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

2. 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

3. Staphylococcus aureus Gram positive cocci –facultative anaerobe Causes a variety of human diseases 7 sequenced strains Well defined molecular biology

4. Multiple human Gram positive pathogens kill C. elegans Time (hours) 050100150 0 25 50 75 100 S. aureus E. faecalis E. faecium S. pyogenes B. subtilis S. pneumoniae Survival (percent)

5. Multiple S. aureus laboratory strains kill C. elegans Time (hours) 050100 0 25 50 75 100 E. faecium *NCTC 8325 RN6390 *COL Reynolds Newman Survival (percent)

6. S. aureus accumulates in the C. elegans intestinal lumen 48 hours of feeding on S. aureus 8325

7. GFP labeled S. aureus accumulate in the C. elegans intestine S. aureus (RN6390) - GFP 24 hours - 63x magnification E. coli (DH5  ) - GFP 24 hours - 63x magnification

8. analysis time 050100125 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

9. A S. aureus V8 protease mutant is attenuated 050100 0 25 50 75 100 Survival (percent) Time (hours) RN6390B (wt) SP6391 (sspA-)

10. Conclusions C. elegans can be used to model S. aureus infection. S. aureus mutants attenuated in mammalian models are also attenuated in C. elegans

11. Transposon mutagenesis of S. aureus Choice of bacterial strain Choice of transposon vector Induction and selection of transposants

12. 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

13. pLTV1

14. RORF Transposon mutagenesis of S. aureus BlaermTn917 tet pLTV1 ColE1 pE194Ts BlaermTn917 ColE1 RF RO 42°C, erm(5) O/N incubation 32 96-well plates generated. 15% glycerol frozen stocks

15. 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?

16. 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

17. Problems with liquid transfer

18. O/N culture of S. aureus transposant Library 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 Protocol for Screen

19. RORF Plasmid Rescue protocol BlaermTn917 tet pLTV1 ColE1 pE194Ts BlaermTn917 ColE1 RF RO 42°C, erm(5) Genomic prep EcoRI digestion Bla ColE1 RO Ligation Transformation Bla ColE1 RO Sequence

20. Screen results I Number of mutants screened~2950 Number of mutants tested in secondary screen 145 (5%) Number of mutants sequenced 22 (~1%)

21. Screen results II MutantsGene identityFunction 3E1, 4D8, 8D9, 10B10, 22A5, 28C12, 29E1 OdhA + 2-oxoglutarate dehydrogenase 25G6, 29B8, 29G6OdhB *,+ dihydrolipoamide succinyltransferase 3H1DinG + Putative DNA helicase 5F15’ BraBBranched chain amino acid transporter 6A5SA0790Similar to N-acetyl-glucosamine catabolism homologue 7G12CitGFumarate hydratase, class II 15G12SA1241 + Similar to nitric-oxide reductase 28G125’ SA0467Similar YacA(B. subtilis)/HrpT (Listeria) 30A5PyrAAcarbamoyl-phosphate synthase small chain (pyrimidine/arg synthesis) 31B11??Downstream BraB

22. Representative results analysis time 020406080 0.00 0.25 0.50 0.75 1.00 3E1 3H1 4D8 5F8 6911 6A5 8325 5F1

23. Distribution of Insertion sites S. aureus chromosome 0 2.8 Mb 28G12 6A5 30A5 31B11 5F1 15G12 25G6 29B8 4D8 8D9 3E1 10B10 29E1 22A5 28C12 28C11 1.35 – 1.36 Mb 3H1 7G12 29C3

24. Other strategies Deletion mutagenesis Anti-sense RNA Modification of existing transposons Creation of a uni-gene transposon library

25. 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

26. 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

27. Acknowledgement s 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