Chaetomium and Stachybotrys indoors
and their metabolites
Campus in Nuuk, Greenland (Peter Barfoed)
Estate in Nuuk, Greenland (Birgitte Andersen)
Birgitte Andersen
Section for Eukaryote Biotechnology
Department of System Biology
Technical University of Denmark
E-mail: [email protected]
The Project
Natural habitats
Fungal Consortia II:
Contamination
Growth physiology
Control and Prevention
funded by
Birgi@e Andersen Nature
Fungal contamination routes
Indoor air
Building Material Manufacturer
The building envelope
Dissemination
Chemistry
Detection
Genetics
Home
Ina Dosen Anna Lewinska Jakob B Nielsen Geo Clausen Ruut Peuhkuri Kris<an F Nielsen Carsten Rode Collaboration between DTU Civil Engineering & DTU Systems Biology
Chaetomium and Stachybotrys in DK
C. globosum
also common
on plywood
and OSB
(unpublished)
Frequency:*
16 % in total
25 % on concrete
Gypsum/
plywood wall
Glue
Floor boards
S. chartarum
Frequency:*
4 % in total
25 % on gypsum
Concrete
floor
Træinfor-­‐ ma<on.dk * Andersen et al. (2011) Appl Environ Microbiol 77: 4180–4188
Metabolome Platform at DTU
Agilent Technologies Thought Leader Lab
Head of platform: Kristian F. Nielsen
1 Agilent 6550 Accurate-Mass Quadrupole
Time-of-Flight (Q-TOF) LC-MS system with
1200+ in-house MS/HRMS of metabolites
7500+ metabolite data in database
2 Agilent 6490 Triple Quadrupole UHPLC-MS
6 Agilent 1100 HPLC-RI-UV
2 Agilent 1100 HPLC-UV-FLD
1 Agilent GC-MS for
13C
1 Bruker maXis G4 Q-TOF UHPLC-MS system
2 Dionex IC systems that can be interfaced with maXis G4 Q-TOF
1 Dionex UHPLC-UV-FLD
2 preparative HPLC-UV for purification of fungal metabolites
1 Thermo GC-MS system
analyses
Metabolite Library and databases
Fungal extract:
Metabolite standards
pure culture or environmental samples
Identification
1200+
Verification
LC-QTOF
MS/MS
Database search
7500 +
1.  List of compounds
2.  Find by formula
10 eV
Acquired MS
Library MS
100
20 eV
50
40 eV
0
2
100
4
6
8 10 12 14 16 18
Time (min)
505.9864
Library search
Accurate Mass
Accurate mass
50
1012.9875
0
500
m/z
QUANT software
1000
Agilent Masshunter
100
Isotope pattern
50
0
506
507
m/z
screening of many samples
fragments as wells as qualifier ions
508
Kildgaard et al. (2014) Mar Drugs 12: 3681–3705. Sample 1:
CH3
C. globosum inoculated on plywood
with different wallpapers and glues
CH3
H3C
H3C
H
H
N
H
N
H
CH3
O O
Prochaetoglobosin I H3C
H3C
CH3
N
H
CH3
N
H
CH3
O O
O
Prochaetoglobosin II H3C
H3C
O
O
H
H
N
H
N
H
CH3
O O
Prochaetoglobosin III 5
2
Prochaetoglobosin IV
2) 
Prochaetoglobosin III a
3) 
Prochaetoglobosin III b
4) 
Prochaetoglobosin II
5) 
Prochaetoglobosin I
O
Prochaetoglobosin IV Automated
database search
1) 
CH3
N
H
CH3
N
H
3
1
CH3
O O
4
Sample 1:
C. globosum inoculated on plywood
with different wallpapers and glues
Library search on accurate mass for verification: + UV-spectrum and retention time on own LC-QTOF
1.
2.
3.
4.
5.
Chaetoglobosin G, D, U or V
Chaetoglobosin G, D, U or V
Chaetoglobosin A
Chaetoglobosin G, D, U or V
Chaetoglobosin C
3
MS spectrum
5
1
2
4
UV spectrum
MS spectrum
UV spectrum
Metabolite Library for Stachybotrys
MPF Library # 465 466 485 409 1097 1095 462 463 1096 1094 464 458 459 453 1090 -­‐ 454 452 460 1089 1091 456 455 -­‐ 721 -­‐ 461 457 Name Accurate mass [M+H+] Satratoxin G 545.2381 Iso-­‐satratoxin F 543.2225 Roridin L-­‐2 531.2589 Satratoxin H 529.2432 K-­‐76 derivative compound 6 516.2956 K-­‐76 derivative compound 1 516.2592 Roridin E 515.2639 Roridin H 513.2483 K-­‐76 derivative compound 4 502.2799 F 1839F acid; Mer-­‐VGF 724B; K-­‐76 derivative compound 3 488.2643 Verrucarin J 485.2170 Atranone G 463.2326 Atranone H 449.2170 Atranone B 447.2377 Stachybotryamide 430.2588 Spirodihydrobenzofuranlactam II 430.2588 Atranone C 417.2272 Atranone A 417.2272 Atranone J 405.2272 Mer-­‐NF-­‐5003-­‐B 405.2272 F-­‐1839-­‐A 402.2275 Atranone E 387.2530 Atranone D 387.2530 Stachybotrydial 387.2166 Stachybotrylactam 386.2326 Spirodihydrobenzofuranlactam I 386.2326 3.4-­‐Epoxy-­‐6-­‐hydroxy-­‐dolabella-­‐7E 319.2268 Atranone F -­‐ RT/min 7.995 8.837 7.834 8.191 10.040 8.650 10.291 11.508 9.890 8.690 10.697 10.104 9.729 10.062 8.810 8.860 10.354 9.459 8.414 8.500 8.250 10.436 10.074 9.970 8.883 8.270 7.509 9.725 Additional data
on standards:
UV-Vis spectrum
Fragmentation
pattern
Sample 2:
Q-tip scrape from water-damaged gypsum
(drywall) with S. chartarum in a kindergarten
O
HO
2
Accurate mass
library search
3
1) 
2) 
3) 
4) 
Stachybotryamide
Stachybotrylactame
F-1839-D
Roridin E
O
H3C
4
HO
1
O
H3C
H
CH3
Stachybotrydial
CH3
Metabolites on real samples
OSB+ and Gypsum+: uninoculated control samples from other experiment
(16 moths at 73 %RH then 25 % H2O by weight)
Plywood and Drywall: samples from water damaged buildings
Fungal metabolite Chaetoglobosins A + C + G, D, U or/and V Chaetomugilins Chaetoviridins Cochliodons A + B Dihydrochaetoglobosin A Prochaetoglobosins I + II + III + IV Chaetospirone Chartarlactames F-­‐1839-­‐E + D Spirodihydrobenzofuran-­‐
lactame V Stachybotryamide Stachybotrylactame Stachybotrydial Roridin E Meleagrin Roquefortine C paper wallpaper OSB + wood chip wallpaper Gypsum board + paper wood chip glass fibre Plywood wallpaper wallpaper wallpaper Drywall construction glass fibre wallpaper + + + + + -­‐ -­‐ -­‐ + + -­‐ -­‐ + + -­‐ -­‐ + + -­‐ + -­‐ -­‐ + + + + -­‐ -­‐ + + + -­‐ + -­‐ + -­‐ -­‐ -­‐ -­‐ -­‐ + + + + + -­‐ + -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ + + -­‐ -­‐ -­‐ -­‐ + + + + + + + -­‐ + + -­‐ -­‐ + -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ + + -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ + + + + + + 

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