1. Presented by Danielle A. Garsin Massachusetts General Hospital Harvard Medical School Modeling Gram-Positive Pathogen/Host Interactions Using Enterococcus and C. elegans

2. Advantages of using C. elegans Well characterized genetics and development Dies when fed a variety of bacterial and fungal pathogens. Factors involved found to be relevant to mammalian infection. Easy to mutagenize, many well-characterized mutants, RNAi library available Easy to grow, large # of progeny, three-day generation time Small and transparent

3. Enterococcus Facts Gram-positive cocci, related to Streptococcus urinary tract infections, bacteremia and endocarditis Genetic elements harboring drug resistant determinants Including vancomycin Little known about virulence factors and host defense Easy to grow in laboratory and amenable to genetic manipulation

4. Summary of Data Describing E. faecalis/C. elegans Model System E. faecalis, but not E. faecium kills C. elegans. Antibiotic-treated E. faecalis does not kill. E. faecalis kills males (not only a matricidal effect). A very small amount of E. faecalis can establish a persistent and deadly infection. The presence of two known enterococcal virulence factors relevant to mammalian pathogenesis (cytolysin and the fsr operon’s gene products) increases the rate of C. elegans death.

5. Group Meeting 5/28/03 Making an Enterococcus library with an insertion in all non-essential genes. –Background - initial screen –Numbers/Statistics thus far –Hot spot problem Understanding the relationships between germ-line, pathogenicity and longevity by studying C. elegans mutants. –Review of insulin signalling (daf) and germ-line proliferation mutants (glp) resistance to pathogens –Possible mechanism for extended lifespan of glp mutants (Dennis Kim)

6. Pathogen Random Transposon Mutagenesis Identification of Bacterial Virulence Factors by Screening for Attenuated Mutants Test for mutants attenuated in the nematode in a variety of mammalian models Screen mutants for those that don’t kill nematodes

7. Why Tn917? Used successfully in Enterococcus, Streptococcus, Bacillus and other Gram-positives. Stable once integrated. Easier to work with than Tn916 because it is smaller (5.2 kb vs. 16 kb).

8. pTV1-OK: temperature-sensitive carrier plasmid for Tn917 Tn917 pTV1-OK repAts-pWVO1 aph3 (kan) erm

9. How Screen Carried Out E. faecalis pTV1-OK Kan 28°C Erm 28°CErm 48°C transformants transposants Put nematodes on a lawn of each transposon mutant and took two time points. Those that caused longer than normal survival were assayed more carefully.

10. Mutants Attenuated in C. elegans DNA Repair Biosynthesis Transcriptional Regulation Quorum Sensing Sucrose Utilization # found Function(s) Homolog

11. Mouse Peritonitis Model skin peritoneal membrane organs Inject bacteria Results in systematic bloodstream infection and death.

12. 010203040 0 25 50 75 100 Time (hrs) % Survival scrB wildtype scrB Attenuated in Mouse Peritonitis Model

13. Mutants Attenuated in C. elegans DNA Repair Biosynthesis Transcriptional Regulation Quorum Sensing Sucrose Utilization # found Function(s) Homolog

14. Summary of Initial Tn917 Screen Screened 1038 transposants and obtained 20 mutants that were avirulent in C. elegans. Mutants had no growth defects and only one had a double insertion (determined by southern). By using primers specific to Tn917 and arbitrary primers, was able to obtain flanking sequence by PCR.

15. A collection of E. faecalis strains containing a disruption in each non-essential open reading frame (ORF) in the E. faecalis genome. Wild typeMutant #1Mutant #2 E. faecalis Ordered Insertion Library 1. Sequence large collection of transposants to create library 2. Screen library

16. TnP 1ARB PCR 1 Tn917 genomic E. faecalis DNA product TnP 2 ARB2 PCR 2 product How Library is Being Sequenced TnP 3 Sequence

17. Plans for Screening Every Non-essential Gene in E. faecalis StrainV583OG1RF Genome Size3.2 MB2.8 MB Non-essential gene estimate (#genes x 0.87) 2,8002,400 Size of Library to be screened (#non-essential x 5) 14,00012,000 Size of Library to be screened (sequencing inefficiencies 22%) 17,80015,300

18. Current Progress on Non-Essential Library # of OG1RF Transposants14,282 # Sequenced Thus Far5,496 # Trimmed Sequences4,087 # Sequences with Bit Score > 602,669 # Distinct Genes Hit377

19. Statistics on Current Progress % of Library Sequenced35.9% % Trimmed Sequences74.4% % Sequences with Bit Score > 60 out of total sequenced 48.6% % Sequences with Bit Score > 60 out of total trimmed sequence 65.3% # Distinct Genes Hit377 % of non-essential genes hit out of estimated total 16%

20. 1.5-1.6 MB 1894/2669 or 70% of Insertions are here # of Transposon Insertions over Genome (100 KB bins)

21. # of Transposon Insertions over Genome (10 KB bins)

22. Genome Location of Previously Identified Mutants Gene HomologLocation# Hits in Library photolyaseEF1598159 recQEF15450 scrREF160392 scrBEF1604107 sacUEF15700 oppAEF15130 dipeptidaseEF11570 cynR? lysR?EF13021 pai1EF15905 Shikimate 5- dehydrogenase EF156111 tcaAEF15421

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. Conclusions from Current Analysis of Library The major problem hampering the library construction is a 100 KB hot spot where 70% of the transposons are inserting. Due to the hot spot, at least twice as many mutants will need to be sequenced to get good (> 80%) coverage. Though the hot spot hampers library construction, it shows the importance of making this library if we ever hope to do a screen that reasonably saturates the genome.

25. Cold Spots: OG1RF compared to V583 25% of the V583 genome found to consist of mobile elements. It is reasonable to assume that OG1RF does not have many of the same mobile elements and this may account for some of the differences in genome size. Indeed, many of the “cold spots” are occuring where these mobile elements are located in V583.

26. Hot Spot Hits over 100 KB Regions Pathogenicity Island Vancomycin Resistance Plasmid Integration Phage Insertion

27. Part II of Group Meeting 5/28/03 Understanding the relationships between germ- line, pathogenicity and longevity by studying C. elegans mutants. –Review of insulin signaling (daf) and germ-line proliferation mutants (glp) resistance to pathogens –Possible mechanism for extended lifespan of glp mutants (Dennis Kim) –Can we extend what we have learned in C. elegans to the mouse?

28. Will the difference in lifespan of C. elegans longevity mutants disappear when grown on B. subtilis? Long-lived C. elegans Mutants = Enhanced Resistance to Pathogens Mutants (erp)?

29. Guarente & Kenyon, Nature (2000)

30. 0 10203040 0 25 50 75 100 wildtype daf-2 % Survival Time (days) E. coli B. subtilis Longevity of daf-2 on E.coli and B. subtilis

31. % Lifespan Extension Relative to wildtype daf-2 age-1 150 100 0 50 E. coli B. subtilis Lifespan Extension for Longevity Mutants is not as Dramatic on B. subtilis

32. Will the difference in lifespan of C. elegans longevity mutants disappear when grown on B. subtilis? Partially

33. Long-lived C. elegans Mutants = Enhanced Resistance to Pathogens Mutants (erp)? Will enhanced lifespan mutants be resistant to pathogen killing?

34. 05101520 0 25 50 75 100 % Survival Time (days) daf-2 age-1 wildtype Longevity Mutants Resistant to Killing by E. faecalis

35. 050100150 0 25 50 75 100 daf-2 age-1 wildtype % Survival Time (hours) Longevity Mutants Resistant to Killing by S. aureus C. Sifri & J. Begun

36. 050100150 0 25 50 75 100 daf-2 age-1 wildtype % Survival Time (hours) Longevity Mutants Resistant to Killing by P. aeruginosa D.Kim & J.Villaneuva

37. Guarente & Kenyon, Nature (2000)

38. 05101520 0 25 50 75 100 daf-16 daf-2 daf-2/16 wildtype % Survival on E. faecalis Time (days) daf-16 Blocks daf-2 Pathogen Resistance

39. Glp-1- Notch signaling receptor. Germ-line fails to proliferate. The few that exist enter meiosis and become sperm. Glp-4 - Not mapped. Germ-line fails to proliferate or enter meiosis. Stuck in prophase of the mitotic cell cycle. Recently it has been shown that both mutants increase lifespan by about 30%. Adapted from Austin and Kimble. Cell. 1987. Glp Mutants have Germ-Line Proliferation Defects

40. Glp Mutants are Resistant to Pathogens

41. K. Lin et al. Nature Genetics. 2001. Vol. 28. Germ-line Ablated Mutants Have More DAF-16 Localization to the Intestinal Cells

42. glp-4/daf-2 Resistance to E. faecalis Killing 010203040506070 0 50 100 wildtype glp-4 daf-2 daf-2/glp-4 Time (Days)

43. Part II Conclusions Insulin signaling/longevity mutants are more resistant to pathogens in a daf-16 dependent manner. Germ-line proliferation/longevity mutants are more resistant to pathogens possibly in a daf-16 dependent manner also. Longer lifespan in the glp mutants might be solely due to pathogen resistance.

44. M. Holzenberger et al. Nature. 2003. Vol. 421. Insulin-Growth Factor (daf-2 homolog) Mutant Mice Have a Longer Lifespan Will They Also be More Resistant to Pathogens?????

45. Acknowledgements Jas Villaneuva Jonathan Urbach Jake Begun Dan Lee Dennis Kim Nikki Liberati Costi Sifri Terry Moy Fred Ausubel postdoctoral funding: Irvington Institute for Immunological Research