“PepTag”

It All Began with...

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It All Began with...

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Looks like a Lung Infection…

It All Began with...

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Looks like a Lung Infection…

but Antibiotics don’t work!

Later on at House‘s Office

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Later on at House‘s Office

1.5 Billion Fungal Infections in

Humans/Year

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Later on at House‘s Office

Candida causes around400,000 septicaemiawith a 40% mortality

rate

1.5 Billion Fungal Infections in

Humans/Year

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Later on at House‘s Office

A. fumigatus causes Aspergillosis

mortality rate as highas 90%

1.5 Billion Fungal Infections in

Humans/Year

Candida causes around400,000 septicaemiawith a 40% mortality

rate

3

Later on at House‘s Office

1.5 Billion Fungal Infections in

Humans/Year

Candida causes around400,000 septicaemiawith a 40% mortality

rate

A. fumigatus causes Aspergillosis

mortality rate as highas 90%

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“PepTag”

A small change for man, a giant pain for germkind!4

Current TherapeuticStrategies

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Current TherapeuticStrategies

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Current TherapeuticStrategies

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Current TherapeuticStrategies

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Current TherapeuticStrategies

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Current TherapeuticStrategies

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Current TherapeuticStrategies

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Our Vision

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Our Vision

Make Fungi Visible to the Immune System

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Our Vision

Make Fungi Visible to the Immune System

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Our Vision

Make Fungi Visible to the Immune System

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Our Vision

Make Fungi Visible to the Immune System

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

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How to Make Fungal Cells Visible?

PepTag

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How to Make Fungal Cells Visible?

PepTag

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Immune System Modulator

How to Make Fungal Cells Visible?

PepTag

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Antibody + Signal Molecule

Immune System Modulator

How to Make Fungal Cells Visible?

PepTag

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Immune System Modulator Antibody + Signal Molecule

Radiolabel

How to Make Fungal Cells Visible?

PepTag

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Immune System Modulator Antibody + Signal Molecule Radiolabel

¬ Located on outer surface

¬ No / little glycosylations

Criteria for Suitable Fungi Surface Proteins

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Candida albicans Saccharomycescerevisiae

Candida glabrata Toxoplasma gondii

Aspergillus fumigatus

Aspergillusnidulans

Selection of 35 Different Surface Proteins

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PCR Amplification

Cloning

attB1 GOI attB2

PCR and Cloning

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Yeast Two-Hybrid Assay

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Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

2. Mating of the twoYeast Strains

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Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

2. Mating of the twoYeast Strains

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Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

3. Mated CellsTransferred to Selection Medium

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

3. Mated CellsTransferred to Selection Medium

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

3. Mated CellsTransferred to Selection Medium

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2. Mating of the twoYeast Strains

Yeast Two-Hybrid Assay

1. Cultivation of Prey Library and Bait Yeast Strains

3. Mated CellsTransferred to Selection Medium

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2. Mating of the twoYeast Strains

In Our Lab

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In Our Lab

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Establishing Contact with Our Community

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Establishing Contact with Our Community

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Establishing Contact with Our Community

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Establishing Contact with Our Community

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Establishing Contact with Our Community

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Establishing Contact with Our Community

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Results are needed!

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Results 2nd Yeast-Two-Hybrid Assay

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Results 2nd Yeast-Two-Hybrid Assay

Sequencing and Homology Modeling

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Sequencing and Homology Modeling

25 Amino AcidSequence

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Sequencing and Homology Modeling

25 Amino AcidSequence

Interacting PeptideGB1 Scaffold16

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Aspergillus fumigatus

PepTagαSun1

Label

In vivo Experiments

SignalMolecule

+Antibody

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Aspergillus fumigatus

PepTagαSun1

Label

In vivo Experiments

CalcofluorBright Field Labeled PepTag Overlay

SignalMolecule

+Antibody

Are Specific PepTagsPossible?

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Are Specific PepTagsPossible?

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Labeled PepTag

A. fumigatusαSun1 PepTag

Are Specific PepTagsPossible?

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Labeled PepTag

A. fumigatusαSun1 PepTag

A. nidulansαSun1 PepTag

Yeast BioBricks

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Name Description Size (kb)

BBa_K1430007 Tool to tag yeast proteins with GFP 4304

BBa_K1430008 Tool to tag yeast proteins with mRFP 4685

Yeast BioBricks

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Name Description Size (kb)

BBa_K1430007 Tool to tag yeast proteins with GFP 4304

BBa_K1430008 Tool to tag yeast proteins with mRFP 4685

TEF2 ADH1

gene of interest

BamH1BglII

Yeast BioBricks

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Name Description Size (kb)

BBa_K1430007 Tool to tag yeast proteins with GFP 4304

BBa_K1430008 Tool to tag yeast proteins with mRFP 4685

Use the PepTag as a Diagnostic Tool

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Ask the Experts

Dr. U. Groβ Dr. S. Krappmann

Dr. J. Wagener

University Medical Center Göttingen

University Medical Center Erlangen

Ludwig-MaximiliansUniversity of Munich

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Ask the Experts

Dr. U. Groβ Dr. S. Krappmann

Dr. J. Wagener

University Medical Center Göttingen

University Medical Center Erlangen

Ludwig-MaximiliansUniversity of Munich

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High specificity

Reliability

Easy application

Low cost

Ask the Experts

Dr. U. Groβ Dr. S. Krappmann

Dr. J. Wagener

University Medical Center Göttingen

University Medical Center Erlangen

Ludwig-MaximiliansUniversity of Munich

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High specificity

Reliability

Easy application

Low cost

Ask the Experts

Dr. U. Groβ Dr. S. Krappmann

Dr. J. Wagener

University Medical Center Göttingen

University Medical Center Erlangen

Ludwig-MaximiliansUniversity of Munich

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High specificity

Reliability

Easy application

Low cost

Ask the Experts

Dr. U. Groβ Dr. S. Krappmann

Dr. J. Wagener

University Medical Center Göttingen

University Medical Center Erlangen

Ludwig-MaximiliansUniversity of Munich

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High specificity

Reliability

Easy application

Low cost

Ask the Experts

Dr. U. Groβ Dr. S. Krappmann

Dr. J. Wagener

University Medical Center Göttingen

University Medical Center Erlangen

Ludwig-MaximiliansUniversity of Munich

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High specificity

Reliability

Easy application

Low cost

Outlook Make It Even Faster!

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Outlook Make It Even Faster!

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GFP ScaffoldsFragmented in 3

Regions

Using a GFP Scaffold

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GFP ScaffoldsFragmented in 3

Regions

Using a GFP Scaffold

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GFP ScaffoldsFragmented in 3

Regions

Using a GFP Scaffold

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Fusion PCR

GFP ScaffoldsFragmented in 3

Regions

Using a GFP Scaffold

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Fusion PCR

Plasmidconstruction

GFP ScaffoldsFragmented in 3

Regions

Using a GFP Scaffold

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Ultimate Goal

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Ultimate Goal

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Ultimate Goal

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Ultimate Goal

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Ultimate Goal

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Ultimate Goal

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Ultimate Goal

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What about Emma?

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What about Emma?

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It’s Fumigatus!

Special Thanks to...

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Questions?

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