demys&fying medicine 29 march 2016...dobell, c. antony van leeuwenhoek and his “little...
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Demys&fyingMedicine29March2016
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TheHumanMicrobiomeProjectca.1690
Antony van Leeuwenhoek Three important points 1. The first direct observation of
bacteria was of those from within the human mouth.
2. No association between microbes and disease
3. This first observation revealed human-associated microbial communities to be complex
"...[T]heres<ckethorgrowethbetweensomeofmyfront[teeth]andmygrinders...aliDlewhitemaDer,whichisasthickasif'twerebaDer.Onexaminingthis...Imostalwayssaw,withgreatwonder,thatinthesaidmaDerthereweremanyveryliDlelivinganimalcules,verypreLlya-moving”
Dobell, C. Antony Van Leeuwenhoek and His “Little Animalcules.” London. Constable & Co. 1932. Print.
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TheHumanMicrobiomeProjectca.2016
HumanMicrobiome
HumanGenome
100xthehumangenome
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TheHumanMicrobiomeProject2013
NetworkInferenceCo-occurrenceorco-exclusionobserva<onsminedtoiden<tysta<s<callysignificantrela<onships.TaxaclusterbasedonbodysiteIsthisabiologicallyrelevantspa<alscales?
Faust, et al. 2012. PLoS Comput. Biol. 8:e1002606
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Spa<aldistribu<onofmicrobes:relevantscales
MowatandAgace.2014.Nat.Rev.Immunol.14:667-685. Grice and Segre. 2011. Nat. Rev. Microbiol. 9:244-253
Gutmicrobiota Skinmicrobiota
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Ahypothesisforthestructureofdentalplaquebiofilms
Kolenbrander,etal.2010NatRevMicrobiolJabra-Rizk,etal.1999JClinMicrobiol
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MicrobialFluorescenceinsituHybridiza<on
Nature Reviews | Microbiology
Sample
Fixed cells arepermeabilized
Fixation
Ribosome
Fluorescently labelled oligonucleotides (probes)
Hybridized cells
Quantification
Fluorescencemicroscopy
Probe
Target (ribosomal RNA)
Hybridization
Washing
Amann, R. & B. Fuchs. 2008 Nat. Rev. Microbiol. 6:339-348
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Spa<aldistribu<onofmicrobes:relevantscales
MowatandAgace.2014.Nat.Rev.Immunol.14:667-685. Grice and Segre. 2011. Nat. Rev. Microbiol. 9:244-253
Gutmicrobiota Skinmicrobiota
![Page 9: Demys&fying Medicine 29 March 2016...Dobell, C. Antony Van Leeuwenhoek and His “Little Animalcules. ” London. Constable & Co. 1932. Print. The Human Microbiome Project ca. 2016](https://reader033.vdocument.in/reader033/viewer/2022060217/5f064cf77e708231d4174db5/html5/thumbnails/9.jpg)
Spa<aldistribu<onofmicrobes:relevantscales
Gutmicrobiota Skinmicrobiota
hDp://irp.nih.gov/our-research/research-in-ac<on/the-microbiome-when-good-bugs-go-bad
Hairfollicle
bacteria
fungi
Propidiumiodide(Eukaryo<cDNA)Propidiumiodide(BacterialDNA)Calcafluorwhite(fungalcellwall)Unknownmatrixmaterial
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FluorescenceImaging:Highspecificity;Lowmul<plicity
Emission Spectra
0
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475 525 575 625 675 725
Wavelength (nm)
Bodipy-Fl
Rhodamine Red-X
green filter
FISHontwomixturesofE.colilabeledwithtwodifferentfluorophores
red filter
Emission Spectra
0�
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475� 525� 575� 625� 675� 725�
Wavelength (nm) �
AF-555�RhodamineRed-X �
red filter
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Sequencingvs.Imaging
High LowHigh LowLow HighLow High
Breadth
Independenceofpriorknowledge
DynamicRange/Detec<onLimit
Abilitytoprovidespa<alinfo.
SequencingAssay ImagingAssay
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With8fluorophores,thereexist28uniquebinarycombina<ons
CLASI-FISHCombinatorialLabelingand
SpectralImaging-Fluorescenceinsitu
Hybridiza<on
Ribosome
Microbe
High mag. detail
Field of labeled
microbes
Red fluorophore
Green fluorophore
Blue fluorophore
Thepowerofcombina<ons Eachbacteriumislabeledwithexactly2fluors
CombinatorialLabeling
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498 548 598 648 698
Inte
nsity
(A.U
.)
Wavelength (nm)
Thisallowsfluorophoreswithhighlyoverlappingemissionspectratobedis<nguished—eveniftheyarepresentin
thesamepixelintheimage.
observed pixel spectrum
375 425 475 525 575 625 675 725 775Wavelength (nm)
known fluorophore spectra
FindBestFit
LinearUnmixing
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Observedpixelspectrum
Knownfluorophorespectra
Abundanceofeachfluorophoreatthatpixel
Noise
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ImagingproofofprinciplewithE.coli
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Establishing Biological Proof of Principle MIxtureofcellsof15laboratorygrownoraltaxa
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Alexa fluor 488Alexa fluor 514Alexa fluor 555
Rhodamine Red XAlexa fluor 594Alexa fluor 647
Structural Analysis of a Natural Community: Human Dental Plaque
Imageofafieldofviewofsemi-dispersedhuman
dentalplaque
Selenomonas
Veillonella
Fusobacterium PorphyromonasPasteurellaceae CapnocytophagaNeisseriaceae Streptococcus
Prevotella
Gemella
Treponema Actinomyces
CampylobacterRothia
Leptotrichiaunknown
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Raw spectral image merge
Taxon-assigned segmented image
Rhodamine Red X
Sele
Veil
Fuso PorpPast CapnNeis Strep
Prev
Geme
Trep Acti
CampRoth
Leptunkn
Raw spectral image merge
Taxon-assigned segmented image
Rhodamine Red X
Sele
Veil
Fuso PorpPast CapnNeis Strep
Prev
Geme
Trep Acti
CampRoth
Leptunkn
Structural Analysis of a Natural Community: Human Dental Plaque
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Structural Analysis of a Natural Community: Human Dental Plaque
Model images of randomly placed cells
Selenomonas
Veillonella
Fusobacterium PorphyromonasPasteurellaceae CapnocytophagaNeisseriaceae Streptococcus
Prevotella
Gemella
Treponema Actinomyces
CampylobacterRothia
Leptotrichiaunknown
Observed image of plaque
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Structural Analysis of a Natural Community: Human Dental Plaque
Streptococcus
Prevotella
Porphyromonas
Actinomyces
Fusobacterium
Rothia
Neisseriaceae
Gemella
Veillonella
Pasteurellaceae
Capnocytophaga
Campylobacter
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Biogeographyofthehumanmicrobiomeatthemicronscale
MarkWelch,etal.2015.PNAS
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Hedgehogstructureindentalplaque
MarkWelch,etal.2015.PNAS
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Corncobstructuresattheborderofhedgehogstructures
MarkWelch,etal.2015.PNAS
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Modelhypothesisofhedgehogstructure
tooth$anoxic$
CO2,$lactate,$acetate,$H2O2$
O2,$saliva,$sugars$
Crevicular$fluid$
v$
annulus$ perimeter$base$
Corynebacterium . ....Porphyromonas . ...Fusobacterium . .other$Streptococcus . . ....Neisseriaceae . ...Leptotrichia.Haemophilus/Aggr.. ....Capnocytophaga . ...Ac=nomyces.
MarkWelch,etal.2015.PNAS
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Hedgehogstructuresasanecosystem
Gene<callydis<nctorganisms
occupyingnichesTrophicinterac<onsEnvironmentalinfluenceRoleofcell-to-cellcontact
MarkWelch,etal.2015.PNAS
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Frameworkfororalmicrobiomefunc<onalstudy
KiDelmann,etal.2013.PLoSOne8:e47879
Ribosome
Microbe
High mag. detail
Field of labeled
microbes
Red fluorophore
Green fluorophore
Blue fluorophore
Microbialmetagenomics
SystemsImaging
Improvedmodelsof
microbialco-occurrence
Testablehypothesesregardingecosystemstructureandfunc<on
Duran-Pinedo,etal.2015.ISMEJ
Metatranscriptomics
daSilva,DorresteinandQuinn.2015PNAS
MetabolomicsKolenbrander,etal.2010.Nat.Rev.Microbiol
Invitrobiofioms
ExploratorySurveys LaboratoryExperiments
Steenackers,etal.2016.FEMSMicrobiolRev.
Computa<onalmodeling
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Acknowledgements• NICHD
– JenniferLippincoDSchwartzLab
• NHGRI– JulieSegreLab
• ForsythIns<tute– GaryBorisyLab
• MarineBiologicalLab– RudolfOldenbourg– JessicaMarkWelch