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Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis

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

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Caenorhabditis elegans as a model for Staphylococcus aureus pathogenesis

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  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 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)

  5. Multiple 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)

  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 (DH5a) - GFP 24 hours - 63x magnification

  8. 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 50 100 125 analysis time

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

  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. pE194Ts tet pLTV1 Bla erm Tn917 ColE1 Bla erm Tn917 ColE1 RF RO Transposon mutagenesis of S. aureus 42°C, erm(5) O/N incubation RORF 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. 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 High throughput liquid transfer assay

  17. Problems with liquid transfer

  18. 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 • Incubate at 25 degrees • Score at 48 hours • Identify disrupted genes by arbitrary PCR or plasmid rescue Transfer synchronized L4 worms manually (~15/plate)

  19. pE194Ts tet pLTV1 RF RO Bla erm Tn917 ColE1 Bla erm Tn917 ColE1 RO Bla ColE1 Plasmid Rescue protocol 42°C, erm(5) RORF Genomic prep EcoRI digestion Ligation RO Bla ColE1 Transformation Sequence

  20. Screen results I

  21. Screen results II

  22. Representative 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

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

  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. 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

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