The Oral Microbiome Meets Cell Biology and Periodontal Disease

Niki Moutsopoulos, DDS, PhD NIDCR/NIH We are “made up” of bacteria

•>10x more bacteria than human cells Microbial • Colonization begins at birth Cells Human cells • Adult-like complexity is attained by 1 year of age

If humans are thought of as a composite of microbial and human cells, and the human genetic landscape as an aggregate of the genes in the human genome and microbiome, creating a “super-organism” The oral cavity is a major ecological niche in the human microbiome

Human Microbial Niches Blood/Eye <1%

Nasal Nasal 14% Oral 26 % Oral

Skin 21% Gastrointestinal

Skin

Urogenital GI tract 29% Urogenital 9 %

Gastrointestinal

Urogenital Oral

PC2 (4.4%) Skin

Nasal

PC1 (13%)

The Human Microbiome Project Consortium* Unique ecological niches within the oral cavity

Abundant phyla

Firmicutes Actinobacteria Bacteroidetes Proteobacteria Fusobacteria

Anterior Buccal Supra- Tongue Stool Nares Mucosa gingival Dorsum Plaque

Abundant species Corynebacterium accolens Corynebacterium kroppenstedtii Prevotella copri Lactobacillus jensenii Prevotella amnii Lactobacillus gasseri Lactobacillus iners Streptococcus mitis Propionibacterium acnes Lactobacillus crispatus

The Human Microbiome Project Consortium* Role of Microbiome in Health

• Prevent invasion of pathogens

• Shape the immune response of the host

• Provide nutrients for the host

What is the role of the oral microbiome in human health and disease ?

We know: • Distinct and diverse microbial communities in health

• Shifts in microbial composition with disease

We don’t know: • What factors influence the development of the oral microbiome?

• How do shifts in microbiome occur with disease?

• How does the oral microbiome participate in shaping oral health/disease ? Factors that affect the Human Microbiome

Lifestyle Host genotype

Core Human Medication Immune system Microbiome

Environment Health/Disease

Disease

Host

?

Disease Periodontitis is one of the most common human diseases

Dysbiotic Microbial Community

Severe Tissue inflammation

Resorbed bone

Adapted from Hajishengallis, Nature Reviews in Immunology, 2015

CDC report, 2012 Eke et al., J Perio 2012 Periodontitis; Loss of Tooth Supporting Structures

Health Disease Radiographic evidence

Mild Severe Mild Moderate Severe Periodontitis; a microbiome triggered inflammatory disease

Periodontal Biofilm Excessive Inflammation Bone Resorption

Osteoclast Dysbiotic microbiome in Periodontitis

Abusleme, 2012 ISME Dysbiotic microbiome in Periodontitis

Microbial Clusters of Periodontitis Global examination of Periodontal Microbiome

Socransky and Hafajee

• Increased Bacterial Burden • Increased Species detected/Richness/Diversity • Overrepresentation of Periodontitis-Associated Microbes

Periodontal microbiome- trigger for systemic disease ?

Atheromatic plaques • Periodontal microbes in atheromatic plaques

Placenta/Cord Blood

Systemic Translocation • Placental Microbiome ≈ Oral Microbiome Periodontal • Preterm birth > antibodies to periodontal microbes Biofilm RA Joint

• Periodontal microbes in synovia fluid • High titers of antibodies to Pg in RA • Pg- linked to ACP

Swallowing GI track • Provotella Copri

• Fusobacterium Nucleatum- Colon Cancer

Adapted from Hajishengallis, Nature Reviews in Immunology, 2015 Microbiome in Periodontitis; trigger or consequence

We know that the microbiome is a disease trigger: - Standard of care: Mechanical removal of biofilm to arrest disease - Effect of antibiotics on periodontitis

We don’t know: - Is it the initial trigger? Or does it develop in a favorable environment ? - How is one susceptible to an exaggerated response to microbial triggers? - Is a particular host susceptible to colonization with an altered microbiome?

Microbiome ?

Host

How does the microbiome become dysbiotic in periodontitis?

Lifestyle Host genotype ?

Core Human Medication Health/Disease Microbiome

Environment Immune system Monogenic defects; A window to human immunity

Moutsopoulos et al., JDR 2015

Degranulation

NETs c

Phagocytosis

Neutrophil Control of Infection of Control Neutrophil

Leukocyte adhesion deficiency (LAD -I); A defect of neutrophil transmigration

Selectins • Rare autosomal recessive disease.

Integrins • Caused by mutations on CD18 in leukocytes.

• Defective neutrophil transmigration.

• Clinical characteristics; - Frequent life-threatening infections Rolling Capture Adhesion Transmigration - Skin infections - Periodontitis - Recurrent Oral Ulcers - Colitis (later in life) 13 year old female with LAD Microbial Colonization/Burden in LAD-I patients

Gram stain/Bacterial Detection H&E stain/Histology

10x

63x 10x 2x

Moutsopoulos et al. 2014, Sci. Transl. Med The LAD oral microbiome is distinct

PC2 (17.5%)

Bacterial Burden Species Detected

Moutsopoulos PLOS Pathogens, 2015 Health LAD Microbiome contribution in triggering immunopathology

Gram Stain LPS Stain

Moutsopoulos PLOS Pathogens, 2015 Immunostimulatory potential of LAD- microbiome

APC • Increased inflammatory response with LAD microbiome

• IL-23/IL17 signature

Moutsopoulos PLOS Pathogens, 2015 IL-17 dominated signature in LAD periodontitis

Perio perio LAD Gingivitis Severe

Signature of a heightened IL23/IL17 response

IL-17 staining

Moutsopoulos et al. 2014, Sci. Transl. Med IL-17 in barrier immunity and inflammation

IL-17 RANKL Epithelial Surveillance- Barrier Integrity MΦ Activated Osteoclast IL-17

IL1-β IL-6 Neutrophil Recruitment/ TNF-α Granulopoiesis

Fibroblasts

Bone Matrix Metalloproteinases Destruction (MMPs)

Our Understanding of LAD-periodontitis

Host Susceptibility

Dysbiotic Microbiome Destructive Inflammation ?

? How should we treat ? periodontitis? Can we target/prevent the formation of dysbiotic microbial communities?

Health- associated Disease- associated Microbial communities Microbial communities • Key microbes that facilitate transition to disease? • Who is there ? • Key microbes in dysbiosis • How do members interact? • What is their role microbial • Which interactions are key ? community formation? • Which microbes interact in vivo with the host?

What more can we learn about periodontal biofilm formation, microbial interactions and in vivo behavior to educate our therapeutic interventions? Acknowledgments

Moutsopoulos Lab NIAID CCR/Heidi Kong Loreto Abusleme LCID Gloria Calderon Holland Lab/Clinic Nicolas Dutzan Steve Holland Hajishengallis Lab Teresa Wild Gulbu Uzel Toshiharu Abe

Alexandra Freeman George Hajishengallis OP-1 Clinic Staff Christa Zerbe Laurie Brenchley Mojgan Sarmadi Lionakis Lab Kelly Betts Natalia Chalmers Mihalis Lionakis UManchester Carol Bassim Tim Break Joanne Konkel Pam Gardner

Tammy Yokum LPD OP1 Staff Belkaid Lab NIDCR Leadership NIDCR collaborators Yasmine Belkaid UCONN SD: Robert Angerer Rob Palmer Nicolas Bouladoux Patricia Diaz ID: Martha Somerman Thomas Bugge

Ilias Alevizos

The oral microbiome: we know who’s there, but what are they doing?

R. J. Palmer Jr., Ph.D. NIDCR/NIH Antonie van Leeuwenhoeck 1632 - 1723

Selenomonas

Treponema

Leptotrichia

"a little white matter, which is as thick as if 'twere batter." The Great Plate-count Anomaly (1970s) seawater

allow bacterial colonies to develop

petri dish = bacteria per unit volume seawater stain nucleic acid (see everything)

examine in microscope

filter disc = bacteria per unit volume # bacteria counted in microscope is >> # of colonies seen on plates ca. 1% of bacteria had been cultivated CARL WOESE 1929 – 2012

MacArthur Fellow Crafoord prize Leeuwenhoek medal US Nat’l Academy Sci Royal Society conserved variable

ribosome is a “molecular clock” 1) universal 2) conserved and variable regions 3) small subunit (16S, 18S) ideal size for sequencing

THREE ^ Walsh and Doolile (2005) Curr Biol 15:R237-240

diplomonads, parabasalids, trypanosomads radiolaria, formanifera animals fungi dinoflagellates, diatoms Sample

Microbiome The Great Plate-count Anomaly is solved by RNA-based taxonomy “Unculture-able” bacteria can be detected and classified, but what does this mean to bacterial taxonomy? valid bacteriological species pure culture physiology sequence data molecular species defined by sequence data SLOTU (Species Level Operational Taxonomic Unit) OTU taxon

all valid bacteriological species are molecular species but NOT vice versa – cultivated organism required The microbiome of healthy skin is well described by cultivation.

182 OTUs

85% are cultivated

15% are yet to be cultured

Gao et al. (2007) The microbiome of the healthy gut is not well described by cultivation.

395 OTUs + 1 archeal OTU 80% yet-to-be cultured

stool is not mucosa

Eckburg et al. (2005) Tooth surface 52 OTUs 44% yet-to-be cultured Firmicutes - Bacilli Subgingival plaque 347 OTUs 52% yet-to-be cultured Firmicutes - other Entire oral cavity ca. 700 OTUs ca. 60% yet-to-be cultured

Actinobacteria Synergistes Spirochaetes Fusobacteria

Proteobacteria TM7 Bacteroidetes

subgingival tooth plaque Aas et al. (2005) Paster et al. (2006) Flora of diseased periodontal pockets differs from that of healthy pockets

29 periodontally healthy subjects

29 subjects with chronic periodontitis shallow pockets (“healthy” sites) deep pockets (“diseased” sites)

direct sequencing of 16s PCR amplicons Proportions of species in diseased sites differ from those in healthy sites

Griffen et al. 2012 Individuals are ecosystems

Griffen et al. 2012 Coaggregation: a driver of spatiotemporal community assembly?

KolenbranderKolenbrander et al.,et al. 2002 2002

Coaggregation is an in vitro assay of cell-cell recognition.

add sugar 1 2 1+2 or protease How can one assess the relevance of coaggregation to biofilms in vivo ?

Microscopy provides spatio–temporal data on developing biofilms. Cell-cell recognion in vivo

S. oralis serotype 1 RPS A. naeslundii T2 fimbriae

S. oralis serotype 1 RPS S. oralis serotype 1 RPS S. gordonii S. gordonii other other

an-RPS

an-Sg (whole cell)

an-fimA

tooth surface ALL BACTERIA STREPTOCOCCAL 4 hrs RECEPTOR POLYSACCHARIDE ADHESIN-BEARING STREPTOCOCCUS

ALL BACTERIA STREPTOCOCCAL 8 hrs RECEPTOR POLYSACCHARIDE ADHESIN OF ACTINOMYCES

Nyvad 1987 Palmer 2003 ALL BACTERIA STREPTOCOCCAL RECEPTOR POLYSACCHARIDE ACTINOMYCES

12 hrs

STREPTOCOCCAL RECEPTOR POLYSACCHARIDE ACTINOMYCES ADHESIN

Nyvad 1987 Palmer 2003 Summary

Classical bacteriology has taught us very much about the microflora of easily accessible human body sites.

Molecular taxonomy has increased that knowledge and provided a way to rapidly obtain complete community descriptions – individuals are ecosystems.

The microflora of diseased sites differs from that of healthy sites primarily in proportions of various OTUs – the community is the pathogen.

Oral biofilms are spatially differentiated multispecies communities from the earliest stages of development.

Cell-cell recognition (coaggregation) plays a role in community assembly in vivo.

It sure would be nice to analyze communities using more than 3 fluorophores...... Acknowledgements Paul Kolenbrander (NIDCR – retired) John Cisar (NIDCR – retired)

Antonie van Leeuwenhoek Demysfying Medicine 29 March 2016 The Human Microbiome Project ca. 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

"...[T]here scketh or groweth between some of my front [teeth] 3. This first observation revealed and my grinders...a lile white maer, which is as thick as if human-associated microbial 'twere baer. On examining this...I most always saw, with great wonder, that in the said maer there were many very lile living communities to be complex animalcules, very prely a-moving”

Dobell, C. Antony Van Leeuwenhoek and His “Little Animalcules.” London. Constable & Co. 1932. Print. The Human Microbiome Project ca. 2016

Human Microbiome

Human Genome

100x the human genome The Human Microbiome Project 2013

Network Inference

Co-occurrence or co- exclusion observaons mined to identy stascally significant relaonships.

Taxa cluster based on body site

Is this a biologically relevant spaal scales?

Faust, et al. 2012. PLoS Comput. Biol. 8:e1002606 Spaal distribuon of microbes: relevant scales

Gut microbiota Skin microbiota

Mowat and Agace. 2014. Nat. Rev. Immunol. 14:667-685. Grice and Segre. 2011. Nat. Rev. Microbiol. 9:244-253 A hypothesis for the structure of dental plaque biofilms

Jabra-Rizk, et al. 1999 J Clin Microbiol Kolenbrander, et al. 2010 Nat Rev Microbiol Microbial Fluorescence in situ Hybridizaon

Probe

Sample

Fixation Target (ribosomal RNA)

Fluorescence Fixed cells are microscopy permeabilized Ribosome

Fluorescently labelled oligonucleotides (probes) Hybridization Quantifcation

Washing

Hybridized cells Amann, R. & B. Fuchs. 2008 Nat. Rev. Microbiol. 6:339-348 Nature Reviews | Microbiology Spaal distribuon of microbes: relevant scales

Gut microbiota Skin microbiota

Mowat and Agace. 2014. Nat. Rev. Immunol. 14:667-685. Grice and Segre. 2011. Nat. Rev. Microbiol. 9:244-253 Spaal distribuon of microbes: relevant scales

Gut microbiota Skin microbiota

fungi bacteria

Hair follicle

Propidium iodide (Eukaryoc DNA) hp://irp.nih.gov/our-research/research-in-acon/ Propidium iodide (Bacterial DNA) the-microbiome-when-good-bugs-go-bad Calcafluor white (fungal cell wall) Unknown matrix material Fluorescence Imaging: High specificity; Low mulplicity

FISH on two mixtures of E. coli labeled with two different fluorophores

red filter green filter red filter

1

1 0.9

0.9 0.8

0.8 0.7

0.7 0.6 AF-555 0.6 Bodipy-Fl 0.5 RhodamineRed-X 0.5 Rhodamine Red-X 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 475 525 575 625 675 725 475 525 575 625 675 725 Wavelength (nm) Wavelength (nm) Emission Spectra Emission Spectra Sequencing vs. Imaging

Sequencing Assay Imaging Assay

Breadth High Low

Independence of prior knowledge High Low

Dynamic Range / Detecon Limit Low High

Ability to provide spaal info. Low High Combinatorial Labeling

The power of combinaons Each bacterium is labeled with exactly 2 fluors

Red fluorophore Green fluorophore Blue fluorophore Ribosome Microbe High mag. detail

Field of labeled microbes

CLASI-FISH Combinatorial Labeling and Spectral Imaging- With 8 fluorophores, there exist 28 Fluorescence in situ unique binary combinaons Hybridizaon Linear Unmixing

observed pixel spectrum known fluorophore spectra

1

0.8

0.6

0.4 Find Best Fit

0.2 Intensity (A.U.) (A.U.) Intensity 0 498 548 598 648 698 375 425 475 525 575 625 675 725 775 Wavelength (nm) Wavelength (nm)

! % ! % ! % y1 ( + x1 n1 # # m11  mp1 # # # # # y # * - # x # # n # " 2 & = *    -⋅" 2 &+" 2 & #  # * - #  # #  # m1q  mpq # y # )* ,- # x # # n # $ q ' $ p ' $ q '

Observed pixel Known fluorophore Abundance of each Noise spectrum spectra fluorophore at that pixel

This allows fluorophores with highly overlapping emission spectra to be disnguished—even if they are present in the same pixel in the image. Imaging proof of principle with E. coli Establishing Biological Proof of Principle MIxture of cells of 15 laboratory grown oral taxa Structural Analysis of a Natural Community: Human Dental Plaque

Image of a field of view of semi-dispersed human dental plaque

SelenomonasAlexa fluor 488CampylobaRhodaminecter Gemella Red X FusobaAlexacter iumfluor 514 PorphyromonasAlexa fuorR othia594 PasteuAlexarella cfluoreae 555 CapnocytophagaAlexa fuorP r647evotella Neisseriaceae Streptococcus Veillonella Treponema Actinomyces Leptotrichia unknown Structural Analysis of a Natural Community: Human Dental Plaque Raw spectral Taxon-assigned Raw spectral Taxon-assigned image merge segmented image image merge segmented image

Sele Camp GemeSele Camp Geme Fuso Porp RothFuso Porp Roth Past Capn PrePvast Capn Prev Rhodamine Red XRhodamineNeis RedStr Xep VeilNeis Strep Veil Trep Acti LepTtrep Acti Lept unkn unkn Structural Analysis of a Natural Community: Human Dental Plaque

Observed image of plaque Model images of randomly placed cells

Selenomonas Campylobacter Gemella Fusobacterium Porphyromonas Rothia Pasteurellaceae Capnocytophaga Prevotella Neisseriaceae Streptococcus Veillonella Treponema Actinomyces Leptotrichia unknown Structural Analysis of a Natural Community: Human Dental Plaque

Campylobacter Fusobacterium Gemella

Capnocytophaga Prevotella

Actinomyces

Veillonella Porphyromonas Neisseriaceae Pasteurellaceae

Rothia Streptococcus Biogeography of the human microbiome at the micron scale

Mark Welch, et al. 2015. PNAS Hedgehog structure in dental plaque

Mark Welch, et al. 2015. PNAS Corncob structures at the border of hedgehog structures

Mark Welch, et al. 2015. PNAS Model hypothesis of hedgehog structure

O2,$saliva,$sugars$

CO2,$lactate,$ acetate,$H2O2$

anoxic$ tooth$ v$

base$ annulus$ perimeter$

Crevicular$fluid$

Corynebacterium . ....Porphyromonas . ...Fusobacterium . .other$ Streptococcus ...... Neisseriaceae . ...Leptotrichia. Haemophilus/Aggr...... Capnocytophaga . ...Ac=nomyces. Mark Welch, et al. 2015. PNAS Hedgehog structures as an ecosystem

Genecally disnct organisms occupying niches

Trophic interacons

Environmental influence

Role of cell-to-cell contact

Mark Welch, et al. 2015. PNAS Framework for oral microbiome funconal study Meta Exploratory Surveys transcriptomics Laboratory Experiments

Microbial In vitro biofioms metagenomics

Systems Duran-Pinedo, et al. 2015. ISMEJ Imaging Metabolomics

Red fluorophore Green fluorophore Kolenbrander, et al. 2010. Nat. Rev. Microbiol Blue fluorophore Ribosome Microbe High mag. detail

Field of labeled microbes

da Silva, Dorrestein and Quinn. 2015 PNAS

Computaonal modeling

Testable hypotheses Improved regarding ecosystem models of structure and funcon microbial co- Steenackers, et al. 2016. FEMS Microbiol Rev. occurrence

Kielmann, et al. 2013. PLoS One 8:e47879 Acknowledgements

• NICHD – Jennifer Lippinco Schwartz Lab • NHGRI – Julie Segre Lab • Forsyth Instute – Gary Borisy Lab • Marine Biological Lab – Rudolf Oldenbourg – Jessica Mark Welch