Modeling model organisms in model systems? The case for

Diphtheria

Dr Paul A Hoskisson,Institute of Pharmacy and Biomedical Sciences,

University of StrathclydeEmail: paul.hoskisson@strath.ac.uk

Corynebacterium diphtheriae• Aetiological agent of Diphtheria – phage conversion

• Controlled by vaccination since 1945

• Still causes ~5000 deaths per year worldwide• Resurgence in Eastern Europe in mid-1990’s• Emergence of non-toxigenic disease causing strains

Non-toxigenic C. diphtheriae• Causes persistent sore throats, pharyngitis,

deep tissue infections, osteomyelitits, endocarditis in immuno-compromised

• Increasing infections in immuno-competent patients

• Can be invasive

Why are we interested in non-toxigenic C. diphtheriae?

• Increasing numbers of cases in UK – no explanation why• We know little about colonisation, persistence and invasion in hosts,

carriage levels etc• Unusual antibiotic resistances• We know little about virulence factors outside of the toxin• We know little about genome and population structure in C. diphtheriae

Why are we interested in non-toxigenic C. diphtheriae?

• Increasing numbers of cases, limited testing, 27 case in Grampian region in the last 5 years

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How are we approaching this problem?

• Identification of novel virulence factors– Transposon mutagenesis– Promoter-probe libraries– Gene dosage libraries

• Understanding colonisation (adhesion & Invasion)– Novel tractable models

• Understanding population and genome structure

Our model system – C. diphtheriae- Caenorhabditis

elegans model

• 3 R’s

• Genetically tractable

• Treatment model/ drug screening model

Optimisation of the worm model

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Bacterial load increases over time

Worm survival is impaired following infection

C. diphtheriae localise to the pharynx- adhesion and persistence in non-invasive strains

Optimisation of the worm model: Infection of C. elegans with invasive and non

invasive C. diptheriae strains

C. elegans infected with invasive C. diptheriae (ISS3319) – 2 d

C. elegans infected with non-invasive C. diptheriae (DSM43988) – 2 d

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Screening libraries of multicopy vectors

• Genomic fragments of DSM43988 (~3Kbp) in pNV18

Incubated with C. elegans and survival monitored

Amenable to high-throughput screens

Acanthamoeba polyphaga can be used to assay bacterial virulence

• A. polyphaga is a free-living amoeba found in soil and water

• Associations between Acanthamoeba and bacteria are known in the environment– M. ulcerans – Buruli Ulcer– Legionella – Microbial gymnasia

• Used as a macrophage model- similar survival strategies

Avirulent strain

Virulent strain

Media concentration

100-10%

Amoebae numbers

10,000-10

Amoeba model allows the study of adhesion and invasion

Amoebae (Brightfield)

Fluorescent C. dip with amoebae

Merged C. dip with amoebae

DSM43988 – non-invasive

ISS3319 – ‘invasive’ Aberdeen strain 1 –invasive

Attachment and invasion of D562 mammalian cells is

variable too

Difference in strains• Strains supposed to be highly similar –

pathogenicity differences due to the presence of bacteriophage

• View is changing – microarray studies show at least 30 loci different in an outbreak strain vs vaccine strain

• Recent MLST analysis shows high levels of strain variation

• Phenotypic variation –inability to ferment sucrose diagnostic

Variation in cell surfaces

What would we like to do?• Cells in C. elegans all mapped and the

developmental process

• Genetic tools available for C. diphtheriae– e.g. Toll mutant

• Lends its self perfectly to study colonisation, persistence, invasion and disease progression

• Amenable to high throughput screens

• Develop models of infection in models- mathematical? Exploit image processing technology?

Acknowledgements• Ashleigh McKenzie• Teresa Baltazar• Dr Alison Hunt • Dr Rebecca Edwards

• Prof Andreas Burkovski – University of Erlangen– Andrea Bischof– Sabine Rodel

Dr Maria Sanchez-Contreras – University of Bath

Dr Jonathon Pettit & Dr Neale Harrison – University of AberdeenCaenorhabditis Genetic Centre – University of Minnesota

Society for General Microbiology