Caenorhabditis elegans as a model for
Staphylococcus aureus pathogenesis
Jakob BegunAusubel Lab -
MGH
Staphylococcus 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 >50% of S. aureus infections
• VRSA strains isolated in US
Staphylococcus aureus
• Gram positive cocci– facultative anaerobe
• Causes a variety of human diseases
• 7 sequenced strains• Well defined molecular
biology
Multiple human Gram positive pathogens kill C. elegans
Time (hours)
0 50 100 1500
25
50
75
100
S. aureusE. faecalis
E. faecium
S. pyogenes
B. subtilis
S. pneumoniaeSu
rviv
al (p
erc
en
t)
Multiple S. aureus laboratory strains kill C. elegans
Time (hours)
0 50 100
0
25
50
75
100
E. faecium *NCTC 8325RN6390*COLReynoldsNewman
Su
rviv
al (p
erc
en
t)
S. aureus accumulates in the C. elegans intestinal lumen
48 hours of feeding on S. aureus 8325
GFP labeled S. aureus accumulate in the C. elegans
intestine
S. aureus (RN6390) - GFP24 hours - 63x magnification
E. coli (DH5) - GFP24 hours - 63x magnification
analysis time0 50 100 125
0.00
0.25
0.50
0.75
1.00
6390
6911 (agr)
ALC488 (sar)
BS
The regulator agr acts a virulence factor in C. elegans
A S. aureus V8 protease mutant is attenuated
0 50 100
0
25
50
75
100
Su
rviv
al (p
erc
en
t)
Time (hours)
RN6390B (wt)SP6391 (sspA-)
Conclusions
• C. elegans can be used to model S. aureus infection.
• S. aureus mutants attenuated in mammalian models are also attenuated in C. elegans
Transposon mutagenesis of S. aureus
• Choice of bacterial strain• Choice of transposon vector• Induction and selection of
transposants
Sequenced 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
pLTV1
RORF
Transposon mutagenesis of S. aureus
Bla erm Tn917
tet
pLTV1
ColE1
pE194Ts
Bla erm Tn917
ColE1
RFRO
42°C, erm(5)O/N incubation
32 96-well plates generated.15% glycerol frozen stocks
Setting 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?
High throughput liquid transfer assay
Wash off L4’s and plate on Staph TSA
Egg prep gravid adults (bleach
treatment)Allow eggs to hatch overnight in M9W
Incubate for 48 hours on OP50 plates @ 25º
Plate out synchronized L1’s on
OP50
Problems with liquid transfer
Liquid transfer vs. picking on 8325
0
0.2
0.4
0.6
0.8
1
liq tx A liq Tx B liq Tx C liq Tx D pick A pick B
Perc
ent k
illing 8325 -48 hrs
8325 -72hrs
O/N culture ofS. aureus transposantLibrary in TSA (erm 5)
•Incubate at 25 degrees•Score at 48 hours•Identify disrupted genes by arbitrary PCR or plasmid rescue
1:10 dilution
3 hour incubation on killing plates
Transfer synchronized L4 worms manually (~15/plate)
Final Protocolfor Screen
RORF
Plasmid Rescue protocol
Bla erm Tn917
tet
pLTV1
ColE1
pE194Ts
Bla erm Tn917
ColE1
RFRO
42°C, erm(5)
Genomic prep
EcoRI digestion
Bla
ColE1
RO
Ligation
TransformationBla
ColE1
RO
Sequence
Screen results INumber of mutants screened
~2950
Number of mutants tested in secondary screen
145 (5%)
Number of mutants sequenced
22 (~1%)
M utant Discovery Distribution
0
1
2
3
4
5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
Plate
Num
ber o
f mut
ants
Frequency distribution of m utants /plate
0
2
4
6
8
10
12
14
16
18
20
0 1 2 3 4 5
number of mutants/ plate
Screen results IIMutants Gene
identityFunction
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
Representative results
analysis time0 20 40 60 80
0.00
0.25
0.50
0.75
1.00
3E1
3H1
4D8
5F8
6911
6A5
8325
5F1
Distribution of Insertion sites
S. aureus chromosome
0 2.8 Mb
28G12 6A5 30A5
31B115F115G1225G629B84D88D93E110B1029E122A528C1228C11
1.35 – 1.36 Mb
3H1 7G12 29C3
Other strategies
• Deletion mutagenesis• Anti-sense RNA• Modification of existing
transposons• Creation of a uni-gene transposon
library
Conclusions
• 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
Future 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
AcknowledgemAcknowledgementsents
Massachusetts General Hospital
Ausubel LabDanielle GarsinDan LeeSachiko MiyataAndrew DienerEdward KazyanskayaSam GoodmanFred Ausubel
Calderwood LabCosti Sifri
Ruvkun Lab
Dartmouth Medical School
Ambrose Cheung