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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

26

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)

39

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