Presented at the 3rd Microbiome R&D and Business Collaboration Congress: Asia. To find out more, visit: www.global-engage.com Using modern genomic techniques Niranjan Nagarajan, Associate Director and Group Leader at A*STAR, has identified key perturbations in the resident skin microflora that could act as triggers for eczema flares. Niranjan presents a new form of the hygiene hypothesis for explaining the increasing incidence of eczema and revealing, in the process, novel therapeutic targets for this disease.

1. The Role of the Skin
Microbiome in Eczema
Niranjan Nagarajan
Associate Director and Group Leader
Computational & Systems Biology

2. Epidermal Hydration
Mechanical Barrier
Permeability Barrier
Waterproof Barrier
Anti-microbial Barrier
Anti-oxidant Barrier
Anti-UV Barrier
Initiation of inflammation
The human skin is an effective barrier
Slide credit: John Common

3. Infection transmission via skin
Conjuctivitis, Acne, Staph/Strep infections,
Herpes, Yeast infections, Ebola, RSV, Hand-
foot-mouth disease, Athlete’s foot, …

4. http://www.genome.gov/pressDisplay.cfm?photoID=20169
Microbial Communities on Skin
107 cells/cm2

5. Skin Microbiome
§ Co-evolved with us
§ Homeostatic and protective
function?
Credit: Matej Bajzer & Randy J. Seeley. Nature 444, 1009-1010 (21 December 2006)
Gut Microbiome
§ Nutrient Acquisition
§ Drug Metabolism
§ Development of Immune
System
How do skin bacteria contribute to host health?

6. http://www.google.com.sg/url?
sa=i&source=images&cd=&docid=79U3NUAL
WEbBrM&tbnid=3aSgFcIo_0tB0M&ved=0CAg
QjRw&url=http%3A%2F
%2Fwww.dampproofingcyprus.com%2Fstop-
asthma-in-children%2F&ei=NtMiVOz-
EoadugTV4oDoCg&psig=AFQjCNHQUM_kT-
EVpCoCdESIs-
vcHubK_w&ust=1411654838367396
Disease Phenotype: Atopic Dermatitis (AD)
Dry and itchy skin
Broken skin barrier
Inflamed lesions that are
prone to bacterial infection
b
Slide credit: John Common

7. http://www.google.com.sg/url?
sa=i&source=images&cd=&docid=79U3NUAL
WEbBrM&tbnid=3aSgFcIo_0tB0M&ved=0CAg
QjRw&url=http%3A%2F
%2Fwww.dampproofingcyprus.com%2Fstop-
asthma-in-children%2F&ei=NtMiVOz-
EoadugTV4oDoCg&psig=AFQjCNHQUM_kT-
EVpCoCdESIs-
vcHubK_w&ust=1411654838367396
The Atopic Epidemic
International study on Asthma and Allergies in childhood
Slide credit: John Common

8. http://www.google.com.sg/url?
sa=i&source=images&cd=&docid=79U3NUAL
WEbBrM&tbnid=3aSgFcIo_0tB0M&ved=0CAg
QjRw&url=http%3A%2F
%2Fwww.dampproofingcyprus.com%2Fstop-
asthma-in-children%2F&ei=NtMiVOz-
EoadugTV4oDoCg&psig=AFQjCNHQUM_kT-
EVpCoCdESIs-
vcHubK_w&ust=1411654838367396
What causes atopic dermatitis?
Complex interplay between
• Skin Barrier
• Immune system
• Environment
- Microbiome
Slide credit: John Common

9. On visually normal, undamaged skin
Normals
no history of AD or allergy
SPT+
Allergy, no history of AD
AD
Active AD patients
Sampling Area:
Antecubital fossa
High-throughput
DNA Sequencing
Relative abundance
and statistical analysis
Cohorts
Analysis
Microbiome-wide Association study for AD
Chng KR, Tay ASL, Li C, Ng AHQ, …, Lane EB, Chew FT, Common JEA#, Nagarajan N# "Whole
metagenome profiling reveals skin microbiome-dependent susceptibility to atopic dermatitis flare"
Nature Microbiology 2016 1:16106 doi:10.1038/nmicrobiol.2016.106

10. Control-Case Case-Case
0.0
0.2
0.4
0.6
0.8
1.0
Yue-ClaytonThetaIndex
p-value = 2.4×10-6
Control (Normal)
Case (AD)
SPT+
Bacterial Diversity on Normal Skin

11. Enrichment of opportunistic pathogens in AD
0
2
4
6
8 Streptococcus
**
RelativeAbundance(%)
Gemella
0.0
0.2
0.4
0.6
0.8
1.0
**
0.0
0.5
1.0
1.5
2.0 Haemophilus
*
Staphylococci produce
factors that promote the
growth of Haemophilus
Known to cause skin
and oral infections.
Changes specific to α-
hemolytic group
Can aggravate
symptoms in
Cystic Fibrosis
Control
Case
SPT+

12. Depletion of metabolically versatile bacteria
0.0
0.5
1.0
1.5
2.0
3.0
2.5
Dermacoccus
**
RelativeAbundance(%)
0.0
0.1
0.2
0.3
0.4 Deinococcus
*
0.0
0.2
0.4
0.6
0.8
1.0 Methylobacterium
*
Actinomycetales are known for
producing secondary metabolites
with anti-inflammatory and anti-
microbial properties
Radiation tolerant and
metabolically versatile
Found commonly
on human feet
Control
Case
SPT+

13. Viruses & Eukaryotes
RelativeAbundance(%)
Malasseziaceae
0
2
4
6
8
10
***
§ No association for Viruses
§ Significant depletion of Malassezia
§ Lipid dependent and adapted to skin
§ Associated with Dandruff, Seborrhoeic
dermatitis, Tinea Versicolor
Reference genome and annotation for all 14 species and 24 strains of the genus
Malassezia Genome Database
RESEARCH ARTICLE
Genus-Wide Comparative Genomics of
Malassezia Delineates Its Phylogeny,
Physiology, and Niche Adaptation on Human
Skin
Guangxi Wu1
, He Zhao2
, Chenhao Li1
, Menaka Priyadarsani Rajapakse1
, Wing
Cheong Wong3
, Jun Xu4
, Charles W. Saunders4
, Nancy L. Reeder4
, Raymond A. Reilman4
,
Annika Scheynius5
, Sheng Sun6
, Blake Robert Billmyre6
, Wenjun Li7
, Anna
Floyd Averette6
, Piotr Mieczkowski8
, Joseph Heitman6
, Bart Theelen9
, Markus
S. Schröder10
, Paola Florez De Sessions1
, Geraldine Butler10
, Sebastian Maurer-Stroh3,11
,
Teun Boekhout9
, Niranjan Nagarajan1
*, Thomas L. Dawson, Jr.12
*
1 Computational and Systems Biology, Genome Institute of Singapore, A*STAR, Singapore, 2 Procter &
Gamble Singapore Innovation Center, Singapore, 3 Bioinformatics Institute, A*STAR, Singapore, 4 Procter
& Gamble Mason Business Center, Mason, Ohio, United States of America, 5 Translational Immunology Unit,
Department of Medicine Solna, Karolinska Institutet and University Hospital, Stockholm, Sweden, 6 Duke
University Medical Center, Durham, North Carolina, United States of America, 7 National Center for
a11111
RESEARCH ARTICLE
Genus-Wide Comparative Genomics o
Malassezia Delineates Its Phylogeny,
Physiology, and Niche Adaptation on H
Skin
Guangxi Wu1
, He Zhao2
, Chenhao Li1
, Menaka Priyadarsani Rajapakse1
, Wi
Cheong Wong3
, Jun Xu4
, Charles W. Saunders4
, Nancy L. Reeder4
, Raymon
Annika Scheynius5
, Sheng Sun6
, Blake Robert Billmyre6
, Wenjun Li7
, Anna
Floyd Averette6
, Piotr Mieczkowski8
, Joseph Heitman6
, Bart Theelen9
, Mark
S. Schröder10
, Paola Florez De Sessions1
, Geraldine Butler10
, Sebastian Ma
Teun Boekhout9
, Niranjan Nagarajan1
*, Thomas L. Dawson, Jr.12
*
a11111
Control
Case
SPT+

14. Association with specific Malassezia species
RelativeAbundance(%)
M. dermatis
0
2
4
6
8
CaseControl SKP+
*
SKP+
M. restricta
0
20
40
60
80
100
CaseControl
M. globosa
CaseControl SKP+
**
0
10
20
30
40
50
60
M. sympodialis
CaseControl SKP+
0
1
2
3
4
RelativeAbundance(%)
RelativeAbundance(%)
RelativeAbundance(%)
SPT+ SPT+
SPT+ SPT+

15. HOST-MICROBIOME INTERACTIONS
Cause vs Effect in relation to AD
1. Direct cell-to-cell interactions
Microbe-human, microbe-microbe
2. By altering the microenvironment
3. Immune-system mediated

16. AD-associated microbiome inhibits S. aureus
Staphylococcus
aureus
Dermacoccus
Methylobacterium
AllSubjects
Staphylococcus
capitis
Streptococcus
mitis
ADSubjects
Bacterial Inhibition Assay – S. aureus culture
A – Streptococcus mitis
B – Staphylococcus epidermidis
C – BHI Media

17. AD associated bacteria elicit distinct immune responses
1. Flare associated bacteria (S.
epidermidis and S. aureus)
induce strong immune
response
– TNF-driven myeloid activating and
Th1 polarizing cytokine signature
2. Depleted bacteria (D.
nishinomiyaensis and D.
radiodurans) elicit minimal
response
0.0 18.0
Log2 Conc.
DR0.001%
DP0.01%
SE0.1%
SE0.01%
SE0.001%
SA0.1%
SA0.01%
SA0.001%
BF0.1%
BF0.01%
BF0.001%
DR0.1%
DR0.01%
DP0.1%
DP0.001%
DN0.1%
DN0.01%
DN0.001%
MCP-3
IL-1a
VEGF
IL-1b
IL-6
EGF
Eotaxin
IL-10
TGF-a
MIP-1a
MDC
MCP-1
IL-8
TNFa
PDGF-AA
TNFb
G-CSF
IP-10
Flt-3L
GRO
FGF-2
PDGF-AB/BB
IFNa2
Fractalkine
IL-12p70
sCD40L
MIP-1b
IL-12p10
RANTES
IL-1RA
SE:
Staphylococcus
epidermidis
SA:
Staphylococcus
aureus
BF:
Bacillus
firmus
DR:
Deinococcus
radiodurans
DN:
Dermacoccus
nishinomiyaensis
DP:
Dermacoccus
profundi
Supernatants on Dendritic cells
Bacteria
Human cells
(NHEKs, MoDCs)

18. AD skin selects for S. aureus strains
DNA Sequence data
AD susceptible skin = geh Arg 373
Normal skin = geh Thr 373
Dry skin sites = geh Arg 373
Moist skin sites = geh Thr 373
Moist
Dry
Healthy skin
AD-susceptible skin
geh gene - Lipase gene
(glycerol ester hydrolase) is
important for virulence.
Cases vs Controls in our study
Dry vs Moist sites in Oh et al, Nature 2014

19. Perturbed microbial metabolism on AD skin
EC 3.5.3.6
Arginine Deaminase
Ammonia
Arginine
Citrulline
H2O
Ammonia
Carbamoyl-P
Ornithine
Pi
(pH )
ATP + CO2
ADP
(pH )
EC 2.1.3.3
Ornithine
Carbamoyltransferase
EC 2.7.2.2
Carbamate Kinase
Depleted in
Cases
Enriched in
Cases
CaseControl CaseControl CaseControl
RelativeAbundance(×10-4)
0
2
4
6
8
10 EC 3.5.3.6
EC 2.1.3.3
EC 2.7.2.2
Combined p-
value = 0.0029
*
***
*

20. Risk
factors
2.
pH
change
Normal
Skin
AD
flare
Skin
3.
Increase
in
pathobionts
ColonizaCon
by
opportunisCc
pathogens
Immune
ac3va3on,
Inflamma3on
Summary
1. Predisposing
microbiome
Altered
microenvironment