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Iden%fica%on and Characteriza%on of Secondary Metabolic Systems Using
Genomic and Metagenomic Approaches
David H. Sherman, Ph.D. Life Sciences Ins6tute
University of Michigan – Ann Arbor
American Society for Microbiology Distinguished Lecturer Program
Evolving Microbial Resistance
Clatworthy, A. E.., Pierson, E., Hung, D. T. Nat. Chem. Biol. 2007, 3, 541-548 CDC. Antibiotic Resistance Threats in the United States, 2013; http://http://www.cdc.gov/drugresistance/threat-report-2013/, Accessed 2/27/2013
United States Annual Statistics 23,000 Deaths 2,000,000 Illnesses $20,000,000,000 ($20B)
Why natural products? • Natural products are the basis of
many important medicines, including penicillin, aspirin, morphine and steroids
• Between 1981 and 2009, almost half of the FDA-approved cancer treatments were derived from natural products
• Recent surge in interest in natural products from pharmaceutical companies
• New technology opens new potential
NATURAL PRODUCTS as Therapeutic Agents
Youyou Tu won a Nobel prize in 2015 for the discovery of a
Artemisinin, a powerful malaria drug derived from
Artemisia annua, or sweet wormword Avermectins
Artemesinin
Daptomycin/Cubicin®
Biodiversity Initiatives for Discovery of New Natural Products
• Brazil (University of Sao Paulo) • Costa Rica (National Biodiversity Institute/UCR) • China (Shanghai Jiaotong University) • Papua New Guinea (University of PNG) • Peru (Universidad Nacional Agraria La Molina - Lima) • Israel (LSI/Weizmann Institute Partnership) • Saudi Arabia (King Abdulaziz University) • Ghana (Kwame Nkrumah University of Science &
Technology)
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Photograph: Michael Marie Schofield
Marine Invertebrates are Rich Sources of Bioac6ve Compounds
• Potent natural products are thought to serve as a chemical defense for sessile invertebrates
• Many of these compounds are thought to be produced by bacterial symbionts
• Many symbionts remain incapable of being cultured in the laboratory.
• Culture-‐independent methods are needed
Ecteinascidia turbinata and ET-‐743
• Mangrove tunicate
• Producer of chemotherapeu6c ET-‐743 (Yondelis®)
• The drug is currently produced in a lengthy semi-‐synthe6c process
• Thought to be produced by an uncul6vable bacterial symbiont
Ecteinascidia turbinata and ET-‐743
• Mangrove tunicate
• Producer of chemotherapeu6c ET-‐743 (Yondelis®)
• The drug is currently produced in a lengthy semi-‐synthe6c process
• Thought to be produced by an uncul6vable bacterial symbiont
3. Saframycin Mx1 in Myxococcus xanthus 4. Safracin in Pseudomonas fluorescens
1. ET-‐743 in E. frumentensis 2. Saframycin A in S. lavendulae
Metagenomics
Elucida8ng the origin of ET-‐743
Previous Evidence for E. frumentensis
Moss et. al. (2003) Marine Biology 143(1):99-‐110
Universal 16S probe
E. frumentensis 16S specific probe
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Metagenomic Sequencing Efforts
Tunicate
Metagenomic Sequencing Efforts
Tunicate
PacBio sequencing at UM core (1 kB library)
MDA single cell sequencing
Illumina and PacBio (10 kB) combined sequencing at JGI X X X
Metagenomic Sequencing Efforts
Tunicate
PacBio sequencing at UM core (1 kB library)
MDA single cell sequencing X X Illumina Metagenomic Sequencing at JGI
Overview of the Metagenome
15233 15306 19872 21664
Total Assembled Sequences
427549 466685 493145 465849
Total Bases 808986041 839356773 847549657 837783164
Protein Coding Genes 1588700 1658335 1683129 1648896
Largest Con8g (bp) 97417 391789 163783 171962
Smallest Con8g (bp) 200 200 200 200
SM COGS 1102 1160 1200 1160
Sequenced Tunicate Samples
From Clusters to Genomes • Do we have the complete genome?
• What could the genome tell us about this organism – Direct bacterial link to ET-‐743 produc6on
– Endosymbiosis – Cul6va6on – Host and symbiont evolu6on
ESOM puts bacterial DNA into dis6nct bins
Sunit Jain
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ESOM puts bacterial DNA into dis6nct bins
Sunit Jain
An Ideal ESOM Map
Sunit Jain
Possible Bacterial Bins
Sunit Jain
Bin 1
Bin 2 More
Bins
Sunit Jain
ET
Cyano More Bins
Sunit Jain
Genomic indicators of endosymbiosis
• Phylogeny • Genome size • GC content • Coding density • Presence of pseudogenes • Absence of essen6al genes • Presence of viral genes
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ET-‐743 Yondelis
Rath, C. M. et al. 2011. ACS Chem. Biol. 6: 1244–1256. M. Schofield et al., Environ. Microbiol. 2015. 17(10): 3964-3975
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Amino Acid Transport and MetabolismCarbohydrate Transport and MetabolismCell Cycle Control, Cell Division, andChromosome Partitioning Cell Wall, Membrane, and Envelope Biogenesis Coenzyme Transport and MetabolismEnergy Production and ConversionInorganic Ion Transport and MetabolismIntracellular Trafficking, Secretion, and Vesicular TransportLipid Transport and MetabolismNucleotide Transport and MetabolismPosttranslational Modification, Protein Turnover, and ChaperonesReplication, Recombination, and RepairSignal Transduction MechanismsTranslation, Ribosomal Structure, and Biogenesis
Outermost Circle
Candidatus Endoecteinascidia
frumentensis631,345 bp
• EMEA approval for relapsed ovarian cancer
• Phase II trials for prostate, breast and pediatric cancers
• FDA approved; soft tissue sarcoma
ectA
ectB
ectC
E. coli
E. coli
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ect
ect
Refactoring and Industrial Scale Production
M. Schofield et al., Environ. Microbiol. 2015 DOI: 10.1111/1462-2920.12908)
In collaboration with National Biodiversity Institute, Costa Rica
New Targets and Therapies for MRSA and Anthrax
Costa Rica ICBG
INBio/UM/HMS International Cooperative Biodiversity Group
Discovery of Natural Product based Drugs and Bioenergetic
Materials from Costa Rican Biota
David Sherman University of Michigan
Ann Arbor, MI
Giselle Tamayo INBio San Jose, Costa Rica
Jon Clardy Harvard Medical School Boston, MA
International Cooperative Biodiversity Groups: Guiding Principles
• Improve human health through the discovery of bioactive natural products from Costa Rica’s rich biodiversity using ecologically-driven approaches.
• Focus natural product and biosynthetic enzyme-related research on unexplored and under explored microorganisms such as marine bacteria and insect microbial endosymbionts.
• Improve the research capacity and economic opportunities for Costa Rica and contribute to its National Biodiversity Strategy
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http://www.lsi.umich.edu/labs/david-sherman-lab
Isla del Coco, Costa Rica
INBio
Technical Office of the CONAGEBIO
Areas where sampling will be done
Project approved
Technical Guide
1
PIC
2
PIC
3
4
Resolution
Guidelines of genetic resources access/permits Natural Product Drug Discovery
• Searching for new drug leads from diverse microbes • Culture previously unidentified bacteria and fungi from a variety of biodiversity resources • Microorganisms are grown in liquid culture • Extracts are fractionated and screened for bioactivity • Extracts with interesting activity are purified to isolate the bioactive metabolite
Collection Primary Sources
Secondary Isolation
Fermentation
Extraction Bioassays & high-throughput screening
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Chemical Diversity Resources and Natural Product Drug Discovery
SciF
inde
r Go
ogle
NMR: 1D, 2D
Exact Mass
Organism Producer
HPLC
HP
LC
1. Active Extract
2. Active Fractions
3. Active Compound
KNOWN COMPOUND
CHECK OTHER FRACTIONS EXACT MASS
NMR: 1D and 2D
NEW COMPOUND
COMPARE WITH SIMILAR ONES
HPLC Retention
time Absorbance
Antibiotic Discovery Program Biodefense • B. anthracis project
responsive to engineered MDR anthrax attack
• Focuses on inhibition of siderophore virulence factor (AsbA) biosynthesis
• Characterization of new antibiotics
Emerging Infectious Diseases • S. aureus staphyloferrin
virulence factor • Screening for inhibitors of
SbnE biosynthesis • Characterization of new
broad spectrum antibiotics • Also active against Gram-
negative pathogens (E. coli, Shigella, Salmonella)
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Targeting Iron Acquisition
• Iron is an essential cofactor in all organisms. • Siderophores are Fe3+-specific chelating and transport agents
secreted into the environment. • Scavenge free iron, prior to active re-uptake via specific transporters. • Essential virulence factors for pathogenicity.
Fe
Biosynthesis
Uptake
Export Fe
Chelate Fe
Dale et al. Infect. Immun. 2004, 72, 29-37 Cheung et al. Molec. Microbiol. 2009, 74, 594-608
Targeting Iron Acquisition
• Inhibitors of siderophore-dependent iron acquisition via biosynthesis targets – Potential to affect a broad spectrum of microbes that use NIS synthetases. – Deletion of siderophore biosynthesis genes shows growth defect for B. anthracis and S.
aureus in iron depleted conditions – Shows severe attenuation in a mouse model
• Inhibition of uptake of siderophore-iron complex – Active transport required for growth in iron depleted conditions
Fe
Biosynthesis
Uptake
Export Fe
Chelate Fe
Dale et al. Infect. Immun. 2004, 72, 29-37 Cheung et al. Mol. Microbiol. 2009, 74, 594-608
Why Investigate Staphylococcus aureus?
• Is a causative agent of: – Impetigo – Skin abscesses – Food poisoning – Pneumonia – Meningitis – Toxic shock syndrome
• Methicillin-resistant S. aureus (MRSA) – Superbug – S. aureus develops resistance to β-lactam
antibiotics – Prevalent in hospitals, nursing homes, schools,
and prisons – Difficult to treat
MRSA-‐associated infec8on
©CDC PUBLIC HEALTH IMAGE LIBRARY #11159
S. aureus cells
©CDC PUBLIC HEALTH IMAGE LIBRARY #14927
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Why Investigate Bacillus anthracis? • Causative agent of anthrax
– Rapidly fatal disease and serious bioterrorism threat
• Potential bioweapon
• Problems with treatment – Must treat immediately after the onset of symptoms – Risk of naturally acquired antibiotic resistance – Risk of engineered antibiotic or vaccine resistant
strains. B. anthracis Spores
©ANTHRAX VACCINE IMMUNIZATION PROGRAM/GETTY IMAGES
©WWW.VISUALPHOTOS.COM
B. anthracis cells in spleen
Siderophore Virulence Factor Biosynthesis in S. aureus and B. anthracis
Staphyloferrin B in S. aureus Petrobactin in B. anthracis
Lee, J.Y. et al., (2007) J. Bacteriol. 189:1698-1710; Pfleger, B.F. et al., (2007) Biochemistry 46:4147-4157; Liu, H. et al., (2007); J. Am. Soc. Mass Spectrom. 18:842-849; Passalacqua, K.D., et al., (2007) J. Bacteriol. 189:3996-4013; Pfleger, B.F., et al., (2008) Proc. Natl. Acad. Sci. 105(44): 17133-17138; Carlson, P.E. Jr., et al., (2010) Mol. Microbiol. 75(4): 900-909; Himpsl, S.D., et al., (2010) Mol. Microbiol. 78(1): 138-157; Lee, J.Y., et al., (2010) PLoS One 6(6): e20777; Nusca, T.D., et al. (2012) J. Biol. Chem. 287(19): 16058-16072;
O-acetyl-serine
SbnASbnB
H2N OHNH2
O
HO OH
OOH
CO2H
O
citrate
DAP
SbnE(ATP, Mg+2)
HN OH
OOH
CO2H
O
NH2
O
HO
SbnH
PLP
CO2
HN OH
OOH
CO2H
O
NH2
L-Orn
L-Pro
O-acetyl-serine
SbnASbnB
L-Orn
L-Pro
H2N OHNH2
O
SbnF(ATP, Mg+2)
HN NH
OOH
CO2H
O
NH2
OH
O
NH2
DAP(diaminopropionate)
HO OH
O
O
O
α-ketoglutarate
NH
NH
OOH
CO2H
O
OH
O
NH2
HN
OHO
O
O
Staphyloferrin B
SbnC
(ATP, Mg+2)
HO OH
OOH
CO2H
O
citrateH2N N
HNH2
spermidine
H2N NH
NH2
spermidine
O CO2H
OHHO
3,4-dehydroshikimate
AsbA
AMP + PPi
NH
NH
NHO
OHHO
AsbF,AsbCDE
O
CO2HHO
CO2HN1-[3,4-dihydroxybenzoyl]-N8-citryl-spermidine-N13-spermidine
NH
NH
NHO
OHHO
O
CO2HHO
ATPAsbB
AMP + PPi
O
H2N NH
NH
NH
NH
HN
O
OH
OH
OHO2C
OH
O
HN
NH
NH
O
HOOH
Petrobactin N1-[3,4-dihydroxybenzoyl]-N8-citryl-spermidine-N13-spermidine
AsbF,AsbCDE
Isolation of the Novel Molecules (BmcA and BmcB)
OH OH OH O
OH
HO
OH OH OH O
OH
HO
Baulamycin A (Bmc A)
Baulamycin B (Bmc B)
Tripathi, Schofield et al. J. Amer. Chem. Soc. 2014. 136(4):1579-1586
Bioactivity of Baulamycins A and B In-vitro Bioactivity
Microbial Strain Classification Targeted Siderophore Pathway
Associated NIS synthetases (Classification)
BmcA IRM IC50 (µM)
BmcA IDM IC50 (µM)
S. aureus (Newman) Gram-positive Staphyloferrin B SbnE (A), SbnC (B), SbnF (C) 85.55 69.10 MRSA (USA 300) Gram-positive Staphyloferrin B SbnE (A), SbnC (B), SbnF (C) 133.2 127.9
B. anthracis (Sterne 34F2)
Gram-positive Petrobactin AsbA (A), AsbB (C) 119.8 107.8
S. typhimurium Gram-negative Aerobactin IucA (A), IucC (C) > 1000 > 1000 E. coli (MC 1061) Gram-negative Aerobactin IucA (A), IucC (C) 145.2 3.761 S. flexneri (BS103) Gram-negative Aerobactin IucA (A), IucC (C) 45.73 20.01
AsbA
SbnE
AsbB
Bacterial Culture Bioactivity
Tripathi, Schofield et al. J. Amer. Chem. Soc. 2014. 136(4):1579-1586
Understanding Mode of Inhibition
Enzymatic Target NIS Synthetase Classification
Associated Microbial Strain
Associated Siderophore BmcA IC50 (µM)
BmcB IC50 (µM) BmcA Ki (µM)
SbnE Type A S. aureus Staphyloferrin B 4.819 18.76 129.97
AsbA Type A B. anthracis Petrobactin 276.4 505.2 140.23
AsbB Type C B. anthracis Petrobactin > 1000 > 1000 ND
SbnE
AsbA
Tripathi, Schofield et al. J. Amer. Chem. Soc. 2014. 136(4):1579-1586
Opportunities for Natural Product Drug Discovery & Development
• Mapping the Microbiome from International Biodiversity Resources
• Develop a National Microbial Library Resource based at UM
• UM Microbial Extract Library • National and Global Partnerships for Target
Validation and Screening • Partnerships for Drug Development/
Commercialization
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Broad Collection Efforts
High-throughput sequencing
Bioinformatic assembly, chemical probe synthesis, biochemical
validation
Synthetic Biology & Heterologous
expression
Extraction
High-throughput structure elucidation
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Emerging NP Discovery Model
• Explore new drug leads from diverse pure culture & unculturable microbial consortia • Assemble genomes and express biosynthetic systems from diverse biodiversity resources • High throughput screening for bioactivity against novel disease targets • Pursue priority molecules for molecular probe development and drug discovery
Acknowledgements University of Michigan • Dr. Ashu Tripathi • Michael Schofield • Center for Chemical
Genomics • Prof. Chuanwu Xi (SPH)
INBIO-UCR • Prof. Giselle Tamayo • Prof. Adrian Pinto • Gabriel Vargas Asensio
Funding • NIH U01 TW007404 • Joint Genome Institute • Life Sciences Institute • Hans W. Vahlteich Professorship • U-M College of Pharmacy LSI Communications/Development • Laura Williams • Erin Grimm • Aaron Westfall • David Doneson
Contributors/Acknowledgements • U-M Life Sciences Institute,
Medicinal Chemistry – Dr. Ashu Tripathi – Michael-Marie Schofield – Dr. Sung Ryeol Park – Dr. George Chlipala – Pam Schultz – Isaiah Yim – Dr. Jung Yup Lee – Dr. Brian Pfleger • Earth Sciences and Environment – Prof. Greg Dick – Sunit Jain
• U-M Center for Chemical Genomics – Dr. Vince Groppi – Martha Larsen – Tom McWade
• U-M Microbiol. & Immunol. – Prof. Phil Hanna – K. Passalacqua – P. Carlson
• NIH GLRCE • NIH FIC ICBG • UM Life Sciences Institute
Current Group at LSI
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