Genomic approaches to metabolite exploitation from ... · • no or very old compounds (side...

project number 223328

Genomic approaches to metabolite exploitation from Xenorhabdus/Photorhabdus

Helge B. Bode Molecular Biotechnology

Institute of Molecular Bio Sciences Goethe University Frankfurt, Germany

[email protected]

project number 223328 2

Outline...

•  why do we need novel therapeutics for infectious diseases?

•  why should we use natural products?

•  where should we look for novel natural products?

•  where is GameXP looking for novel natural products?

•  how is GameXP looking for novel natural products?


Why do we need novel therapeutics?

•  no or very old compounds (side effects etc)

•  expensive drugs

•  increasing global resistance

•  climate change


Why should we use natural products?

•  natural products are made by an organism

•  they are complementary to synthetic compounds

•  they were quite useful in the past •  50-75% of all antibiotics and anti-cancer compounds

are either natural products or derivatives thereof



Artemisia annua (plant)

Streptomyces roseosporus (Gram-positive bacterium)

Acetylsalicylic acid (natural product derivative)



•  natural products are made for a reason (a specific target)

•  What is their natural function? •  not made for selling them in a drugstore…

•  What can we use them for? •  can we sell them in a drug store? •  can we use them to treat “related” human diseases?



Mycangimycin (Streptomyces sp.) Artemisia annua (plant)

... made by a bacterium that is carried inside a beetle to protect the food source fungus of the beetle larvae from antagonistic fungi ...

Clardy and Currie groups (2009)


Where should we look for novel natural products?

•  everywhere…

•  everywhere we have not looked yet....

•  in interactive environments....


Where is GameXP looking for novel natural products?


Photorhabdus/Xenorhabdus

•  members of the family Enterobacteriacae

•  Gram-negative

•  rod shaped

•  motile (peritrichous)

•  entomopathogenic against several insect larvae

•  associated with Heterorhabditis or Steinernema nematodes


Photorhabdus‐infectedM.sextalarvae

•  members of the family Enterobacteriacae

•  Gram-negative

•  rod shaped

•  motile (peritrichous)

•  entomopathogenic against several insect larvae

•  associated with Heterorhabditis or Steinernema nematodes

•  Photorhabdus is bioluminescent

Photorhabdus‐infectedG.melonellalarvae

Photorhabdus/Xenorhabdus


IJs emerge from insect cadaver

nematode growth and development

protection of the cadaver

IJs infect insect larvae

bacteria emerge from the nematode

bacteria kill insect larvae

LifecycleofPhotorhabdusandXenorhabdus


IJs emerge from insect cadaver

nematode growth and development

protection of the cadaver

IJs infect insect larvae

bacteria emerge from the nematode

bacteria kill insect larvae

smallmolecules

LifecycleofPhotorhabdusandXenorhabdus


The GameXP project...

nematodes

small molecules Xenorhabdus/ Photorhabdus

•  bioassays •  genome sequencing •  heterologous expression •  (manipulation) •  (derivatisation)


TheGameXPproject

≥250 strains

Other bacteria

Xenorhabdus

Photorhabdus


Straincollec;onofPhotorhabdus/Xenorhabdus

Narisara Chantratita, Sharon Peacock

Photorhabdus Xenorhabdus


Compoundisola;on

96 compounds

Other bacteria

Xenorhabdus

Photorhabdus


Compoundisola;on


10 20 Time[min]

UV(200‐600nm)

S;lbene

Anthraquinones

How do we find them...


10 20 Time[min]

UV(200‐600nm)

MS(+,100‐1100)

S;lbene

Anthraquinones

Look carefully (and use HPLC/MS)...


How to find bioactive compounds?

Organism with good bioactivity profile OR

Organism that produces several possibly bioactive compounds

5 10 15 20 25 30 Time [min]


Organism with good bioactivity profile OR

Organisms that produces several possibly bioactive compounds

5 10 15 20 25 30 Time [min]

????

The most interesting compound … might be a trace compound … … might not be detectable at all … might be masked by other compounds …

How to find bioactive compounds?


Bleed

Wash and dilute ≥3 passages (P)

End sequence

Pool and inject

Waterfield et al. (2008) PNAS, 105, 15967-15972.


Rapid virulence annotation of P. asymbiotica

nematode

amoeba

insect

insect

Waterfield et al. (2008) PNAS, 105, 15967-15972.

•  very sensitive assays (ng-µg/L compound) •  other screens are possible


control

pRVA14

GameXPeptides in E. coli

•  potent anti-cancer activity •  similar compounds have been

tested as anti-cancer agents


Genome of Photorhabdus luminescens

Duchaud et al. (2003) Nat. Biotechnol., 21, 1307.

•  6.5%isdedicatedtosecondarymetabolism

•  morethan23biosynthesisgeneclusters

–  mostlyNRPS,afewPKS,afewPKS/NRPShybridclusters

H. Goodrich-Blair


heterologous expression in E. coli

Genome of Photorhabdus luminescens


E. coli E. coli/pUindAC

Heterologous expression in E. coli

Indigoidin

Alexander Brachmann

from Erwinia: Reverchon et al., J. Bacteriol. 2002 from Streptomyces: Takahashi et al., J. Biol. Chem. 2007


Genomemininganddiversifica;on

XNC1_2300

module 1 module 2

C C A MT T C A MT T

XNC1_2299

Xenortide A

Decarboxylase


Mutasynthesis

XNC1_2300

module 1 module 2

C C A MT T C A MT T

XNC1_2299


Mutasynthesis


Outlook...

Bioactivity testing of

compound library

Industrial partners

GameXP assays

Other consortia


Summary...

•  there are still many natural products awaiting their identification

•  Xenorhabdus and Photorhabdus are a very rich source for novel natural products

•  several of these natural products show biological activity against relevant pathogens

•  chemical diversity can be increased by heterologous expression and/or mutasynthesis

•  GameXP is looking for partners for testing these compounds


Thanks to…

Sharon Peacock (Mahidol University, Thailand; Cambridge University, UK) Narisara Chantratita (Mahidol University, Thailand) Vera Siegmund (EURICE, Germany) Long Phan Ke (Vietnamese Academy of Science and Technology, Vietnam) Nick Waterfield (University of Bath, UK) Richard ffrench-Constant (University of Exeter, UK)

and all GameXP consortium members

Genomic approaches to metabolite exploitation from ... · • no or very old compounds (side...

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