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Modeling model organisms in model systems? The case for Diphtheria

Modeling model organisms in model systems? The case for Diphtheria

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Modeling model organisms in model systems? The case for Diphtheria

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  1. Modeling model organisms in model systems? The case for Diphtheria Dr Paul A Hoskisson, Institute of Pharmacy and Biomedical Sciences, University of Strathclyde Email: paul.hoskisson@strath.ac.uk

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Our model system – C. diphtheriae- Caenorhabditis elegans model • 3 R’s • Genetically tractable • Treatment model/ drug screening model

  8. % Survival of C. elegans post infection Time (h) Optimisation of the worm model Worm survival is impaired following infection C. diphtheriae localise to the pharynx- adhesion and persistence in non-invasive strains Bacterial load increases over time

  9. 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

  10. % Survival of C. elegans post infection Time (h) Screening libraries of multicopy vectors • Genomic fragments of DSM43988 (~3Kbp) in pNV18Incubated with C. elegans and survival monitored Amenable to high-throughput screens

  11. 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 Acanthamoeba polyphaga can be used to assay bacterial virulence Media concentration 100-10% Amoebae numbers 10,000-10 Virulent strain Avirulent strain

  12. 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

  13. Attachment and invasion of D562 mammalian cells is variable too

  14. 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

  15. Variation in cell surfaces

  16. 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?

  17. 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 Aberdeen Caenorhabditis Genetic Centre – University of Minnesota Society for General Microbiology