mapping microbiomes at the micron scale -...
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Mapping Microbiomes at the Micron Scale
Whitehead Institute Gary BorisyPartnership for Science Education June 05, 2017
Microbes live in communities
Microbiome—”the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease” Lederberg, 2001
Defining a Healthy MicrobiomeTaxonomy—organism centric viewpoint
Lloyd-Price et al (2016) Genome Medicine 8:51
Defining a Healthy MicrobiomeFunction—gene centric viewpoint
Lloyd-Price et al (2016) Genome Medicine 8:51
Defining a Healthy Microbiome
Lloyd-Price et al (2016) Genome Medicine 8:51
Dynamics—Energy Landscape
The Importance of Mapping Microbiomes at the Micron Scale
• Microbes live and work at the micron scale• Their neighborhoods—microbiomes—are at the micron scale• The DNA revolution has opened new research avenues• Metagenomics—culture-independent analysis• But a gap remains• Need to know “who is next to who”; and “who is next to what”
to understand how a community works• Need to know biogeography at the micron scale
• Imaging• Develop multiplexed, spectral imaging• Go beyond the limitations of band pass filters; use all the
information available in fluorescent probes• Go beyond individual fluorescent probes; use combinatorial
labeling to create fluorescent “signatures”
• Genomics• Use genomic information to design taxon-specific probes • Use metagenomics for culture-independent analysis• Use single-nucleotide resolution to guide imaging priorities
• Putting them together—Imaging Genomics• Correlative imaging and genomics on the same samples
How to Map Microbiomes
Plan of Talk• Introduction to the Oral Microbiome• The importance of single-nucleotide resolution in
microbial taxonomy• Visualizing complexity through spectral imaging • Imaging Proof-of-Principle • Biological Proof-of-Principle • Mapping oral microbiomes• Future prospects
Why to Study the Human Oral Microbiome
Kolenbrander, P.E., et al 2002. Microbiol. Mol. Biol. Rev. 66:486-505
Hypothesis for oral biofilm structure
Leuwenhoek, 1680
• First microbes seen• Accessible, portal to body• Curated database available (HOMD)• ~700 species-level entries• Many taxa cultivated (67%)• Many genomes available (58%)• Human Microbiome Project (HMP)
provides data at 9 oral sites
Oral Microbiome is a good test bed
Microbial Identification Relies on a16S rRNA Gene Barcode
Nature Structural Biology9, 750 - 755 (2002)
Single nucleotide resolution reveals diversity of oral taxa and habitats
SUB
SUP
• 16S RNA gene • V1-V3 region• 493 oligotypes—significant sequence variants• Each oral site has unique oligotype signature
• Eren et al 2014 PNAS 111: E2875-84• Mark Welch et al 2014 Frontiers Microbiol 5: 568• Utter et al 2016 Frontiers Microbiol 7: 564
SUB
SUP
Spectral Image Data Cube3-D data set
Solution is Spectral ImagingFluorescence microscope
spectral detector
Emitted light from specimen
Diffraction grating D
etector
Deconstruct emission signatures by linear unmixing
Proof-of-Principle Test of Spectral Imaging
• Use E. coli as a “test bead”• Label E. coli with 16s rRNA-specific oligonucleotide
conjugated to a single fluor• Label with a binary combination of oligo-fluor probes• n fluorophores; n(n-1)/2 unique binary combinations• Make mixture of differently labeled populations• Spectrally image• Quantify abundance of label types• Compare to input
Bacterial cellrRNA
Fluorescent Oligo Probe
5’- GCT GCC TCC CGT AGG AGT-3’
Probes hybridize against
complementary sequences of the
ribosomal RNA (rRNA)
Fluorescence in situ hybridization (FISH)
Delong, Wickham & Pace 1989. Science 243: 1360-1363
With 8 fluorophores, there exist 28 unique binary combinations
CLASI-FISHCombinatorial Labeling and
Spectral Imaging-Fluorescence in situ Hybridization
Valm et al., 2011 PNAS 108: 4152-57Valm et al., 2012 Syst Appl Microbiol 35: 496-502Valm et al., 2016 PLOS doi: 10.1371/journal.pone.0158495
8 fluorophores
Conjugated to a DNA FISH probe that labels most bacteria
28 tubes of E. coli suspended in hybridization buffer
Add one fluorophore to each tube......then a second to give 28 unique binary combinations
Allow probe to hybridize
Wash cells to remove excess probe
Add an equal volume of each label-type to a single tube
Spot this mixture on a slide and
image
Imaging Proof of Principle with E. coli
Biological Proof-of-Principle with Oral Microbes• Streptococcus
• Prevotella
• Fusobacterium
• Selenomonas
• Rothia
• Gemella
• Actinomyces
• Campylobacter
• Capnocytophaga
• Leptotrichia
• Treponema
• Veillonella
• Porphyromonas
• Pasteurellaceae
• Neisseriaceae
1. Identified 15 of the most abundant genera in the mouth
2. Designed probes for these 15 genera
3. Grew representative species in the lab
4. Validated probes individually and as a set
5. Made an artificial mixture of the 15 species in a test tube
6. Performed CLASI-FISH on the mixture
Actinomyces 476
Rothia 492
Gemella 572
Pasteurellaceae 111
Prevotella 392
Capnocytophaga 371
Veillonella 488
Streptococcus 405
Leptotrichia 568
Selenomonas 60
Porphyromonas 1160
Neisseriaceae 1030
Fusobacterium 714
Treponema 684
Campylobacter 1021
15 Oligonucleotides Targeting Oral Genera Labeled with Binary Combinations of 6 Fluorophores
Human Dental Plaque Microbiome
20 mm
2016 Feb 9;113(6):E791-800. doi: 10.1073/pnas.1522149113. Epub 2016 Jan 25.
https://www.statnews.com/2016/05/04/mouth-full-bacteria-blooming-beautiful/
LIVE on Air in France: Le Magazine de la Santé
News CoverageDiscovery Channel Daily Planethttp://review.bellmedia.ca/view/962622322
Nature Reviews Microbiology RESEARCH HIGHLIGHTS FISHing in the oral microbiota Published online 8 Feb 2016; doi:10.1038/nrmicro.2016.21
Trends in Microbiology SPOTLIGHTOral Biofilm Architecture at the Microbial ScaleFerrer, D.M & Mira, A. Published online http://dx.doi.org/10.1016/j.tim.2016.02.013
NATURE METHODS | VOL.14 NO.1 | JANUARY 2017 | 39Microbiology: The return of culture
STAT: Bacteria in your mouthBoston Globe, video, 2016
CorynebacteriumStreptococcusCapnocytophagaFusobacteriumLeptotrichiaActinomycesPasteurellaceaeNeisseriaceaePorphyromonas
“Fly-through” plaque structure in 2 mm steps
Step 1
CorynebacteriumStreptococcusCapnocytophagaFusobacterium
Capnocytophaga, Fusobacterium are located in a sub-perimeter annulus
CorynebacteriumStreptococcusCapnocytophagaFusobacteriumLeptotrichiaActinomycesHaemophilus/AggrNeisseriaPorphyromonas
Haemophilus/Aggregatibacter, Porphyromonas, Neisseria are at
perimeter
Taxa can be identified to species level
CorynebacteriummatruchotiiStreptococcus cristatus/mitusAggregatibacterPorphyromonaspasteri
8 μm
“Corn-cobs” come in several types
CorynebacteriumStreptococcusHaemophilus/AggrPorphyromonas
1.1 um
0.4 um
0.4 um
1.7 um
1.1 um
toothanoxic
CO2, lactate, acetate, H2O2
O2, saliva, sugars
Crevicular fluid
v
annulus perimeterbase
Corynebacterium Porphyromonas Fusobacterium otherStreptococcus Neisseriaceae LeptotrichiaHaemophilus/Aggr. Capnocytophaga Actinomyces
Map of Plaque Microbiome
Conclusions• Microbiome biogeography matters• Microbiome structures are like “organs”• It matters “who is next to who and to what”• Imaging strategy is generalizable • Applicable to any microbiome• The challenge now is to understand how the
microbiome “organs” work; and how they are formed and maintained.