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Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH
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Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Dec 25, 2015

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Page 1: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Caenorhabditis elegans as a model for

Staphylococcus aureus pathogenesis

Jakob BegunAusubel Lab -

MGH

Page 2: 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

Page 3: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Staphylococcus aureus

• Gram positive cocci– facultative anaerobe

• Causes a variety of human diseases

• 7 sequenced strains• Well defined molecular

biology

Page 4: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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)

Page 5: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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)

Page 6: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

S. aureus accumulates in the C. elegans intestinal lumen

48 hours of feeding on S. aureus 8325

Page 7: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

GFP labeled S. aureus accumulate in the C. elegans

intestine

S. aureus (RN6390) - GFP24 hours - 63x magnification

E. coli (DH5) - GFP24 hours - 63x magnification

Page 8: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 9: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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-)

Page 10: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Conclusions

• C. elegans can be used to model S. aureus infection.

• S. aureus mutants attenuated in mammalian models are also attenuated in C. elegans

Page 11: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Transposon mutagenesis of S. aureus

• Choice of bacterial strain• Choice of transposon vector• Induction and selection of

transposants

Page 12: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 13: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

pLTV1

Page 14: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 15: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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?

Page 16: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 17: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 18: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 19: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 20: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 21: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 22: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 23: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Distribution of Insertion sites

S. aureus chromosome

0 2.8 Mb

28G12 6A5 30A5

31B115F115G1225G629B84D88D93E110B1029E122A528C1228C11

1.35 – 1.36 Mb

3H1 7G12 29C3

Page 24: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

Other strategies

• Deletion mutagenesis• Anti-sense RNA• Modification of existing

transposons• Creation of a uni-gene transposon

library

Page 25: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 26: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

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

Page 27: Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis Jakob Begun Ausubel Lab - MGH.

AcknowledgemAcknowledgementsents

Massachusetts General Hospital

Ausubel LabDanielle GarsinDan LeeSachiko MiyataAndrew DienerEdward KazyanskayaSam GoodmanFred Ausubel

Calderwood LabCosti Sifri

Ruvkun Lab

Dartmouth Medical School

Ambrose Cheung