Modeling model organisms in model systems? The case for Diphtheria Dr Paul A Hoskisson, Institute of Pharmacy and Biomedical Sciences, University of Strathclyde

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-1990s 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

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 Rs Genetically tractable Treatment model/ drug screening model

Optimisation of the worm model Time (h) % Survival of C. elegans post infection 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

Time (h) % Survival of C. elegans post infection 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 % 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 – invasiveAberdeen 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 Aberdeen Caenorhabditis Genetic Centre – University of Minnesota Society for General Microbiology