Please note that the following presentation may change slightly
before the Summer School starts, but it has been provided now for
those of you leaving home soon, and who need to print it out.
If you print out at three slides per page, you will have space adjacent
to each slide to add notes.
Looking forward to seeing you all in Split.
Best regards,
Mervyn Bibb
Mining Biodiversity for New Antibiotics
Mervyn Bibb
Department of Molecular Microbiology
John Innes Centre, Norwich
There is a very real and
urgent need for new antibiotics
New approaches are needed
Metagenomics
High-throughput culturing
A wealth of unexplored microbial diversity
Microorganisms in the Environment
Habitat
Seawater
Freshwater
Soil
Activated Sludge
Gut
Cultured (%)
0.001 - 0.1
0.25
0.3
1.0 - 15.0
1.0 – 50.0
A wealth of unexplored microbial genomes
• Less than 1% of all microorganisms have been cultured under laboratory
conditions: wealth of unexplored biochemistry and enzymology
• Many of these uncultivated microbes exist in complex, competitive communities
- potentially source of novel biologically active compounds
Hot springs
Soil
Deep ocean
tube worms
Sponges
• How can these organisms and communities be explored and potentially
exploited?
Metagenomics
What is metagenomics?
Academic
Culture-independent genomic analysis of microbial communities and their
physiology
Environmental or Community Genomics
Nature 428: 37-47 (2004)
• Produces acid mine drainage
• Worldwide environmental problem
•
•
•
•
Acidophilic biofilm
pH 0.5, 40oC, FeS2
Bacteria (Leptospirillum)
Archaea (Ferroplasma)
• Environmental genomics
• Shotgun sequence
• 2 “complete” and 3 partial
genomes of uncultivated
microbes
• Reconstruct metabolism
• Shared metabolic roles
in biofilm formation and
sustainability
• Explore community
metabolic network
• Culture and explore
uncultivated microbes
Environmental or Community Genomics
Agricultural soil
Deep-sea whale fall carcasses
Human gut
Science 312: 1355-1359 (2006)
Science 308: 554-557 (2005)
What is metagenomics?
Industrial
Culture-independent exploitation of untapped microbial biodiversity
Works well for enzyme discovery
Metagenomics for enzyme discovery
Environmental DNA
Cloned (small inserts) in E. coli
HT screen for enzyme activity
Gene/enzyme readily
characterised and manipulated
GigaMatrix™
100,000 wells in the
footprint of a 96-well plate
Nitrilases in the Public Database
Eukaryotic
nitrilases
Fungal
cyanide
hydratases
24
Bacterial
nitrilases
Nitriles
Acids
Diversa Corporation
New Nitrilase Diversity
Eukaryotic
Microbial
236
Fungal
cyanide
hydratases
Bacterial
nitrilases
Robertson et al. (2004)
AEM 70:2429/2436
Diversa Corporation
What is metagenomics?
Industrial
Culture-independent exploitation of untapped microbial biodiversity
Works well for enzyme discovery
Can it work effectively for small molecule (antibiotic) discovery?
Traditional approach to antibiotic discovery
New clinically
useful antibiotics
Accessing uncultured microbial diversity - Metagenomics
Isolate environmental DNA (e-DNA)
Insert e-DNA into convenient bacterial host
Streptomyces
coelicolor
Screen for antibiotic activity
Readily characterized and manipulated
Metagenomics can yield new natural products
Turbomycin A ☺
Violacein
Terragine A ☺
Turbomycin B☺
Indirubin
Deoxyviolacein
Fatty dienic alcohol isomers
Long chain N-acyl amino acids
☺
Can it be de done on an industrial scale and yield complex, biologically
active molecules in the numbers required to support drug discovery?
What is needed?
What do you need for an efficient metagenomic
approach to new natural products?
• “Secondary metabolites” are products of complex pathways, derived from
common precursors
• Biosynthetic genes clustered
• Biosynthetic clusters range from ca. 15 kb to >120 kb
• Cloning requires specialized fosmid and BAC/PAC vectors
• Expression of pathway genes requires appropriate host (e.g. Streptomyces sp.)
• Relaxed expression requirements
• Genetically manipulable – enhance natural product biosynthesis
• Understanding of the physiology of the organism (for precursor supply)
• Understanding of the regulatory circuitry to effect efficient expression
• High-throughput screening
• Requires expertise in natural product isolation and characterisation
The host is critical: knowledge-based improvement of
antibiotic production in S. diversaTM
Used to express large gene clusters from
a variety of actinomycetes
and pseudomads
Actinorhodin is a blue-pigmented Type II polyketide – ca. 25 kb cluster with ca.
20 genes
Directed mutations enhanced productivity 60 to 250 fold above wild type
(depending on heterologous pathway expressed)
Diversa Corporation
Screening metagenomic libraries for anti-microbial activity
E. coli conjugative
fosmid/PAC library
“Environmental” sample
Agar plug assay
Pintool
Colony
pick
Expression host
e.g. Streptomyces
diversaTM
Master plate
96 well agar plate
Liquid: solvent extracts
Screen against, e.g. E. coli, Str. pneumoniae
Sta. aureus and C. albicans
Diversa Corporation
Where should we look?
• Unbiased approach – environmental DNA
• Issues:
• High molecular weight and digestible DNA
• Representative libraries – dominant species – normalised libraries?
• Large numbers to screen
• Targeted approach
• Proven, but intractable sources of novel chemical diversity
- quasi-metagenomics (Quasi: Resembling, seeming, virtual)
Antibiotics from microbes
Origin
Number
Actinomycetes
9120
Other bacteria
1640
Fungi (moulds)
4140
Antibiotics made by Actinomycetes
Medicine
Agriculture
Application
Examples
Application
Examples
Anti-bacterial
Erythromycin
Tetracyclines
Kanamycin
Chloramphenicol
Cephamycin
Vancomycin
Livestock
rearing
Monensin
Tylosin
Virginiamycin
Anti-fungal
Canesten
Anti-parasitic
Avermectin
Anti-cancer
Doxorubicin
Fungicide
Polyoxin
Kasugamycin
Immunosuppression
FK 506
Herbicide
Basta
Where should we look?
• Unbiased approach – environmental DNA – issues:
• High molecular weight and digestible DNA
• Representative libraries – dominant species
• Large numbers to screen
• Proven, but intractable sources of novel chemical diversity – quasi-metagenomics
• Rare (difficult to culture) actinomycetes - rifamycins, erythromycin,
teichoplanin, vancomycin, gentamicin, ramoplanin, dalbavancin
• Marine actinomycetes
PAC Library Screening in Streptomyces diversaTM
Strain
Total
Clones
Mean Insert
Size (kb)
>100kb
Actinomycete (unknown)
1536
120
85%
Pseudonocardia
2304
76
45%
Saccharopolyspora
1920
95
65%
Streptosporangium
1536
90
50%
Microbispora
1536
110
75%
Saccharothrix
1536
125
90%
Amycolatopsis
1920
83
40%
Actinoplanes
2304
80
40%
Amycolatopsis
1920
125
N.D.
Saccharopolyspora
1920
95
N.D.
Actinomycete mixture
4608
115
75%
Actinomycete mixture
4608
91
70%
PAC Library Screening in Streptomyces diversaTM
Strain
Clones
screened
E. coli
(ESS) hits
S. aureus
hits
Pseudonocardia
1500
23
2
Saccharopolyspora
1500
3
0
Streptosporangium
1500
20
1
Microbispora
1500
1
N.D.
Amycolatopsis
1300
16
N.D.
E. coli hit
S. aureus hit
Novel metabolites made by “marine” actinomycetes 2003–2005
Compound
Source
Abyssomicins
Verrucosispora sp.
Aureoverticillactam
Streptomyces aureoverticillatus
Bonactin
Streptomyces sp.
Caprolactones
Streptomyces sp.
Chandrananimycins
Actinomadura sp.
Chinikomycins
Streptomyces sp.
Chloro-dihydroquinones
Novel actinomycete
Diazepinomicin (ECO-4601)
Micromonospora sp.
3,6-disubstituted indoles
Streptomyces sp.
Frigocyclinone
Streptomyces griseus
Glaciapyrroles
Streptomyces sp.
Gutingimycin
Streptomyces sp.
Helquinoline
Janibacter limosus
Himalomycins
Streptomyces sp.
IB-00208
Actinomadura sp.
Komodoquinone A
Streptomyces sp.
Lajollamycin
Streptomyces nodosus
Marinomycins
‘Marinispora’
Mechercharmycins
Thermoactinomyces sp.
MKN-349A
Nocardiopsis sp.
Salinosporamide A (NPI-0052)
Salinispora tropica
Sporolides
Salinispora tropica
Trioxacarcins
Streptomyces sp.
Activity
AB
AC
AB, AF
AC
AA, AB, AC, AF
AC
AB, AC
AB, AC, AI
AC
AB
AB
AB
AB
AB
AC
NA
AB
AB, AC
AC
Unknown
AC
Unknown
AB, AC, AM
AB antibacterial, AC anticancer, AF antifungal, AI anti-inflamatory, AM antimalarial, NA Neuritogenic activity
Lam - Current Opinion in Microbiology 9: 245-251 (2006)
Where should we look?
Marine actinomycetes
Mining marine microorganisms as a source of new antimicrobials and antifungals
Bernan VS, Greenstein M, Carter GT
Curr Med Chem – Ant-Infective Agents 3: 181-195 (2004)
Tropical terrestrial actinomycetes
Journal of Industrial Microbiology & Biotechnology (1999) 23, 178–187
Where should we look?
Back garden?
Diversity of Actinoplanes and related genera isolated from an Italian soil
Mazza P, Monciardini P, Cavaletti L, Sosio M, Donadio S
Vicuron Pharmaceuticals, via R. Lepetit 34, 21040 Gerenzano (VA), Italy.
Actinoplanes and related genera are good producers of bioactive secondary metabolites. However, many strains within these genera present similar
morphological characteristics, and this prevents an effective discrimination of replicate strains during industrial isolation and screening programs.
Using PCR-RFLP analysis of the 23S rDNA gene and of the 16S-23S intergenic spacer, we have analyzed 182 strains of Actinoplanes and related
genera obtained through a selective isolation method from a single Italian soil. Combining the 23S and IGS data, 99 unique profiles were observed,
and morphologically undistinguishable strains were discriminated. Further analyses on a restricted number of strains through 16S sequencing and
hybridization to a probe for secondary metabolism established a good correlation between strain diversity seen by PCR-RFLP and that seen by the
other methods. Overall, the data indicate the presence of a high diversity of Actinoplanes and related genera isolated from a single Italian soil.
Microbial Ecology 45: 362-372 (2003)
Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes
Busti E, Monciardini P, Cavaletti L, Bamonte R, Lazzarini A, Sosio M, Donadio S
Vicuron Pharmaceuticals, via R. Lepetit 34, 21040 Gerenzano, Italy
The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since
extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the
probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera
Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia,
have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and
type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously
reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be
detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil
(Jospeh et al., Appl Environ Microbiol 69, 7210–7215, 2003). Thus, these previously uncultured actinomycetes share several features with
Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics.
Microbiology 152: 675–683 (2006)
Where should we look?
Sources of polyketides and non-ribosomal peptides
Donadio S, Busti E, Monciardini P, Bamonte R, Mazza P, Sosio M, Cavaletti L
Vicuron Pharmaceutical, Gerenzano, Italy
Ernst Schering Research Foundation Workshop 51:19-41 (2005)
Where should we look?
• Microbial symbionts – sources of potent anti-tumour compounds (PKS-NRPS)
Uncultured Pseudomonas sp.
(beetle symbiont)
Joern Piel
Uncultured sponge symbionts
Piel et al. (2004)
PNAS 101:16222/16227
• Myxobacteria – many difficult to culture
Sensitive high-throughput screening of metagenomic
libraries for biological activity
• Use a gene reporter system to detect a transcriptional response to an antibacterial agent
Co-encapsulation of a tetracycline (Tc) producer with a Tc-responsive GFP reporter
Tc+
Tc-
• Greater sensitivity than growth inhibition assay
• Amenable to high-throughput FACS-based screening
• Screens can be directed towards particular aspects of cell physiology
(mode of action) e.g. cell wall biosynthesis – fuse specific stress-induced
promoters to GFP
Diversa Corporation
What is metagenomics?
Industrial
Culture-independent exploitation of untapped microbial biodiversity
Works well for enzyme discovery
Can it work effectively for small molecule (antibiotic) discovery?
Yes – requires technology development, concerted multi-disciplinary
effort, and financial and lengthy commitment
Metagenomics can yield new natural products
Turbomycin A ☺
Violacein
Terragine A ☺
Turbomycin B☺
Indirubin
Deoxyviolacein
Fatty dienic alcohol isomers
Long chain N-acyl amino acids
Many more to come
☺
Genome scanning – the Ecopia way
•
Focus on cryptic pathways in actinomycetes
•
Construct small and large (cosmid and BAC) insert libraries
•
Sequence one end of small inserts (up to 700 nt) – Genome Sequence Tags
– 1000 GSTs of 8.5 Mb genome – sample every 8.5 kb on average
– 20 – 200 kb cluster represented 2 – 20 times
•
Compare sequences to microbial natural product (NP) gene/enzyme database
•
Use small inserts containing NP biosynthetic gene(s) to probe large insert library
•
Sequence large insert(s)
•
Reconstruct biosynthetic gene cluster in silico and predict structure
•
Screen for growth conditions that induce expression of novel compounds
Genome scanning – the Ecopia way
•
Used to identify over 450 actinomycete natural product gene clusters :
• Enediyne anti-tumour compounds (Type 1 PKS)
• Dynemicin (Micromonospora chersina)
Calicheamicin
Dynemicin
C-1027
• Macromomycin (Streptomyces macromyceticus)
(8/50 actinomycetes not known to make enediynes contained enediyne-like gene
clusters – widely distributed across many genera – likely to encode new classes of
enediynes)
Genome scanning – the Ecopia way
•
Used to identify over 450 actinomycete natural product gene clusters :
• Anti-fungal compounds
• ECO-02301 (Streptomyces aizunensis) – novel linear Type I PKS
derived compound
• E-837 (Streptomyces aculeolatus) and E-492 & E-975 (Streptomyces
sp Eco86) – novel alkenyl furanones (Type I PKS-derived)
Genome scanning – the Ecopia way
References
Zazopoulos E, Huang K, Staffa A, Liu W, Bachmann BO, Nonaka K, Ahlert J, Thorson JS,
Shen B, Farnet CM. A genomics-guided approach for discovering and expressing cryptic
metabolic pathways. Nat Biotechnol 21: 187–190 (2003)
McAlpine JB, Bachmann BO, Piraee M, Tremblay S, Alarco AM, Zazopoulos E, Farnet CM.
Microbial genomics as a guide to drug discovery and structural elucidation: ECO-02301, a
novel antifungal agent, as an example. J Nat Prod 68: 493–496 (2005)
Banskota AH, McAlpine JB, Sørensen D, Aouidate M, Piraee M, Alarco AM, Omura S, Shiomi
K, Farnet CM, Zazopoulos E. Isolation and identification of three new 5-Alkenyl-3,3(2H)furanones from two Streptomyces species using a genomic screening approach. J Antibiot
59: 168–176 (2006)
Banskota AH, Mcalpine JB, Sørensen D, Ibrahim A, Aouidate M, Piraee M, Alarco AM, Farnet
CM, Zazopoulos E. Genomic analyses lead to novel secondary metabolites. Part 3. ECO0501, a novel antibacterial of a new class. J Antibiot 59:533-42 (2006)
There is a very real and
urgent need for new antibiotics
New approaches are needed
Metagenomics
High-throughput culturing
High throughput cultivation
• Growth of previously uncultivated microorganisms under natural conditions
• Throughput of 5-10,000 isolated strains per month
• Yields novel strains AND novel biologically active molecules
• Provides material for the metagenomic approach
High throughput cultivation
•
FACS-based technology
•
Gel micro-droplet (GMD) encapsulation
•
Screening rates of 5,000 GMDs/second
•
Enables HTP isolation and cultivation of
previously uncultured microorganisms
Screen for biological activity
Sorting of gel micro-droplets containing micro-colonies
GMD with
micro-colony
GMD with
single cell
Free bacteria
Empty micro-droplets
5,000 GMDs
/second
Model of the experimental setup
Cultivation of Sargasso Sea microbes
4 Different
Media
4 different types of media:
• Filtered sea water + rich marine medium (1/100)
• Filtered sea water + amino acids
• Filtered sea water + inorganic nutrients
• Filtered sea water
Cultivation of Sargasso Sea microbes
• Encapsulated single cells in a GMD
• Grown for 5 weeks
• 1200 GMDs with micro-colonies FACS sorted into 96 well plates (marine medium)
• 16S rRNA gene library made by PCR; 140 clones characterised by RFLP
• 50 16S rRNA genes sequenced to determine nature of micro-colonies
Cultivation of Sargasso Sea microbes
Four different types of media, 50 16S rRNA gene sequences:
• Sea water + rich marine medium (1/100)
4 species:
Vibrio
Marinobacter
Cytophaga
• Sea water + amino acids
• Sea water + inorganic nutrients
• Sea water
12 species
11 species
39 species
More extensive
analysis of
500 GMDs
16 new clades
Rich marine
medium
Zengler et al. (2002)
PNAS 99:15681/6
Filtered seawater
Mining Biodiversity for New Antibiotics
Supplementary Slides
There is a very real and
urgent need for new antibiotics
Emergence of antibiotic resistant
pathogens
Experts fear superbug
pandemic
1 April 2005
A strain of the MRSA superbug
caught in public places as opposed
to hospitals could spread faster
and wider than first thought,
experts say.
New superbug outbreak sweeps
southern England
20 July 2005
An outbreak of a superbug resistant to antibiotics
has infected more than 1,000 people and caused
dozens of deaths. The bug, which can lead to
blood poisoning, is spreading in southern England
and is more serious than Clostridium difficile,
which hit the headlines last month after a virulent
strain infected 15 hospitals.
Superbug deaths up by nearly a quarter
in year
24 February 2006
· NHS hospitals the most likely source of infection
The number of deaths related to MRSA, the socalled hospital superbug, increased by almost a
quarter, according to the latest figures. MRSA
is now six times more likely to be a factor in the
deaths of people in NHS hospitals than anywhere
else, the Office for National Statistics said yesterday.
Superbug that moves faster than
science
1 March 2005
The world may run out of effective antibiotics by
the end of this decade and faces a gap of at
least five years before new drugs can be
developed to combat superbugs, according to
one of the world's most influential scientists.
Marine killed by scratch and superbug
'Superbug' infections spiralling in
Canadian hospitals
24 May 2005
23 March 2005
A SUPERFIT Royal Marine collapsed and died
within days of scratching his leg on a bush while
on a training run — victim of a mutated
superbug one doctor described as the worst she
had ever seen.
Toronto - Hospitals are failing to control antibioticresistant "superbug" infections that kill as many as
8,000 patients each year and cost health-care
systems at least $100 million annually, a CBC News
investigation has learned.
New strains of superbug can kill
in 24 hours
Lethal superbug hits 44,000 elderly patients
20 February 2005
27 August 2005
Highly virulent strains of the superbug MRSA
which infect healthy young people with no
connection to hospitals are appearing in the UK.
The new varieties cause skin and soft tissue
infections such as boils, abscesses and
inflammation and, in rare cases so far only
seen in other countries, a severe pneumonia
that can kill in 24 hours.
Record numbers of elderly people fell victim last year to a
potentially lethal superbug which is plaguing Britain's
hospitals, according to details of the first complete survey
of the disease. Concerns about the bug, Clostridium
difficile, were first revealed in The Independent in June
following an outbreak of a lethal strain at Stoke
Mandeville Hospital. The figures yesterday showed that
there were 44,488 cases of the bug among people over 65.
Hospital and Community Based Problem
Hospital-Acquired Infections
•
•
•
•
•
Methicillin resistant Staphylococcus aureus (MRSA) – 37%
Vancomycin resistant Enterococci (VRE) – 65%
Vancomycin resistant Staphylococcus aureus (VISA and VRSA)
Cephalosporin resistant Gram-negative bacteria
Azole resistant Candida albicans
Community-Acquired Infections
• Penicillin resistant Streptococcus pneumoniae (PRSP) – 25%
• Multidrug resistant S. pneumoniae
• Multidrug resistant Salmonella, e.g.Salmonella DT104
(AmpR, CmlR, StrR, SulR, TmpR, TetR, KanR, CipR)
• Multidrug resistant Shigella
• Fluoroquinolone resistant gonococci
• Multidrug resistant Mycobacterium tuberculosis
X
Increasing incidence of antibiotic resistance
• Infectious disease mortality
• 1900 to1980: declined 20 fold
• 1980 to1995: increased two fold
• Antibiotic resistance is the major contributor
% incidence
60
50
40
MRSA
30
VRE
20
10
0
1980
1985
1990
1995
2000
Declining numbers of new antibiotics entering the clinic
• Few new antibiotics marketed in the last 40 years
• And yet large pharmaceutical companies are curtailing anti-infective research
and antibiotic discovery programmes
• Why?
Demise of antibiotic discovery in the pharmaceutical industry
• Development of a new antibiotic is expensive and risky: how long will the antibiotic
remain effective and will it be profitable?
• Competition from highly profitable drugs for chronic diseases (high blood pressure,
cholesterol lowering agents, depression etc)
• Led to reduced investment in or elimination of many antibiotic drug discovery
programs
• Discovery of new antibiotics from traditional sources (microbes) has become
increasingly difficult - have they all been found already?
• No – but new and imaginative approaches are required to find them