C30H39N5O6

Hydrolyzed Cyclo(R1R2VF) C30H43N5O5 R1R2VF (31) 31.8 R1+R2 side chains=C12H19N

10


25.8

18.0

18.7

12.7

24.4

23.7

23.1

26.9

13.8

15.4

36.4

29.4 26.2 17.4

19.1

12.3

13.2

21.0

Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ochratoxin A (1)

Ochratoxin B (2)

C20H18NO6Cl

C20H19NO6 C20H30O4 C21H30N4O4

-0.3

404.0894

[M+H]+

Hydrolyzed Cyclo(VR2WV) C30H43N5O6 VR2WV (26) 33.2 R2 side chain=C7H11O

0.2

592.3107

[M+Na]+

15.5

0.1

402.0750

[M-H]-

C31H41N5O5

-0.9

562.3030

[M-H]-

19.2

-0.6

370.1283

[M+H]+

C31H41N5O5

-0.9

562.3030

[M-H]-

21.4

368.1138

[M-H]-

531.2722

[M-2H+Na]-

37.3

333.2062

[M-H]-

510.3554

[M+Na]+

36.9

401.2183

[M-H]-

510.3554

[M+Na]+

38.2 39.5

-0.4 -2.8 -2.8

26.5

C31H42O6

39.4

C31H45N5

35.4

C31H45N5

-1.1 -2.6 -2.6

C21H30N4O4

-2.8

401.2183

[M-H]-

25.8

C32H40O6

-4.3

521.2875

[M+H]+

C21H32N2O2

-1.3

345.2532

[M+H]+

27.0

C32H42O7

-3.7

537.2838

[M-H]-

39.5

C21H32N2O2

-1.3

345.2532

[M+H]+

23.5

C32H42O8

0.5

553.2810

[M-H]-

38.3

C21H32N4O5

-3.3

419.2286

[M-H]-

17.1

C32H44O9

-2.3

571.2899

[M-H]-

36.8

C21H36O3

-4.2

337.2723

[M+H]+

37.1

C34H64O8

-3.5

599.4507

[M-H]-

39.2

449.1793

[M-2H+Na]-

25.4

C38H39N5O7

0.1

676.2778

[M-H]-

36.9

C38H45N5O6

-4.1

668.3415

[M+H]+

23.8

C38H45N5O6

-4.1

668.3415

[M+H]+

24.2

C22H28N4O5

-2.9

C23H26N2O4

-1.0

415.1635 [M-2H+Na]-

17.9

C25H32N4O5

-3.2

469.2430

[M+H]+

21.2

C25H32N4O5

-3.2

469.2430

[M+H]+

24.1

R1GFFV (33) R1 side chain=C11H11 R1GVVF (32) R1side chain=C15H11

218 219

Elemental compositions were identified using a natural products-specific database

220

(AntiBase (2012)23) and the literature regarding Penicillium species. Ochratoxin A (compound

221

1 in Figure S1) and ochratoxin B (compound 2 in Figure S1), two well-known mycotoxins from

222

P. nordicum14,15, were identified at level 1 by comparison with standard compounds. Since

223

reference compounds for many natural products are not available21, several metabolites were

224

putatively assigned by comparison of their analytical properties with those reported in the

225

literature. The substances with elemental compositions of C18H22N4O3 (m/z 343.1761), C6H10O4

226

(m/z 147.0649) and C16H11N3O2 (m/z 278.0922) were identified at level 2 as three metabolites

227

known to be produced by P. nordicum15, namely, anacine (compound 3 in Figure S1),

228

verrucolone (compound 6 in Figure S1) and sclerotigenin (compound 5 in Figure S1),

229

respectively 2425, since their experimental UV and/or mass spectra were in agreement with the

230

spectra reported in the literature24,25. Other metabolites were identified at level 3, namely,

231

aurantiomide C (compound 4 in Figure S1), which is known to be produced by P. nordicum

232

growing on cereal26; the α-pyrones PC-2 and LL-P880γ (compounds 10 and 9 in Figure S1),

11



233

which are also known to be produced by P. nordicum27, and the similar metabolites LL-P880α

234

and LL-P880β (compounds 7 and 8 in Figure S1).

235

To support the level 3 identification, high-resolution CID spectra (Supporting Information,

236

Table S1) were compared with in silico spectra generated by CFM-ID, which is one of the most

237

effective tools for in-silico MS/MS spectral prediction. First, it generates MS/MS spectra

238

considering the structure of the molecule and not only the nature of carbon-carbon bonds.

239

Moreover, since CFM-ID is based on machine learning, it could consider fragmentation ways

240

unreferenced in other tools. Although it should not be considered as proof of identification, this

241

tool provides relevant supporting information. The in silico spectrum of LL-P880α (7) and the

242

experimental product ion spectrum of the metabolite detected at m/z 215.1277 in positive mode

243

in P. nordicum displayed eight common product ions: a major product ion at m/z 197.1170 and

244

seven other product ions in low abundance at m/z 183.1013, 179.1066, 171.1376, 169.1223,

245

155.1066, 151.1116 and 137.0959. Similarly, six product ions were found common between

246

the in silico and the experimental CID spectra of LL-P880β (8) (m/z 213.1120, 199.0662,

247

195.1014, 171.1012, 127.0387 and 71.0488). The in silico spectrum of PC-2 (10) presented 10

248

product ions in common with the experimental CID spectrum (m/z 195.1011, 181.0854,

249

153.0906, 125.0229, 109.0645, 99.0439, 97.0280, 97.0645 and 95.0488). Four out of the five

250

product ions of the experimental CID spectrum of the m/z 229.1067 ion were found in the in

251

silico spectrum of LL-P880γ (9) (m/z 229.1067, 211.0964, 125.0230 and 154.0493). Moreover,

252

two common product ions (m/z 324.1337 and 296.1388) were found in the in silico spectrum

253

of aurantiomide C (4) and in the CID spectra of C18H20N4O3 (m/z 341.1606).

254

Thirteen metabolites in the fungisporin family were also identified (Table 1 and Figure S1,

255

compounds 11 to 23). Among them, fungisporin D (i.e., cyclo(VFWV), m/z 532.2901) (20) and

256

cyclo(YWVV) (m/z 548.2845) (22) were detected. The product ion spectra of these compounds

257

are presented and annotated in Supporting Information Figure S2. The 11 other assigned

12


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258

fungisporin-related metabolites were detected as linear peptides. According to Klitgaard et al.28

259

and Ali et al.29, who characterized these compounds, such linear analogues are a result of the

260

enzymatic degradation of cyclic fungisporins in the growth medium of the fungus and must be

261

considered full-fledged secondary metabolites. Throughout this article, the linear analogues of

262

fungisporins are referred to as “hydrolyzed fungisporins”. These hydrolyzed fungisporins were

263

identified at level 1 by analysis of reference compounds: hydrolyzed fungisporin A (VFFV, m/z

264

511.2906, compound 15 in Figure S1); hydrolyzed fungisporin B (VYFV, m/z 527.2850

265

compound 11 in Figure S1); hydrolyzed cyclo(FFIV) (VFFI, m/z 525.3046, compound 12 in

266

Figure S1); hydrolyzed cyclo(FFVI) (IFFV, m/z 525.3046, compound 13 in Figure S1);

267

hydrolyzed cyclo(YFVI) (IYFV, m/z 541.3039, compound 14 in Figure S1); hydrolyzed

268

cyclo(YFIV) (VYFI, m/z 541.3039, compound 23 in Figure S1); and two isomers of hydrolyzed

269

fungisporin D (WVVF, compound 17 in Figure S1 and VFWV, compound 19 in Figure S1,

270

both at m/z 550.3015) and two isomers of hydrolyzed cyclo(YWVV) (VYWV, compound 18

271

in Figure S1 and WVVY, compound 16 in Figure S1, both at m/z 566.2959). For all these

272

peptides, their first and third residues were L-amino acids, while their second and fourth

273

residues were D-amino acids. The peptide VFVF (m/z 511.2906, compound 21 in Figure S1)

274

was identified at level 2 by comparison of its retention time and product ion spectrum with

275

those reported in the literature20. Assuming that such linear tetrapeptides result from the

276

enzymatic hydrolysis of fungisporins, several isomers with the same amino acid sequence are

277

likely to come from the same cyclic tetrapeptide. Here, VFWV (19) and WVVF (17) are likely

278

to result from the hydrolysis of fungisporin D (20) at two different peptide bonds, and VYWV

279

(18) and WVVY (16) likely both result from the hydrolysis of cyclo(YWVV) (22). Therefore,

280

the 11 linear peptides were likely generated from enzymatic hydrolysis of nine fungisporins

281

(Table 1). To the best of our knowledge, this is the first time that these secondary metabolites

282

have been accurately identified in P. nordicum.

13



283

The identification of 13 known fungisporin-related metabolites suggested that other

284

unknown tetrapeptides could be produced by this fungus. Evaluations of compounds belonging

285

to a given chemical family were performed by MetGem11 (Figure 1) and GNPS Molecular

286

Networking10 (Figure 2) using MZmine 2 to extract MS/MS data from raw data12. Molecular

287

networks were generated from high-resolution targeted product ion spectra collected

288

specifically from each of the 92 metabolites produced by P. nordicum listed in Table 1

289

(Supporting Information Table S1). Spectra of produced metabolites of P. nordicum were

290

processed in GNPS in combination with the metabolome of P. verrucosum, which was

291

investigated in our previous study under the same conditions (cultures of both fungi were

292

performed the same day, with the same batch of labeled and unlabeled wheat grains, with the

293

same culture conditions. Produced metabolites were extracted at the same time by the same

294

operator and analyzed successively on the same mass spectrometer in the same analytical

295

conditions)20. Indeed, since P. verrucosum is genetically the most similar species to P.

296

nordicum, we assumed that their metabolomes should be similar and that processing these data

297

together could help in the assignment of metabolites of interest, in the interpretation of the

298

network (Figure 2), and in the identification of metabolites. However, only 20 produced

299

metabolites were found to be shared by both species (i.e around 20 % of their produced

300

metabolites). A recent work estimates that P. nordicum and P. verrucosum diverged only 2

301

million years ago (mya) (0.946-3.473 mya)30. Taken together, these results show that their

302

secondary metabolisms evolved very quickly within a geological short period by contrast to

303

their morphological characters so similar that both species were considered as only one species

304

for a while31.

14


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LL-P880

PC2

LL-P880

Anacine

Hydrolyzed fungisporin A

Aurantiomide C Ochratoxin A Ochratoxin B

305 306

Figure 1. Molecular network obtained with MetGem from targeted CID product ion spectra

307

achieved on the metabolites detected in P. nordicum. Each node is identified by the m/z value

308

of the precursor ion.

15



Page 16 of 32

Ochratoxin A

Verrucine(s) A/B Aurantiomide C Anacine

Verrucine F

Ochratoxin B

LL-P880 PC-2 LL-P880

Fungisporin D

Hydrolyzed cyclo(VFVF) Hydrolyzed fungisporin A

309 310

Figure 2. Molecular network obtained with GNPS from targeted CID product ion spectra

311

achieved on the metabolites detected in P. nordicum (yellow), in P. verrucosum (blue), or in

312

both (green). Each node is identified by the m/z value of the precursor ion. The nodes size

313

represents the absolute abundance (logarithmic scale) of the parent ions in the mass spectrum.

314 315

The main challenge in constructing the molecular network came from the processing of

316

datasets through a spectral archives approach that involved merging all the similar product ion

317

spectra to create a single consensus spectrum before calculating their similarity scores32.

318

Therefore, this automatic clustering prevents the detection of isomers. To overcome this

16


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319

limitation and to generate a curated network, MZmine 2 could be used to extract MS/MS

320

spectra, eliminate isotopes, in-source fragment ions, or adducts33. However, MZmine 2 does

321

not prevent the detection of ions, which are not specific of the studied samples, such as solvent

322

contaminants or molecules coming from the culture media for example. This lead to the

323

extraction of numerous not specific MS/MS spectra, which complicating obtained molecular

324

networks. Here, by the specific detection of metabolites of interest using stable isotopes,

325

followed by targeted MS/MS only on these specific metabolites, molecular networks are much

326

more informative. To interpret the networks and to note the production of potential new

327

fungisporins by P. nordicum, the corresponding nodes were marked. The linking of similar

328

structures, such as ochratoxins or quinazolines (verrucines, anacine and aurantiomide C), in

329

Figures 1 and 2 validated the networks generation parameters. In these two networks, unknown

330

metabolites are linked with ochratoxins or quinazolines by non-specific fragmentations, such

331

as losses of water, which did not suggest any particular structural similarities. Interestingly,

332

identified fungisporins and their hydrolyzed analogues were linked in a same independent graph

333

in both networks Figures 1 and 2. Structural analyses by MS/MS were performed to characterize

334

these potentially new fungisporins.

335

The unannotated metabolite with a formula of C28H36N4O6 (detected at m/z 525.2682 at

336

25.8 min, compound 25 in Table 1) was linked to fungisporin D (20) in the two molecular

337

networks (Figures 1 and 2). Their product ion spectra were compared to facilitate the

338

characterization of the unknown metabolite. The two product ion spectra showed seven

339

common fragment ions (m/z 346.2115, 247.1436, 219.1485, 199.1439, 171.1485, 120.0809 and

340

72.0804). Moreover, the two product ion spectra both presented two identical neutral losses:

341

C5H9NO and C9H9NO. These two neutral losses correspond to the elimination of valine and

342

phenylalanine, respectively, in the product ion spectrum of fungisporin D. These losses

343

therefore suggested that unknown metabolite C28H36N4O6 contained each of these amino acids.

17



344

The fragment ions at m/z 72.0804 and 120.0809 were attributed to the corresponding immonium

345

ions and supported this hypothesis. It was assumed that the unknown metabolite consisted of a

346

cyclic peptide since a loss of carbon monoxide was observed directly from the precursor ion

347

without previous loss of water (neither in HCD nor in CID mode), similar to what was seen in

348

the product ion spectrum of fungisporin D. The other fragment ions of metabolite C28H36N4O6

349

detected at 25.8 min were interpreted according to the classical fragmentation mechanisms of

350

cyclic peptides34,35 (Figure S3). In addition to the losses of valine and phenylalanine, the product

351

ion spectrum showed a loss of C9H9NO3, putatively representative of a hydroxylated tyrosine

352

residue. The corresponding immonium ion was observed at m/z 152.0701. The observation of

353

direct losses of valine, phenylalanine and of the putative hydroxylated tyrosine supported the

354

hypothesis of a cyclic structure since each amino acid may be lost directly from the precursor

355

ion depending on the cleavage site of the cyclic peptide. All other product ions were interpreted

356

considering these four losses (i.e., CO, C5H9NO, C9H9NO and C9H9NO3) (Figure S3, right

357

side). The presence of two valine residues was supported by the product ions detected at m/z

358

426.2016 and m/z 327.1331, which likely resulted from two consecutive losses of C5H9NO. The

359

secondary metabolite with the formula C28H36N4O6 was therefore identified as a new cyclic

360

tetrapeptide composed of two valines, a phenylalanine and a nonstandard amino acid with a

361

side chain composition of C7H7O2, which might correspond to a hydroxylated tyrosine.

362 363

The structures of the unknown linear peptides highlighted by molecular networking were

364

investigated based on classical peptide fragmentation mechanisms36,35. Complementary

365

information was provided by examinations of both the CID spectra and HCD spectra35

366

(Supporting Information Table S1). The metabolite detected at m/z 477.3060 (compound 25 in

367

Table 1) was characterized as the tetrapeptide VIVF (or VLVF) (Figure 3). To support the

368

manual de novo sequencing, the theoretical fragmentation of VIVF was simulated using the

18


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369

MS-Product tool of ProteinProspector37. Nine out of the 11 product ions detected in the

370

experimental product ion spectrum were proposed by the software, and only one theoretical

371

fragment ion (corresponding to the a3 ion) was not found in the experimental product ion

372

spectrum. To specify the sequence and to investigate the stereochemistry of this metabolite, the

373

experimental retention time and product ion spectra were compared with those of standard

374

compounds corresponding to each viable structural hypothesis (i.e., 16 different stereochemical

375

configurations of VIVF and 16 different stereochemical configurations of VLVF). As a result,

376

this new fungisporin analogue was identified as the tetrapeptide L-valine-D-isoleucine-L-

377

valine-D-phenylalanine (Figure 3). To the best of our knowledge, this is the first time that this

378

peptide has been detected in a fungal extract.

19



379 380

Figure 3. High-resolution CID (NCE 15%) and HCD (NCE 20%) product ion spectra of five

381

unknown linear peptides detected in P. nordicum, according to the nomenclature established by

382

Biemann38.

383 384

Some of the unknown peptides displayed sequences containing one or two nonstandard

385

amino acids. The unknown peptides detected at m/z 592.3107 (RT 15.5 min) (compound 26 in

386

Table 1) and m/z 537.3050 (RT 23.1 min) (compound 27 in Table 1) displayed similar structures.

387

Interpretation of their product ion spectra indicated that they both consisted of two valines (in

388

C-terminal and N-terminal positions) and an aromatic residue in the third position (Figure 3).

20


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389

This aromatic residue was a tryptophan in the first peptide (26) and a phenylalanine in the

390

second peptide (27). The metabolite at m/z 592.3107 (26) contained an amino acid with a side

391

chain of C7H11O in the second position, which might correspond to tetrahydrotyrosine.

392

Similarly, the metabolite at m/z 537.3050 (27) displayed an amino acid with a side chain that

393

might correspond to tetrahydrophenylalanine. This type of tetrahydroamino acid is known to

394

be produced by microorganisms39. Two other isomers of the peptide at m/z 537.3050 were

395

detected at different retention times (compounds 28 and 29 in Table 1), but they produced very

396

weak signals. Their product ion spectra could not be analyzed due to interference from the

397

product ions of the major isomer detected at RT 23.1 min. It is likely that the three isomers of

398

C28H42N4O5 resulted from cleavage of the same cyclic tetrapeptide at different sites and thus

399

were related to the same fungisporin.

400

The metabolite detected at m/z 539.2860 (compound 30 in Table 1) was identified as the

401

linear tetrapeptide R1FFV, with R1 displaying a side chain of C4H7O, which might correspond

402

to a tetrahydrofuran moiety (Figure 3). Finally, a new tetrapeptide was detected at m/z 576.3165

403

(compound 31 in Table 1), and it displayed two nonstandard C-terminal amino acids (Figure

404

3).

405

The two unknown metabolites detected at m/z 668.3415 at RT 24.2 min (compound 32 in

406

Table 1) and 23.8 min (compound 33 in Table 1) are correlated with fungisporins only in the

407

MetGem network (Figure 1), illustrating the complementarity of MetGem and GNPS. The

408

metabolite detected at 24.2 min (32) presented the sequence R1GVVF with R1 as a nonstandard

409

amino acid with a side chain of C15H11. The metabolite detected at 23.8 min (33) was identified

410

as the peptide R2GFFV with a side chain of R2 = C11H11 (Supporting Information, Figure S4).

411

To support the metabolomic detection of fungisporin-related metabolites in P. nordicum

412

and to obtain genetic information about their synthesis, a BLAST analysis was performed. The

413

amino acid sequences of the protein HcpA (AID57164.1) involved in the synthesis of

21



414

fungisporins in a P. rubens strain formerly identified as P. chrysogenum and of all proteins

415

referenced in GenBank were compared. Fourteen Penicillium species presented sequences with

416

high homology (between 90% and 99%) and important recovery (between 99% and 100%),

417

supporting the hypothesis of Nielsen et al. that many Penicillium species are able to produce

418

fungisporins40. In particular, the protein KOS44855.1 (GenBank) of P. nordicum displayed

419

91% homology for a recovery of 100% with the protein HcpA. NRPSpredictor2 was used to

420

detect and compare the adenylation domains of KOS44855.1 and of HcpA41. Four adenylation

421

domains were detected on the sequence of the protein KOS44855.1 of P. nordicum, supporting

422

the assumption that this protein was involved in the synthesis of tetrapeptides. The active sites

423

of the adenylation domains of the two proteins displayed 97% homology (Table S2), and the

424

hypothetical substrates were identical. Therefore, it was suggested that the protein KOS44855.1

425

of P. nordicum was homologous to HcpA and is also involved in the synthesis of fungisporins.

426

The hypothetical substrates of each of the adenylation domains proposed by NRPSpredictor2

427

supported the structural hypotheses mentioned above.

428

It was recently reported that among 24 Penicillium species, the gene responsible for the

429

synthesis of fungisporins is the most frequent biosynthetic gene coding for nonribosomal

430

peptide synthetases (NRPS)40. Moreover, fungisporin A was recently detected on 11

431

Penicillium species growing on different media26. These observations suggest that this cyclic

432

peptide is crucial to growth or survival functions. Other fungisporins and their hydrolyzed

433

analogues have been identified as key secondary metabolites for aerial growth and colonization

434

processes in P. rubens29.

435

The last objective of this study was to evaluate the production of fungisporin-related

436

metabolites by P. nordicum growing on its substrates. To this end, pork ham slices, turkey ham

437

slices and cheese slices were inoculated with P. nordicum. The cultures of P. nordicum were

438

extracted after one week as fungisporins are produced during the first week of culture in P.

22


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439

rubens 29. Then, the known and newly characterized peptides were analyzed using a targeted

440

approach that allows higher detection sensitivity (Supporting Information Table S3). Except for

441

VYFI (23), all known fungisporin-related metabolites were detected on all the substrates either

442

in their cyclic native form or in their hydrolyzed forms. Among the new fungisporin-related

443

metabolites characterized from wheat cultures, two were detected on meat and cheese: VIVF

444

(25) and C30H43N5O5 (m/z 576.3165 at 21.0 min) (31).

445

For each type of culture, separated analyses of the mycelium extract and of the substrate

446

extract were performed by placing cellophane sheet on the cheese or meet slices before

447

inoculation. This method is commonly used to separate fungal mycelium from the substrate,

448

and the cellophane does not interfere with either the growth or the the metabolome produced

449

by the fungus42. The mycelium extract and the substrate extract displayed identical results,

450

suggesting that the tetrapeptides are able to migrate from the mycelium into the substrate.

451

The production of fungisporin-related metabolites by P. nordicum does not in and of itself

452

guarantee that these compounds are involved in the contamination of food stocks by this

453

pathogenic fungus. Indeed, such linear or cyclic tetrapeptides have been demonstrated to be

454

involved in the development processes of Penicillium species by promoting the aerial growth

455

of the fungus29,40. Toxicological analyses would allow us to assess the pathogenicity of

456

fungisporin-related compounds in humans and animals. Subsequent targeted strategies to

457

inhibit the growth of the fungus as well as to control the amount of these tetrapeptides in food

458

may suggest methods to protect the population from the deleterious effect of P. nordicum.

459

These secondary metabolites could also be of industrial interests since a hydrolytic product of

460

fungisporin B displayed antifungal activities similar to those of the industrial fungicide

461

benomyl43. Moreover, Ali et al. suggested that fungisporins may display cardiac ion-channel

462

blocking activities relevant to the treatment of hypertension and angina29.

463



CONCLUSIONS

23



464

Food contamination by fungi is associated with sanitary concerns and economic losses, due

465

to the production of fungal secondary metabolites. Investigation of fungal metabolomes is

466

therefore one of the most direct ways to identify new fungal toxicity issues and to develop new

467

strategies for preventing food destruction. This study aimed at the detection of putative new

468

toxins produced in case of fungal infection of food, that could be ingested by the consumer,

469

whether they are directly of a fungal origin or indirectly produced from the substrate after fungal

470

infection. Analyses by high-resolution mass spectrometry showed that P. nordicum infection

471

of wheat grains lead to the production of at least 92 metabolites, including 69 unknowns. We

472

cannot exclude that some compounds from weakly induced biosynthetic pathways have not

473

been produced in detectable quantities on all three substrates and therefore have not been listed,

474

since 12C, 13C and 13C/15N wheat grains samples might not be identical even though that the

475

wheat plants are from the same seeds and the cultures conditions are identical. Nor can we

476

exclude that some compounds are only slightly modified plant components due to plant biomass

477

degradation. Indeed, our approach does not discriminate the secondary metabolites produced

478

by the fungus from primary metabolites such as acyl-CoA, amino acids or terpenes, as well as

479

plant constituents simply transformed by the fungus. However, given that (i) enzymatic arsenal

480

involved in the plant biomass degradation is conserved between two very close species44, (ii)

481

on the contrary, secondary metabolism is considered as a significant feature for the delineation

482

of fungal species45,46,47, and (iii) only around 20% of their respective metabolites are shared by

483

P. nordicum and its closest known relative P. verrucosum, it is very likely that any modified

484

wheat metabolites constitute a minority among the 69 unknown molecules.

485

Bioinformatic tools enabled the assignment of known metabolites, but the identification of

486

new compounds of interest and the elucidation of their structures is still challenging. The

487

curation of the MS/MS dataset using targeted MS/MS performed only on compound of interest,

488

allowed the generation of an informative molecular networks. Thus, eight unknown

24


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489

fungisporin-related metabolites were characterized. Furthermore, the analysis of P. nordicum

490

growing on pork ham, turkey ham and cheese demonstrated that 10 of the known fungisporin-

491

related metabolites as well as three of the new metabolites were synthesized during fungal

492

growth on its natural substrates.

493



ASSOCIATED CONTENT Supporting Information

494 495

Experimental details on fungal cultures on natural and labeled substrate; HCD and CID product

496

ion spectra of secondary metabolites detected in P. nordicum after culture on wheat grains;

497

Structures of the secondary metabolites identified from P. nordicum; HCD product ion spectra

498

of [C30H37N5O4+H]+ detected at m/z 532.2901 and Rt=36.4 min, identified as fungisporin D and

499

[C30H37N5O5+H]+ detected at m/z 548.2845 and RT=29.4 min, identified as cyclo(YWVV);

500

HCD product ion spectrum (NCE 20%) of [C28H36N4O6+H]+ detected at m/z 525.2682 and

501

Rt=25.8 min; Interpretation of the high-resolution CID product ion spectra of the protonated

502

peptides detected at m/z 668.3415 and Rt=4.2 or Rt=23.8 min; Comparison of the sequences of

503

the active sites of the adenylation domains detected by NRPSpredictor2 for HcpA in P. rubens

504

(GenBank AID57164.1) and KOS44855.1 in P. nordicum; Targeted analysis in the positive

505

ESI mode of the production of fungisporin-related metabolites and pentapeptides when growing

506

P. nordicum on wheat, meet and cheese. ACKNOWLEDGMENT

507



508

PhD doctoral fellowship was co-funded by INRA and French Minister of Higher Education

509

and Research. Authors thank the Team Recherches Appliquées en Phytotechnologie, CEA,

510

IBEB, Cadarache, Saint-Paul-les-Durance, France for labeled wheat production.

511

All MS experiments were performed on the instruments of the MetaToul-AXIOM

512

platform, partner of the national infrastructure of metabolomics and fluxomics: MetaboHUB

25



513

[MetaboHUB-ANR-11-INBS-0010, 2011]. This study was co-funded by INRA and French

514

Minister of Higher Education and Research in the frame of a project supported by French

515

National Agency of Research [Newmyco-ANR-15-CE21-0010-21, 2015].

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Combination of isotope labeling and molecular networking of tandem

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