Mapping Microbiomes at the Micron Scale

Transcription

1 Mapping Microbiomes at the Micron Scale Whitehead Institute Gary Borisy Partnership for Science Education June 05, 2017

2 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

3 Defining a Healthy Microbiome Taxonomy organism centric viewpoint Lloyd-Price et al (2016) Genome Medicine 8:51

4 Defining a Healthy Microbiome Function gene centric viewpoint Lloyd-Price et al (2016) Genome Medicine 8:51

5 Defining a Healthy Microbiome Dynamics Energy Landscape Lloyd-Price et al (2016) Genome Medicine 8:51

6 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

7 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 How to Map Microbiomes 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

8 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

9 Why to Study the Human Oral Microbiome Oral Microbiome is a good test bed 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 Kolenbrander, P.E., et al Microbiol. Mol. Biol. Rev. 66:

10 Microbial Identification Relies on a 16S rrna Gene Barcode Nature Structural Biology 9, (2002)

11 Single nucleotide resolution reveals diversity of oral taxa and habitats SUB SUP 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: E Mark Welch et al 2014 Frontiers Microbiol 5: 568 Utter et al 2016 Frontiers Microbiol 7: 564

12 Spectral Imaging Began in Physics

13 Problem of Standard Fluorescence Microscopy with Band-Pass Filters Spectral bleed-through

14 Solution is Spectral Imaging Fluorescence microscope spectral detector Diffraction grating Detector Emitted light from specimen Spectral Image Data Cube 3-D data set Deconstruct emission signatures by linear unmixing

15 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

16 Fluorescence in situ hybridization (FISH) Fluorescent Oligo Probe 5 - GCT GCC TCC CGT AGG AGT-3 Probes hybridize against complementary sequences of the ribosomal RNA (rrna) rrna Bacterial cell Delong, Wickham & Pace Science 243:

17 CLASI-FISH Combinatorial Labeling and Spectral Imaging- Fluorescence in situ Hybridization With 8 fluorophores, there exist 28 unique binary combinations Valm et al., 2011 PNAS 108: Valm et al., 2012 Syst Appl Microbiol 35: Valm et al., 2016 PLOS doi: /journal.pone

18 Imaging Proof of Principle with E. coli 8 fluorophores Conjugated to a DNA FISH probe that labels most bacteria 28 tubes of E. coli suspended in...then Add one a second fluorophore to give to 28 each unique tube... binary Allow hybridization probe to hybridize buffer combinations Wash cells to remove excess probe Spot this mixture on a slide and Add an equal volume of each label-type image to a single tube

19 Label Type Assignment and Quantification spectral label types merge

20 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

21 15 Oligonucleotides Targeting Oral Genera Labeled with Binary Combinations of 6 Fluorophores Gemella 572 Pasteurellaceae 111 Prevotella 392 Capnocytophaga 371 Actinomyces 476 Veillonella 488 Streptococcus 405 Leptotrichia 568 Fusobacterium 714 Selenomonas 60 Porphyromonas 1160 Treponema 684 Neisseriaceae 1030 Rothia 492 Campylobacter 1021

22 15 oral taxa visualized simultaneously

23 Mapping Microbiomes Dental plaque Tongue dorsum Buccal mucosa Hard palate Saliva Mouse gut

24 Human Dental Plaque Microbiome 20 mm 2016 Feb 9;113(6):E doi: /pnas Epub 2016 Jan 25.

25 News Coverage Discovery Channel Daily Planet LIVE on Air in France: Le Magazine de la Santé STAT: Bacteria in your mouth Boston Globe, video, 2016 NATURE METHODS VOL.14 NO.1 JANUARY Microbiology: The return of culture Nature Reviews Microbiology RESEARCH HIGHLIGHTS FISHing in the oral microbiota Published online 8 Feb 2016; doi: /nrmicro Trends in Microbiology SPOTLIGHT Oral Biofilm Architecture at the Microbial Scale Ferrer, D.M & Mira, A. Published online

26 Fly-through plaque structure in 2 mm steps Corynebacterium Streptococcus Capnocytophaga Fusobacterium Leptotrichia Actinomyces Pasteurellaceae Neisseriaceae Porphyromonas Step 1

27 Step 2

28 Step 3

29 Step 4

30 Step 5

31 Corynebacterium Streptococcus Streptococcus perimeter surrounds filamentous Corynebacterium core

32 Corynebacterium Streptococcus Capnocytophaga Fusobacterium Capnocytophaga, Fusobacterium are located in a sub-perimeter annulus

33 Corynebacterium Streptococcus Capnocytophaga Fusobacterium Leptotrichia Leptotrichia is internal

34 Corynebacterium Streptococcus Capnocytophaga Fusobacterium Leptotrichia Actinomyces Haemophilus/Aggr Neisseria Porphyromonas Haemophilus/Aggregatibacter, Porphyromonas, Neisseria are at perimeter

35 Taxa can be identified to species level Corynebacterium matruchotii Streptococcus cristatus/mitus Aggregatibacter Porphyromonas pasteri

36 Corn-cobs come in several types Corynebacterium Streptococcus Haemophilus/Aggr Porphyromonas 0.4 um 1.1 um 0.4 um 8 μm 1.1 um 1.7 um

37 Map of Plaque Microbiome O 2, saliva, sugars CO 2, lactate, acetate, H 2 O 2 tooth v anoxic base annulus perimeter Crevicular fluid Corynebacterium Porphyromonas Fusobacterium other Streptococcus Neisseriaceae Leptotrichia Haemophilus/Aggr. Capnocytophaga Actinomyces

38 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.

39 Microbiome Team G. borisy M. eren A. kempchinsky J. schuhmann J. mark welch F. dewhirst S. wilbert D. utter