a forbidden kingdom: the fungal microbiome in skin health, why it's important, what needs to be...

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

The Middle Class: Consumers

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A Forbidden Kingdom

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Microbiome – defining the cutaneous microbiomein health and disease

The Skin Microbiome

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Microbiome – defining the cutaneous microbiomein health and disease

The Skin Microbiome

Presentationslast winter in KL

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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

• 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

example: Dandruff and seborrheic dermatitis

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

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Why Study Dandruff, anyway?

NormalScalp

Flaking

Itching

DandruffScalp

SeborrheicDermatitis

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 when introduced 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 when introduced 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 seborrheic dermatitis and dandruff. The Journal of Investigative Dermatology, 10(3), 194–197.

A three factor causal model

IndividualSusceptibility

MalasseziaSebum

SeborrheicDermatitis


Fragment Analysis by Size

Gemmer, C.M. et al., (2002) Journal of Clinical Microbiology, 40(9), pp.3350–3357.

Amplification

ITS 1 Forward Middle Reverse

ITS 2 ReverseMiddle Forward

ITS1 ITS218s Ribosomal gene 28s Ribosomal gene5.8s gene

18S Forward

28S Reverse

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) and M. pachydermatis (74522)

(newly identified M. nana, M. yamatoensis, and M. dermatis also uniquely identifiedwith 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

Is there a causal species?

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Early Microbiome: Malassezia


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.

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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


Days Post Initial Treatment

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Placebo vehicle

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The model:How Malassezia Cause Flaking (in susceptible individuals)

Consumption ofSaturated Lipids

Penetra-tion of

,unsaturated fatty acids

Hydro-lysis ofTrigly-cerides

Skin Surface

MalasseziaFungal Colony 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

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M. restrictaM. globosaM. sympodialisM. slooffiaeM. equinaM. dermatisM. obtusaM. pachydermatisM. japonicaM. capraeM. furfurM. cuniculiM. nanaM. yamatoensis

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Malassezia meta-genomics

Reanalysis of NIH dataconfirms Malassezia as dominant skin eukaryotes

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 for eMicrobiomeI nvestigations inS kin

Fook Tim Chew (NUS), clinical cohort managementMarkus Wenk, SLING, (NUS), lipidomicsYY Teo (NUS, GIS), statistics, cohort managenemtChong Yap Seng, (SICS), S-PRESTO cohortGiulia Rancati (IMB), genomics instability, evolution

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

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