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A Forbidden Kingdom: The Fungal Microbiome in Skin Health.Why it’s Important, What Needs To Be Done, and How to Intervene
Thomas L. Dawson,JrSenior Principal InvestigatorAgency for Science, Technology, and Research (A*STAR) Institute of Medical Biology (IMB)Director, Healthy Hair and Skin ProgramDirector, Malassezia Research Consortium
A Forbidden Kingdom: Fungal Microbiome in Skin Health
Agenda:• Asia and the world• Skin and fungi – the “Forbidden Kingdom”
• The skin – an underexplored niche• Fungi – a crucial knowledge gap• Economic impact
• Example: dandruff and seborrheic dermatitis• What it is• Learning from Koch• Early skin mycobiome• Role of microbiome – Malassezia• Current Malassezia genomics
• Key next steps for skin• Summary
Agenda:• Asia and the world• Skin and fungi – the “Forbidden Kingdom”
• The skin – an underexplored niche• Fungi – a crucial knowledge gap• Economic impact
• Example: dandruff and seborrheic dermatitis• What it is• Learning from Koch• Early skin mycobiome• Role of microbiome – Malassezia• Current Malassezia genomics
• Key next steps for skin• Summary
A Forbidden Kingdom: Fungal Microbiome in Skin Health
The Middle Class: Consumers
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Data fromGapminder.com
Agenda:• Asia and the world• Skin and fungi – the “Forbidden Kingdom”
• The skin – an underexplored niche• Fungi – a crucial knowledge gap• Economic impact
• Example: dandruff and seborrheic dermatitis• What it is• Learning from Koch• Early skin mycobiome• Role of microbiome – Malassezia• Current Malassezia genomics
• Key next steps for skin• Summary
A Forbidden Kingdom: Fungal Microbiome in Skin Health
A Forbidden Kingdom
This is from 2014!
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Microbiome publications by year
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Biome publications with skin and fungi
Microbiome – defining the cutaneous microbiomein health and disease
The Skin Microbiome
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Gut Other Oral Vaginal Lung Skin
Microbiome – defining the cutaneous microbiomein health and disease
The Skin Microbiome
Presentations last winter in KL
1/383%
Today: 7/4217%
Cutaneous microbiome in health and disease
Increasingly implicated in common diseases, global direct/indirect costs >$20B USD. So far, investigations primarily gut/bacteria, leaving skin eukaryotes poorly understood. A crucial and exploitable knowledge gap. The microbiome is influenced by or influences everything human.
The Skin Microbiome
example: Dandruff and seborrheic dermatitis
• Dandruff and seborrheic dermatitis account for a substantial percentage of dermatologist visits
• The anti-dandruff shampoo market is roughly $10 Billion USD/year
• In the anti-dandruff business efficacy is point of entry
• Highly competitive market
• Current technology modulates the microbiome by killing fungi -Malassezia
Size of the prize –Skin Microbiome
• Incidence is high globally – huge market
• About 50% of all people are affected
• May vary by ethnicity
Dandruff
Prevalence
(%)
40
20
60
80
100
African A African A Caucasian Caucasian Females Males Females Males
Asian Females
Asian Males
Why Study Dandruff, anyway?
Normal Scalp
Flaking
Itching
Dandruff Scalp
Seborrheic Dermatitis
Flaking
Itching
Visible irritation
What is it?cosmetic manifestations
Dandruff and seborrheic dermatitis
Do we know the “cause”?
1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease whenintroduced into a healthy organism.
4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.
“Koch’s Postulates” (1890):
Heinrich Hermann Robert Koch(11 Dec 1843 – 27 May 1910)
wikipedia
1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms (susceptibility).
2. The microorganism must be isolated from a diseased organism and grown in pure culture (unculturables).
3. The cultured microorganism should cause disease whenintroduced into a healthy organism (ethics).
4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent (see above).
“Koch’s Postulates” (modern issues):
Some difficult examples: viruses, aspergillosis, cholera
Important published historical data1. Malassezia is on everyone (and every warm blooded animal)2. In susceptible individuals
• Remove all fungi, dandruff goes away• Remove bacteria, it does not• Remove both, benefit is the same• Add back resistant Malassezia, dandruff returns
Malassezia meet 3 of 4 of Kochs postulates
Dandruff and seborrheic dermatitis - Koch
Gosse RM, V. R. (1969). The relationship of a nystatin-resistant strain of Pityrosporum ovale to dandruff. J Soc Cosmet Chem, 20, 603–6.
Hay, R. J. (2011). Malassezia, dandruff and seborrhoeic dermatitis: An overview. British Journal of Dermatology, 165(SUPPL. 2), 2–8.Wyk, R. Van der, & Hechemy, K. (1967). A comparison of the bacterial and yeast flora of the human scalp and their effect upon dandruff production. J. Soc. Cosmet. Chem, 639, 629–639.Ro, B. I., & Dawson, T. L. (2005). The role of sebaceous gland activity and scalp microfloral metabolism in the etiology of seborrheicdermatitis and dandruff. The Journal of Investigative Dermatology, 10(3), 194–197.
A three factor causal model
Sebum
Individual Susceptibility
Seborrheic Dermatitis
Malassezia
Dandruff and seborrheic dermatitis
Fragment Analysis by Size
Gemmer, C.M. et al., (2002) Journal of Clinical Microbiology, 40(9), pp.3350–3357.
ITS 1 Forward Middle Reverse
ITS 2 ReverseMiddle Forward
18s Ribosomal gene ITS1 5.8sgene ITS2 28s Ribosomal gene
18SForward
28SReverse
Amplification
The key gene cluster
The first skin mycobiome - tFLP
All Known Malassezia Genotypes in One Sample (and others)
M. furfur (7982),M. globosa(7966),M. globosa(7874),M. globosa(7990),M. obtusa(7968),M. restricta (7991),M. restricta (7877),M. restricta (8742),M. slooffiae (7971),M. slooffiae (7956),M. sympodialis (7977) andM. pachydermatis (74522)
(newly identified M. nana, M. yamatoensis, and M. dermatis also uniquely identified with many control non-Malassezia fungi)
The first skin microbiome - tFLP
Example scalp sample
restricta
globosa
Gemmer, C.M. et al., (2002) Journal of Clinical Microbiology, 40(9), pp.3350–3357.
all fungi (almost?) assessed – only 2 major players
The first skin microbiome - tFLP
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ASFS <10
ASFS >24
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Early Microbiome: Malassezia
Is there a causal species?
Dandruff and seborrheic dermatitis
How could his be?
Malassezia convert sebum triglycerides into irritating free fatty acids in vitro.
The model: sebum
Dandruff and Sebum
Malassezia convert sebum triglycerides into irritating free fatty acids in vivo.
0%
30%
20%
10%
40%
50%
60%
1 6 11 16 21
High Acid (Active Malassezia)
Low Acid (no Malassezzia)
The model: sebum
Dandruff and Sebum
The model: individual susceptibility
What if some people can’t get it?• Recruit 2 populations, with and without dandruff• Treat them – remove Malassezia, return to baseline• Challenge with a “toxic metabolite” – oleic acid
Dandruff and Susceptibility
Flak
ing
Dif
fere
nce
fro
mB
ase
line
-1
0
1
7.5% Oleic Acid
Placebo vehicle
3 4 5 6 7 8 9
Days Post Initial Treatment
The model: individual susceptibility
Some People Get Dandruff
Dandruff and Susceptibility
3 4 5 6 7
Days Post Treatment
8 9
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0
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Placebo7.5% OleicAcid
Flak
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ase
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The model: individual susceptibility
Some People don’t.
Dandruff and Susceptibility
The model:How Malassezia Cause Flaking (in susceptible individuals)
Consumption of Saturated Lipids
Penetra-tion of
,unsaturated fatty acids
Hydro-lysis of Trigly-cerides
Skin Surface
Malassezia FungalColony Sebaceous
Lipids
Expressionof Lipase
Hyperproliferation
Dandruff Pathogenesis – Susceptible Individuals
Individual susceptibility and commensalism influence investigation:• Affected vs unaffected comparisons confuse interpretation.• Cannot assume “unaffected” microbiome is “benign”.• In non-susceptible: entire causal apparatus could be present –
subjects simply cannot respond
Why studies comparing affected to unaffected remain confusing?
Demonstrates need - longitudinal studies in susceptible individuals
Susceptibility – important considerations
Malassezia genomics
PLOS Genetics, 2015• Sequenced 24 strains representing all known species• Clarified the phylogeny• Defined the gene repertoire• Identified a set of HTGs which define the genus• Established lipid dependence in all species• Identified numerous potential therapeutic targets• Provided a database for all future skin microbiome studies
Wu, G., Zhao, H., Li, C., Rajapakse, M. P., Wong, W. C., Xu, J., … Dawson, T. L. (2015). Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin. PLOS Genetics, 11(11), e1005614.
New genomes:• Define 3 main groups:
• furfur clade (A)• common human skin residents (B)• uncommon outgroup (C)
• Indicate HTGs• define the genus• define the human skin group
Malassezia genomics
Species
M.restrictaM.globosaM.sympodialisM. slooffiaeM.equinaM.dermatisM.obtusaM. pachydermatisM. japonicaM.capraeM. furfurM. cuniculiM. nanaM. yamatoensis
Nu
mb
er
of
sam
ple
sN
um
be
ro
f in
div
idu
als
247 17199 1669 1220 413 113 610 2
8 37 36 52 11 10 00 0
Body sites
Rel
ativ
eab
un
dan
ces
00.
20.
40.
60.
81
.0
Malassezia meta-genomics
Reanalysis of NIH data confirms Malassezia as dominant skin eukaryotes
Agenda:• Asia and the world• Skin and fungi – the “Forbidden Kingdom”
• The skin – an underexplored niche• Fungi – a crucial knowledge gap• Economic impact
• Example: dandruff and seborrheic dermatitis• What it is• Learning from Koch• Early skin mycobiome• Role of microbiome – Malassezia• Current Malassezia genomics
• Key next steps for skin• Summary
A Forbidden Kingdom: Fungal Microbiome in Skin Health
Define “Healthy Skin”• Most current data only western Caucasians• Superseded methods• Different locations, disease states
Things to do first, AKA, the hard parts:• Define sampling paradigms• Define reproducible protocols• Define disorder-specific sampling• Build in more replicates• “neutrality, stability, reproducibility, accuracy, precision”• Initiate longitudinal cohorts
The Cutaneous Microbiome – next steps
How A*STAR IMB and SICS are approaching it:• Core focus area• Well placed for Asia – skin health• Divergent pan-Singapore teams• Clinical/Basic science collaboration (IMB/NTU/SSC)
Hypothesis:Intervention in eukaryotic microbiome (mycobiome) can improve skin health
The Cutaneous Microbiome – A*STAR IMB
A siaR egionalT eam foreMicrobiomeI nvestigations inS kin
Maurice van Steensel (IMB), Acne, Sebum, Seb DermMark Tang (NSC), Clinician, Seb Derm, Psoriasis, AcneHazel Oon (NSC), Clinician, Seb Derm, Psoriasis, AcneKimberly Kline (NUS), bacterial virulenceAndrea Camattari (BTI), fungal engineering
John Common(IMB) Atopic Dermatitis
Skin Health
Tom Dawson(IMB)Malassezia
Fungal virulence, Seb Derm
Niranjan Nagarajan(GIS) Bioinformatics
Fook Tim Chew (NUS), clinical cohort management Markus Wenk, SLING, (NUS), lipidomicsYY Teo (NUS, GIS), statistics, cohort managenemt Chong Yap Seng, (SICS), S-PRESTO cohortGiulia Rancati (IMB), genomics instability, evolution
Summary:• Asia is the key developing market for middle class consumers• Skin mycobiome is an exploitable knowledge gap• Dandruff is one good example
• We know mycobiome is involved.• Suitable economic opportunity
• Individual susceptibility must be included in experimental design• New genomics data empowers exploration• We need to sharpen the tools, specific to skin• A*STAR IMB is well placed and looking for collaboration
Summary
Presented at the 4th Microbiome R&D and Business Collaboration Forum: USA.
To find out more, visit: www.global-engage.com
Summary