Lichen symbioses as a bioresource for natural products

Martin Grube1

1Karl-Franzens-University, Graz, Austria

in collaboration within collaboration with

Joel Boustie2, Delphine Parrot3, Sophie Tomasi2

2Université de Rennes 1, France3INRIA, Lyon, France

King George Island, Antarctica

More than 10% of terrestrial surface are dominated by lichensof terrestrial surface are dominated by lichens

What are lichens?

Not all fungal–algal combinations will form lichens !

Specific fungal–algal combinations for mutual support !

lichens:up to 86°S latitudeup to 7400 m altitudephotosynthesis at < 0°C

c. 2300 m alt.

Bacteria develop biofilm-like structures on lichensBacteria develop biofilm like structures on lichens

Cl d i b l L l tU bili i li d iCladonia arbuscula Lecanora polytropaUmbilicaria cylindrica

Grube et al. (2009) ISME J

Erlacher et al. Front Microbiol 2015

Bacteria on lower side seem to form different communities

Image: Ines Aschenbrenner

Reproduction modes of lichens

sexual asexual

Mitotic propagules (soredia)Apothecia (meiotic Ascospores)

Aschenbrenner et al. 2014 Environ Microbiol

Bacterial colonization on vegetative propagules

Volume rendering

Green: Algae

Microbial Cargo: Do bacteria on symbiotic propagules reinforce the microbiome of lichens? (2014) Ines Aline Aschenbrenner1,2, Massimiliano Cardinale1,2, Gabriele Berg1 and Martin Grube2

DOI: 10.1111/1462-2920.12658

3D re-construction

Yellow: Alphaproteobacteria

Red: other eubacteria

Putative new bacterial lineages are present in lichens

Bacteria have a high survival rate in storedmaterial (Cernava et al accepted) !material (Cernava et al. accepted) !

Frondihabitans cladoniiphilum sp novFrondihabitans cladoniiphilum sp. nov., new in ActinobacteriaCells are non-motile, non-spore-forming, aerobic, irregular, short rods (1.2–2.0 μm) that stain Gram-positive Growth occurs at temperature between 1 andpositive. Growth occurs at temperature between 1 and 30°C (optimum temperature, 22−26 °C) and at pH 3–8 (optimum, pH 3.7−5.4). Catalase-positive and oxidase-negative. The type B2β peptidoglycan contains the amino acids ornithine, alanine, glycine, homoserine and glutamic acid in an approximate molar ratio of 1.0 : 0.5 :

Selbmann et al. (2009) Polar BiolCardinale et al. (2011) IJSEM

1.0 : 0.5 : 1.0, respectively. Ornithine is the diagnostic diamino acid. Hydroxyglutamic etc.etc.

From diversity to function: a metagenomic approach

• Metagenomic DNA was extracted for Illumina gnext generation sequencing

DNADNA Extraction

170 mio paired-end reads were obtained pwith an average read length of 100 bp

Analysis and public repository

Tera-BLASTTM (blastN and blastX)

15Metagenomic studies – functional profile

Distribution of particular functions in the Lobaria metagenome and the most abundant taxa therein. Grube et al. (2015) ISME J

16 Metaproteomes – Taxonomic (A) and functional (B) assignment

Grube et al. (2015) ISME J(Voronoi treemaps)

From diversity to function: environmental proteomics

Schneider et al. (2011) Proteomics

A refined model for the lichen symbiosis

Vitamins, Cofactors,growth-promoting hormones Provision of carbohydratesgrowth promoting hormonesDegradation and recycling,Resistance against stress factorsNitrogen fixation,

Provision of carbohydratesProtection from stress

System regulationg ,

phosphate solubilization CO2 - fixation

Lichen secondary metabolitesLichen secondary metabolites

• Compounds from lichen fungi: 854 glichen substances (Elix 2016)

• Compounds from photobionts

C f• Compounds from associated fungi

C d f• Compounds from associated bacteria

Typical compounds from acetate polymalonate pathwayTypical compounds from acetate-polymalonate pathway

orcinols and orsellinic acid are often the building blocks for more complex compounds

Boustie & Grube 2005 Pl Gen Res

optional reducing domains

KS KRMT ACPERDH TEAT ACPPTSAT

Extension ProcessingLoading

type I PKS generalstructure

optional reducing domains

• Evolution of type I polyketide synthases in fungi includes mutation, domain acquisition,fungi includes mutation, domain acquisition, gene duplication, and horizontal transfer frombacteria

• Typically, many more genes are encoded inTypically, many more genes are encoded in fungal genomes than compounds expressed

Kroken et al. 2003 PNAS

Overview of the biosynthesis of dibenzofuran metabolites

Millot et al. 2016 Nat Prod Rev

Usnic acid

OH OO AntioxidantAntiinflamatory O

H3C

HO O

CH3H3C

Antioxidant

Gastroprotective

Cardioprotective

Antiinflamatory

Immunoestimulatory

OCH3

Cardioprotective

Cytoprotective

Antimicrobial

AnticarcinogenicPro-oxidant

• Antimicrobial effects of usnic acid, especially against gram positive bacteria, are widely reported in literature while mechanisms of action remain poorly studiedp y

• the medicinal use of usnic acid remains limited due to its hepatotoxicity

Millot et al. 2016 Nat Prod Rev

Usnic acid actsagainstbiofilm formation ofbiofilm formation ofStaphylocooccusaureus

Confocal images showing S. aureus biofilm on the surfaces of the control polyurethane (A) and (� )-usnicacid-loaded polyurethane (B) disks 3 days postinoculation. The grey arrow indicates a cluster of cocci, and the white arrow indicates the biofilm slime matrix. Bar, 55 � m. S. aureus adhered to the usnic acid-loaded polyurethane disk after 30 min (C) and 24 h (D) stained with the Live/Dead BacLight viability kit. Bar,

Francolini et al. 2004 Appl Env Microbiol

125 � m. The predominance of red cells after 24 h indicated a progressive loss of viability.

Xanthone dimers from lichens

Effective against Staphylococcus aureus and Bacillus subtilis

Wezeman et al. (2015) Nat Prod Rev

Eumitrins A1 and A2 could be used as inhibitors for nitric oxide formation by macrophages and thus be useful for a wide range of illnesses

Lichen compounds: effects on bacteria

Protolichesterinsäure

Active against:Interferes with the QS based communication systems ofgEscherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Proteusvulgaris, Streptococcus faecalis, Bacillus cereus, Bacillussubtilis, Pseudomonas aeruginosa, Listeria monocytogenes, Helicobacter pylori,

Interferes with the QS-based communication systems of Pseudomonas aeruginosa and Burkholderia cenocepacia and represses protease production in both organisms without inhibiting growthMycobacterium aurum, M. tuberculosis

Chromobacter violaceum J26 assay

Vulpinic acidEffects on bacterial gene expression g p

Alterations in secondary metabolism of aposymbiotically grown mycobionts of

voucher specimen aposymbiotically grown mycobionts of

Xanthoria elegans and cultured resynthesis stages

l dcultured mycobiont

resynthesis

Brunauer et al 2007 Pl Phys Biochem

Unusual compounds in lichens

Boustie & Grube 2005 Pl Gen Res

Mycosporines as light protectantsMycosporines as light-protectants

Roullier et al. 2011 Phytochemistry

Nosperin expression in natural thalli of the lichen Peltigera membranacea: the first cyanobacterial trans-AT polyketide biosynthetic pathway in the Nostocy p y y p ysymbiont discovered by metagenomics

Kampa et al. 2013 PNAS

Bioactive compounds of lichen-associated bacteria: an untapped treasure chest

Bioactive compounds of lichen-associated bacteria: an untapped treasure chest

So far only few actinobacterial isolates were analysed for their biosynthetic potential

uncialamycin dinemicin angucylin butenolide JBIR98

cladoniiamides A-Gcladoniiamides A-G

Parrot et al. 2016, Suzuki et al. 2016Coumabiocins A-F, novobiocin, isonovobiocin

Bacterial bioconversion of lichen metabolites

erythtrennes #67-314 RT: 2,24-9,60 AV: 133 SB: 80 26,29-31,07 NL: 5,11E6T: c ESI Full ms [ 50 00 2000 00]

T: - c ESI Full ms [ 50,00-2000,00]167,15T: - c ESI Full ms [ 50,00-2000,00]

3000000

4000000

5000000

ty

271,08

167,15 60000

sity

,

intermediated 0 d + 15

1000000

2000000

3000000

Inte

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421,11149,14 306,98123,15 542,96357,08 422,18175,03 596,06 693 03

20000

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123,12 168,13 359,10312 93 496 21100 200 300 400 500 600 700

0175,03 596,06 693,03

100 200 300 400 500 6000

,312,93 496,21382,60 6107,13 517,63

Esterase Esterase

Orsellinic acid/

MontagnetolErythrin/ [ ] m/z [M-H]- 167m/z [M-H]- 271m/z [M-H]- 421

Parrot et al. ms.

Compounds of lichen-associated bacteria: Marine lichens as a potential source

cyaneodimycin

Streptomyces cyaneofuscatus

usnic acidPhenoxazine Diketopiperazines

cyaneodimycin

Parrot et al. EurJOC accepted

37 Imaging chemistry – LDI-MSg g y

Le Pogam et al. 2015 Anal Chem

38 Lichen symbiosis – chemical variationLichen symbiosis – chemical variation

inherited variation, abiotic and biotic induction of variation...

Islandoquinone

Coculturing will be a fruitful approach in the future to detect the influenceof symbiotic partners

39 Coculturing of fungal, algal and bacterial partners

Muggia et al. submitted

40

Conclusions

Lichens harbor a highly diverse well structured bacterial Lichens harbor a highly diverse, well-structured bacterial microbiome – the 3rd component of the symbiosis

Lichen fungi photobionts associated fungi and bacteriaLichen fungi, photobionts, associated fungi and bacteria produce secondary metabolites of considerable interest

Metagenomic approaches reveal biosynthetic pathways inMetagenomic approaches reveal biosynthetic pathways in various members of the lichen symbioses

Experimental work requires coculturing of symbiotic partnersp q g y pto assess and utilize the extended biosynthetic potential

Acknowledgments

Austrian lichen team:

Gabriele Berg (TU Graz)Tomislav Cernava (TU Graz) ( )Ines Aschenbrenner (KFU Graz)Lucia Muggia (KFU Graz)

French lichen team:

Marcelino Suzuki (CNRS Banyuls)Marylène Chollet-Krugler (Univ. Rennes)Pierre Le Pogam (Univ. Rennes)

42 ConclusionsConclusions Lichens harbor a highly diverse well Lichens harbor a highly diverse, well-

structured bacterial microbiome – the 3rd t f th bi i ?component of the symbiosis?

Specific taxa are well-adapted and Welcome to Graz!p ppredominantly present on various lichens.

The lichen microbiome fulfills essential

Welcome to Graz!

The lichen microbiome fulfills essential functions within the symbiosis.

A combination of –omics and conventional techniques was useful to obtain a holistictechniques was useful to obtain a holistic view on microbial contributions.