Prospecting volatile compounds produced by Brazilian ... · produced by Brazilian cyanobacteria with allelopathic effects ... Geosmin and MIB production comparison by genetic and

Prospecting volatile compounds produced by Brazilian cyanobacteria

with allelopathic effects

Prof. Dr. Ernani PintoSchool of Pharmaceutical Sciences - USP

[email protected] 1

PITE-FAPESP Agilent 2011/51950-3

Introduction

1. Cyanobacteria produce massive blooms in freshwater environment

2. Production of toxins and volatile compounds affecting water quality

2

Cyanobacteria Photosynthetic microorganisms

Introduction - Cyanobacteria

Unicellular, but may form colonies and filaments

The oldest organisms found in fossils, 3,5 billion years -Stromatolite. 3

4

Introduction - CyanobacteriaEnvironmental problem Massive bloom formation

5

Introduction - CyanobacteriaBlooms Input of nutrients - Eutrophication

What can trigger formation of blooms?

Environment x Lab Conditions

6

Introduction - CyanobacteriaEnvironmental problem Volatile compounds and cyanotoxins

2- methyl -isoborneol -

MIB

Geosmin

PITE-FAPESP Agilent 2011/51950-3

Cyanotoxins

Main Cyanotoxins

Microcystins - hepatotoxic

O

NH

O

HN

O

N

OHO

O

O

NH

R2

HN

HN

R1

OOHO

O

HNO

Saxitoxinsneurotoxin

HN

NH

HN

NH

HN

O

H

N

OHOH

H2N O

Anatoxin-a(s)neurotoxin

HN

N

N

HN

OP

O

OHO

Anatoxin-a and homoanatoxin-a

neurotoxins

HN

O

HN

O

Cylindrospermopsincytotoxin

H

NH HN NH

O

O

OH-O3SO

N

H

H

NH+

BMAAneurotoxin

NH2

O

OHHN

?

Environmental problem: blooms of cyanobacteria and presence of toxins

7

8

Billings Reservoir – Sampling procedures

Monitoring São Paulo reservoirs

9

Billings Reservoir – Sampling procedures


10

1

5

10

Billings Reservoir – Harvest, Isolation and cultivation

2

4

3

6


Cyanobacteria and secondary metabolites

1. Billion years adaptation diversity intra and interspecies and production of metabolites

2. Environmental problem (toxins, taste and odor alterations)

3. Are these compounds acting as allelophatic or quorum sensing?

4. Are there any metabolites that trigger rapid growth or “predict” bloom formation?

11

Targeted and Untargeted metabolomics to detect volatile compounds and their connection with bloom formation

Investigation of certain metabolites and quorum sensing and allelopathy

Influence of some metabolites on growth of cyanobacteria

Search for volatile and fixed compounds that are possible biomarkers of bloom

Correlate lab and environment conditions

12

Main aims of this project:

Screening volatile metabolites and implication on cellular growth

13

(mainlib) trans-1,10-Dimethyl-trans-9-decalinol

90 100 110 120 130 140 150 160 170 180 1900

50

100

97

109

112

121

125

135 139149

153 164 182

OH

Strains and environment samples

14

Monitoring of volatile compounds by GC-MS in Brazilian reservoirs

(mainlib) 2-Methylisoborneol

90 100 110 120 130 140 150 160 170 180 190 2000

50

100

93

95

107

110

117121 125

135150

168

OH


15



16


Strains and environmental samples (100 strains and 5 reservoirs)

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Geosmine

2-Methylisoborneol

b-Ionone

a-Cyclocitral

b-Cyclocitral

2-Pentylfuran

2-Ethyl-1-hexanol

2,2,6-Trimethylcyclohexanone

1,1,3-Trimethyl-2-cyclohexanone

2,6-Dimethylcyclohexanol

Tetradecane

Pentadecane

Diisobutyric acid 1-tert-butyl-2-methyl-1,3-

propanediyl ester

Hexadecane (XII)

6,9-Heptadecadiene (XIII)

8-Heptadecene

8-Methylheptadecane

3-Octadecene and moreFusije et al. Journal of Chromatography A2010. 1217(39), 6122-6125

Verify how metabolites vary in two strains of Microcystis aeruginosa (LTPNA 01 and 08)

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Circadian rhythm of two (toxic and non-toxic) of Microcystis

T 0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15

24 hs

Sampling

LTPNA 01(non-toxic)

LTPNA 08(toxic)

Microcystis aeruginosa

Alpha-cyclocitral(L)

B-cyclocitral(R)

B-ionone(L)

1 4 7 10 13 16 19 22

T i m e ( h o u r s )

-2,00E+06

4,00E+06

1,00E+07

1,60E+07

0,00E-01

6,00E+07

1,20E+08

1,80E+08

2,40E+08

3,00E+08

Dark DarkLight

B-c

yc

loc

itral

(Ab

so

lute

are

a)

Alp

ha

-cy

clo

cit

ral

an

d B

-io

no

ne

(A

bso

lute

are

a)

LTPNA 08 - Microcystis aeruginosa – toxic strain

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Alpha-cyclocitral(L)

B-cyclocitral(R)

B-ionone(L)

1 4 7 10 13 16 19 22

T i m e ( h o u r s )

0

2E6

4E6

6E6

8E6

1E7

1,2E7

1,4E7

1,6E7

1,8E7

2E7

-5E7

0

5E7

1E8

1,5E8

2E8

2,5E8

3E8

3,5E8

B-c

yc

loc

itral

(Ab

so

lute

are

a)

Alp

ha

-cy

clo

cit

ral

an

d B

-io

no

ne

(Ab

so

lute

are

a)

Dark Light Dark

20


Vieira et al. Biochemical Systematics and Ecology. 2015

LTPNA 01 - Microcystis aeruginosa – non toxic strain

Cyanobacterial strains assessed for MIB and geosmin biosynthesis

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Genetic and chemical investigation of MIB and geosminbiosynthesis in Brazilian cyanobacterial strains

Strain Species Habitat Sampling Location

CENA21 Nostoc piscinale sediment Uarini, AM

CENA67 Nostoc sp. soil Iranduba, AM

ITEP24 S. torques-reginae freshwater Recife, PE

UFV-E1 Brasilonema octagenarum phyllosphere Timóteo, MG

CENA161 Fischerella sp. spring Piracicaba, SP

CENA114 Brasilonema sennae wet iron pipe Santo André, SP

SPC777 Microcystis aeruginosa freshwater São B. Campo, SP

CENA302 Cylindrospermopsis raciborskii freshwater São B. Campo, SP

CENA595 Aliterella atlantica seawater S. Atlantic Ocean

CENA303 Cylindrospermopsis raciborskii freshwater Lajeado, RS

CENA596 Nodularia spumigena soil Rio Grande, RS

Cyanobacterial strains assessed for MIB and geosmin biosynthesis

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Figure 2. Representative chromatograms of A. MIB (8.641 min) and geosmin(10.881 min) standard solution; and cyanobacterial strains B. CENA114; C.CENA161; D. CENA21; E. CENA302; F. CENA303; G. CENA595; H. CENA596; I.CENA67; J. ITEP24; K. SPC777; L. UFV-E1. Asterisks indicate geosminidentification.

Geosmin synthetase genes (geo) were detected in strains CENA67, CENA114, CENA596 and UFV-E1. Except for UFV-E1, genes for their corresponding transcription regulators (cnb)were also detected, allowing the annotation of complete operons.

Geosmin and MIB production comparison by genetic and chemical approaches

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Strain OrderGenetic approach Chemical approach

Geo MIB Geo MIB

CENA21 Nostocales – – – –

CENA67 Nostocales + – + –





CENA595 Chroococcidiopsidales – – + –


ITEP-024 Nostocales – – – –

SPC777 Chroococcales – – + –

UFV-E1 Nostocales + – + –

Geosmin and MIB production comparison by genetic and chemical approaches

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The use of both genetic and chemical approaches might be usefulin providing reliable data on T&O compounds production, sinceknowledge of the microorganisms responsible for theirbiosynthesis is important for early warning detection andprediction of impending water quality impairment.

Dörr et al. Water Research. submitted. 2017

Treatment: dialysis membrane

• 50 µmol photons.m-2.s-1

• 12h dark 12h light with air

• Temperature (24 oC ± 2)

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Microcystis aeruginosa

(LTPNA 08 - toxic)Microcystis aeruginosa(LTPNA 03 – non-toxic)

Untargeted and targeted metabolomics to determine competition between species and metabolites prospection

Competition experiment: non-toxic x toxic cyanobacterial strains

• Sampling each 3 days – growth curve

GC-MS and LC-MS

Control: separated cultures

26

Competition between species and metabolites prospection


Growth

Control: separated cultures Treatment

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Targeted GC-MS for volatile compounds


Control: separated cultures Treatment (toxic strain)

Geosmine

2-Methylisoborneol

b-Ionone

a-Cyclocitral

b-Cyclocitral

Peaked day 9 ( ≈ 10 times)

Trace amounts after day 9

Control: no variation between

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Targeted GC-MS for volatile compounds


Treatment (toxic strain) – Presence of 2-methylisoborneol

Geranyl diphosphate 2-methyltransferase (GPPMT) and Monoterpene synthase - MIB synthase (MIBS)

Found m/z 168 by GC-MSAnd m/z 167 by LC-MS

Needed to be confirmed

Giglio et al. Environ. Sci. Technol. 2011, 45, 992–998

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Targeted LC-MS for microcistins (LR, RR, LA and YR) and untargeted compounds by LC-QTOF


Control: separated cultures Treatment

Separeted toxic strain presented: microcistins

LR and RR

Unidentified compounds in both cultures

(relevant, small differences)

Peaked day 12 ( ≈ 2 twice)

Toxic strain: slighly increased amount of microcintin-LR

Toxic strain: Production of unknown peptides!

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Oligopeptides in the toxic strain ofMicrocystis aeruginosa LTPNA 08

Oligopeptides in Brazilian cyanobacteria

Microcystis sp – strain LTPNA 08 – peptide profile

Carneiro et al. FEMS Microbiol Ecol 82 (2012) 692–702

[M + 2H]2+ = 520

R1 and R3 = ArgR2 = MeMW = 1038

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Microcystins

Carneiro et al. FEMS Microbiol Ecol 82 (2012) 692–702

R1 = LeuR2 = MeR3 = ArgMW = 994

[M + H]+ = 995

32

Microcystis sp – strain LTPNA 08 – peptide profile

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Microginins - New variants

Carneiro et al. Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp. FEMS Microbiol Ecol. 2012. 82(3): 692-702.

Microcystis sp - strain LTPNA 08 - Peptide Profile

34Carneiro et al. Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp. FEMS Microbiol Ecol. 2012. 82(3): 692-702.

Microginins - New variants

Microcystis sp - strain LTPNA 08 - Peptide Profile

Paiva et al. Toxicon, submitted 2017.

Control: Amastatin

35

Microginins - Protease Inhibition

Microginin 770

Ensaio de Inibição de Aminopeptidase M

Sphaerospermopsis torques-reginae ITEP-024 4 new variants of spumigins.

Sanz et al. Toxicon (2015): 108: 15–18.


- Methods: Structure Analysis

Mass Spec analysis HRMS, MS, MS/MS, MSnMolecular WeightMolecular Formula Fragmentation profile

NMR Analysis Uni and Bi dimensional experiments1H RMN e 13C RMN, 1H-1H COSY, 1H-1H TOCSY, 1H-13C HSQC, 1H-13C HMBC, 1H-13C HSQC e 1H-1H NOESY

Sphaerospermopsis torques-reginae ITEP-024 4 new variants of spumigins.

Fig. 1- Base Peak Chromatogram of a) the aqueous methanolic extract of S. torques-reginae and of b) the NaBH4

reduction extract.

Table 1. Retention time, experimental m/z,, molecular formula, error and sigma values for the peptides identified in the S. toqrues reginae extract

NRT

(min)[M+H]+ MFa

Error

(ppm)mσ

Tentatively

IdentificationRef

1 20.6 599.3168 C30H43N6O7 3.3 2.0 spumigin 598 In this study

2 21.5 613.3347 C31H45N6O7 -0.4 10.4 spumigin 612 In this study

3 25.9 866.5710 C42H76N9O10 -0.1 17.7 anabaenopeptide In this study

4 28.1 562.3222 C28H44N5O7 2.3 7.1 namalide 561a In this study

5 28.8 562.3 221 C28H44N5O7 2.6 3.6 namalide 561b In this study


7 29.3 882.3852 C49H52N7O9 3.5 68.0 anabaenopeptide In this study


9 30.1 576.3392 C29H46N5O7 -0.1 4.7 namalide 575 In this study






15 33.4 808.4605 C42H62N7O9 -0.2 2.0 anabaenopeptin 808 [2]







Cyanopeptide profile of the toxic cyanobacterium Sphaerospermopsis torques-reginae

mycosporine like-amino acid

MAA

Lead Compounds

Spumigin 599 and 597and Spumigin 612 and 610

Sanz et al., 2015. Toxicon,108, 15-18.

Namalide 575 and Namalide 562

Sanz, Salinas and Pinto, Journal Natural Products, accepted, 2017

Table 5. Fragmentation spectra data for namalides (1) and (2)

Product ion assignmenta

1

m/z

Error

(ppm)

2

m/z

Error

(ppm)

1Ile-CO-[Lys-2Ile-Hty] + H+576.3386 1.0 C29H45N5O7

562.3224 -0.3

1Ile-CO-Lys-Hty + H 463.2523 6.5 C23H35N4O6 463.2520 6.61Ile-CO-Lys-Hty-CO + H 435.2577 5.8 C22H35N4O5 435.2592 2.2

[Lys-2Ile-Hty] + H417.2483

6.1C22H33N4O4

-----------

-

[Lys-2Val-Hty] + H C21H31N4O4 403.2314 6.31Ile-CO-Hty - CO + H 350.1686 6.9 C17H24N3O5 350.1684 7.5

Hty-2Ile + H 291.1691 4.1 C16H23N2O3 ----------

C10H21N4O5 277.1503 1.11Ile-CO-Lys 286.1757 1.6 C13H24N3O4

Hty-2Ile - CO + H 263.1746 3.0 C15H23N2O2-----------

C14H21N2O2 249.1600 -0.8

Hty-2Val - CO + H 261.1584 5.3 C15H21N2O2 261.1596 0.71Ile-CO-Lys -CO 258.1809 1.3 C12H24N3O3

1Ile-CO-NH2 + H 173.0901 11.6 C7H13N2O3

Hty immonium ion 150.0907 4.3 C9H12NO 150.0900 8.91Ile-CO-NH2 - CO2 + H 129.0998 2.5 C6H13N2O 129.1012 8.0

[CH2PhOH+H]+ 107.0468 22.1 C7H7O 107.0465 24.3

Ile immonium ion 86.0929 41.2 C5H12N

Lys immonium ion 84.0767 48.8 C5H10N 84.0768 48.2

Ileu2 LysIleu1

Hty ε-NH Arg

Hα-NH

Hβ´-NH

Hβ-NH

Hγ-NH

Conformationalexchange


Peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest

Sanz et al. Mar. Drugs 2015, 13(6), 3892-3919

Total of 38 peptides from 3 different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected and tentatively characterized

Metabolomics is a powerful tool for volatile and fixed compounds search in cyanobacteria

Data mining and post-analyses are time consuming!!!

b-ionone and MIB (precursor) can be candidates for regulating cyanobacterial growth

Different peptides are found in Brazilian cyanobacteria species

Microginins can also be further investigated as biomarkers

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Final considerations

Acknowledgements

Dr. Felipe Dörr and Fabiane Dörr - FCF/USP

Prof. Dr. Roberto Salinas - IQ/USP

Profa. Dra. Marli Fiore - CENA/USP

Undergrad, PhD students, Post-docs adn Staff

Thank you - Obrigado!

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Prospecting volatile compounds produced by Brazilian ... · produced by Brazilian cyanobacteria with allelopathic effects ... Geosmin and MIB production comparison by genetic and