Title: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis
1Caenorhabditis elegans as a model for
Staphylococcus aureus pathogenesis
- Jakob Begun
- Ausubel Lab - MGH
2Staphylococcus aureus is an important pathogen
- In 1995, nosocomial infections cost 4.5 billion
and resulted in 85,000 deaths - S. aureus is the leading cause of nosocomial
infection and a major cause of community acquired
pneumonia - MRSA accounts for gt50 of S. aureus infections
- VRSA strains isolated in US
3Staphylococcus aureus
- Gram positive cocci
- facultative anaerobe
- Causes a variety of human diseases
- 7 sequenced strains
- Well defined molecular biology
4Multiple human Gram positive pathogens kill C.
elegans
100
B. subtilis
E. faecium
75
S. pyogenes
Survival (percent)
50
E. faecalis
S. aureus
25
S. pneumoniae
0
0
50
100
150
Time (hours)
5Multiple S. aureus laboratory strains kill C.
elegans
100
E. faecium NCTC 8325 RN6390 COL Reynolds Newman
75
50
Survival (percent)
25
0
0
50
100
Time (hours)
6S. aureus accumulates in the C. elegans
intestinal lumen
48 hours of feeding on S. aureus 8325
7GFP labeled S. aureus accumulate in the C.
elegans intestine
S. aureus (RN6390) - GFP 24 hours - 63x
magnification
E. coli (DH5a) - GFP 24 hours - 63x magnification
8The regulator agr acts a virulence factor in C.
elegans
1.00
0.75
BS
6911 (agr)
ALC488 (sar)
0.50
6390
0.25
0.00
0
50
100
125
analysis time
9A S. aureus V8 protease mutant is attenuated
RN6390B (wt) SP6391 (sspA-)
100
75
Survival (percent)
50
25
0
0
50
100
Time (hours)
10Conclusions
- C. elegans can be used to model S. aureus
infection. - S. aureus mutants attenuated in mammalian models
are also attenuated in C. elegans
11Transposon mutagenesis of S. aureus
- Choice of bacterial strain
- Choice of transposon vector
- Induction and selection of transposants
12Sequenced S. aureus strains
- NCTC 8325 University of Oklahoma
- MRSA 252 Sanger Center
- MSSA 476 Sanger Center
- COL TIGR
- Mu50 - Juntendo University
- N315 - Juntendo University
- MW2 - Juntendo University
13pLTV1
14Transposon mutagenesis of S. aureus
42C, erm(5) O/N incubation
RORF
32 96-well plates generated. 15 glycerol frozen
stocks
15Setting up a screen for S. aureus virulence
factors
- Desired characteristics
- High throughput
- High sensitivity (negative predictive value)
- Reproducibility
- Size of library to screen
- Based on number of hits?
16High throughput liquid transfer assay
17Problems with liquid transfer
18Final Protocol for Screen
O/N culture of S. aureus transposant Library in
TSA (erm 5)
110 dilution
3 hour incubation on killing plates
- Incubate at 25 degrees
- Score at 48 hours
- Identify disrupted genes by arbitrary PCR or
plasmid rescue
Transfer synchronized L4 worms manually
(15/plate)
19Plasmid Rescue protocol
42C, erm(5)
RORF
Genomic prep
EcoRI digestion
Ligation
Bla
RO
ColE1
Transformation
Sequence
20Screen results I
Number of mutants screened 2950
Number of mutants tested in secondary screen 145 (5)
Number of mutants sequenced 22 (1)
21Screen results II
Mutants Gene identity Function
3E1, 4D8, 8D9, 10B10, 22A5, 28C12, 29E1 OdhA 2-oxoglutarate dehydrogenase
25G6, 29B8, 29G6 OdhB, dihydrolipoamide succinyltransferase
3H1 DinG Putative DNA helicase
5F1 5 BraB Branched chain amino acid transporter
6A5 SA0790 Similar to N-acetyl-glucosamine catabolism homologue
7G12 CitG Fumarate hydratase, class II
15G12 SA1241 Similar to nitric-oxide reductase
28G12 5 SA0467 Similar YacA(B. subtilis)/HrpT (Listeria)
30A5 PyrAA carbamoyl-phosphate synthase small chain (pyrimidine/arg synthesis)
31B11 ?? Downstream BraB
22Representative results
6911
1.00
5F1
0.75
3E1
4D8
0.50
0.25
6A5
5F8
3H1
0.00
8325
0
20
40
60
80
analysis time
23Distribution of Insertion sites
31B11 5F1 15G12 25G6 29B8 4D8 8D9 3E1 10B10 29E1 2
2A5 28C12 28C11
1.35 1.36 Mb
28G12
6A5
30A5
3H1
7G12
29C3
0
2.8 Mb
S. aureus chromosome
24Other strategies
- Deletion mutagenesis
- Anti-sense RNA
- Modification of existing transposons
- Creation of a uni-gene transposon library
25Conclusions
- A 3,000 member transposon insertion library has
been generated - This library has been screened in a C. elegans
model system - Identified mutants have been sequenced
- Site preference for Tn917 has been observed
26Future Plans
- Transduce unique mutants into clean genetic
background and re-test in C. elegans - Use positive transduced mutants to assess
virulence in a murine model - Characterize mutant phenotypes
27Acknowledgements
Massachusetts General Hospital Ausubel
Lab Danielle Garsin Dan Lee Sachiko Miyata Andrew
Diener Edward Kazyanskaya Sam Goodman Fred Ausubel
Calderwood Lab Costi Sifri Ruvkun Lab Dartmouth
Medical School Ambrose Cheung