Download Caenorhabditis elegans as a model for Staphylococcus

Caenorhabditis elegans as a
model for Staphylococcus
aureus pathogenesis
Jakob Begun
Ausubel 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
Survival (percent)
Multiple human Gram positive
pathogens kill C. elegans
100
B. subtilis
E. faecium
75
S. pyogenes
50
S. aureus
E. faecalis
25
S. pneumoniae
0
0
50
100
Time (hours)
150
Multiple S. aureus laboratory
strains kill C. elegans
Survival (percent)
100
E. faecium
*NCTC 8325
RN6390
*COL
Reynolds
Newman
75
50
25
0
0
50
Time (hours)
100
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) - GFP
E. coli (DH5a) - GFP
24 hours - 63x magnification
24 hours - 63x magnification
The regulator agr acts a virulence
factor in C. elegans
1.00
0.75
BS
6911 (agr)
ALC488 (sar)
6390
0.50
0.25
0.00
0
50
analysis time
100
125
Survival (percent)
A S. aureus V8 protease mutant is
attenuated
RN6390B (wt)
SP6391 (sspA-)
100
75
50
25
0
0
50
Time (hours)
100
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
Transposon mutagenesis of S. aureus
pE194Ts
tet
pLTV1
Bla
erm
Tn917
ColE1
RORF
32 96-well plates
generated.
15% glycerol frozen
stocks
RO
42°C, erm(5)
O/N incubation
Bla
ColE1
erm
Tn917
RF
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
Egg prep gravid adults
(bleach treatment)
Allow eggs to hatch
overnight in M9W
Plate out synchronized
L1’s on OP50
Incubate for 48 hours
on OP50 plates @ 25º
Wash off L4’s and
plate on Staph TSA
Problems with liquid transfer
Percent killing
Liquid transfer vs. picking on 8325
1
0.8
0.6
0.4
0.2
0
8325 -48 hrs
8325 -72hrs
liq tx A liq Tx B liq Tx C liq Tx D pick A pick B
Final Protocol
for Screen
O/N culture of
S. aureus transposant
Library in TSA (erm 5)
1:10 dilution
3 hour incubation on
killing plates
Transfer synchronized
L4 worms manually
(~15/plate)
•Incubate at 25 degrees
•Score at 48 hours
•Identify disrupted genes
by arbitrary PCR or plasmid
rescue
Plasmid Rescue protocol
pE194Ts
tet
pLTV1
Bla
erm
RO
Tn917
Bla
Tn917
RF
ColE1
42°C, erm(5)
ColE1
erm
RORF
Genomic prep
EcoRI digestion
Ligation
RO
RO
Bla
ColE1
Sequence
Transformation
Bla
ColE1
Screen results I
Number of mutants screened
~2950
Number of mutants tested in
secondary screen
145 (5%)
Number of mutants
sequenced
22 (~1%)
Mutant Discovery Distribution
4
3
2
1
Plate
31
29
27
25
23
21
19
17
15
13
11
9
7
5
3
0
1
Number of mutants
5
Screen 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
Representative
results
6911
1.00
5F1
0.75
3E1
4D8
0.50
0.25
6A5
5F8
3H1
8325
0.00
0
20
40
analysis time
60
80
Distribution of
Insertion sites
31B11
5F1
15G12
25G6
29B8
4D8
8D9
3E1
10B10
29E1
22A5
28C12
28C11
28G12
6A5
30A5
1.35 – 1.36 Mb
3H1
7G12
0
29C3
2.8 Mb
S. aureus chromosome
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
Acknowledgements
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