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
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 nontoxigenic 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 nontoxigenic C. diphtheriae?
• Increasing numbers of cases, limited testing,
27 case in Grampian region in the last 5 years
Number of laboratory confirmed cases
300
Toxigenic
Nontoxigen
250
200
150
100
50
0
1990
1992
1994
1996
1998
Year
2000
2002
2004
2006
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. diphtheriaeCaenorhabditis elegans model
• 3 R’s
• Genetically tractable
• Treatment model/ drug screening model
% Survival of C. elegans post
infection
Optimisation of the worm model
Time (h)
Worm survival is impaired following infection
7
8x10
C. diphtheriae localise to the
pharynx- adhesion and
persistence in non-invasive
strains
7
7x10
7
6x10
CFU per worm
7
5x10
7
4x10
7
3x10
7
2x10
7
1x10
0
0
20
40
60
80
100
120
Time (h)
Bacterial load increases over time
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
Screening libraries of multicopy
vectors
• Genomic fragments of DSM43988 (~3Kbp) in pNV18
Incubated with C. elegans and survival monitored
Amenable to high-throughput screens
of C. elegans
infection
% Survival
Worm survival
followingpost
infection
(%)
100
95
90
85
80
75
NonInvWT
Clone16
Clone18
Clone21
Clone28
70
65
60
0
20
40
60
Time (h)
Time
(h)
80
100
120
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
Media concentration
100-10%
Amoebae
numbers
10,000-10
Avirulent strain
Virulent strain
Amoeba model allows the study
of adhesion and invasion
Amoebae (Brightfield)
DSM43988 – noninvasive
Fluorescent C. dip with
amoebae
ISS3319 – ‘invasive’
Merged C. dip with
amoebae
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 modelsmathematical? 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