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Modeling Gram-Positive Pathogen/Host Interactions Using Enterococcus and C. elegans Presented by Danielle A. Garsin Massachusetts General Hospital Harvard Medical School Advantages of using C. elegans Small and transparent Well characterized genetics and development Easy to grow, large # of progeny, three-day generation time Easy to mutagenize, many well-characterized mutants, RNAi library available Dies when fed a variety of bacterial and fungal pathogens. Factors involved found to be relevant to mammalian infection. 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 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. 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) Identification of Bacterial Virulence Factors by Screening for Attenuated Mutants Pathogen Random Transposon Mutagenesis Screen mutants for those that don’t kill nematodes Test for mutants attenuated in the nematode in a variety of mammalian models 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). pTV1-OK: temperature-sensitive carrier plasmid for Tn917 repAts-pWVO1 Tn917 pTV1-OK erm aph3 (kan) How Screen Carried Out transformants pTV1-OK E. faecalis Kan 28°C transposants Erm 28°C Erm 48°C 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. Mutants Attenuated in C. elegans # found Homolog Function(s) 2 photolyase DNA repair 1 recQ DNA helicase 9 scrR repressor of sucrose operon 1 scrB sucrose-6-phosphate hydrolyase 1 sacU Quorum Sensing 1 oppA 1 dipeptidase Transcriptional Regulation 1 cynR? lysR ? 1 pai1 DNA Repair Sucrose Utilization 1 Biosynthesis 1 shikimate 5dehydrogenase tcaA (weak homolog) two-component regulator of sucrose utilization oligopeptide binding protein amino acid utilization? oligopeptide processing? positive transcriptional regulator (weak) negative transcriptional regulator of sporulation aromatic amino acid biosynthesis membrane protein, cell wall synthesis? Mouse Peritonitis Model Inject bacteria skin peritoneal membrane organs Results in systematic bloodstream infection and death. scrB Attenuated in Mouse Peritonitis Model % Survival 100 75 50 scrB wildtype 25 0 0 10 20 Time (hrs) 30 40 Mutants Attenuated in C. elegans # found Homolog Function(s) 2 photolyase DNA repair 1 recQ DNA helicase 9 scrR repressor of sucrose operon 1 scrB sucrose-6-phosphate hydrolyase 1 sacU Quorum Sensing 1 oppA 1 dipeptidase Transcriptional Regulation 1 cynR? lysR ? 1 pai1 DNA Repair Sucrose Utilization 1 Biosynthesis 1 shikimate 5dehydrogenase tcaA (weak homolog) two-component regulator of sucrose utilization oligopeptide binding protein amino acid utilization? oligopeptide processing? positive transcriptional regulator (weak) negative transcriptional regulator of sporulation aromatic amino acid biosynthesis membrane protein, cell wall synthesis? 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. E. faecalis Ordered Insertion Library A collection of E. faecalis strains containing a disruption in each non-essential open reading frame (ORF) in the E. faecalis genome. Wild type Mutant #1 Mutant #2 1. Sequence large collection of transposants to create library 2. Screen library How Library is Being Sequenced Tn917 genomic E. faecalis DNA PCR 1 ARB TnP 1 product PCR 2 ARB2 TnP 2 product Sequence TnP 3 Plans for Screening Every Non-essential Gene in E. faecalis Strain V583 OG1RF Genome Size 3.2 MB 2.8 MB Non-essential gene estimate (#genes x 0.87) 2,800 2,400 Size of Library to be screened (#non-essential x 5) 14,000 12,000 Size of Library to be screened (sequencing inefficiencies 22%) 17,800 15,300 Current Progress on Non-Essential Library # of OG1RF Transposants 14,282 # Sequenced Thus Far 5,496 # Trimmed Sequences 4,087 # Sequences with Bit Score > 60 2,669 # Distinct Genes Hit 377 Statistics on Current Progress % of Library Sequenced 35.9% % Trimmed Sequences 74.4% % 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 Hit 377 % of non-essential genes hit out of estimated total 16% # of Transposon Insertions over Genome (100 KB bins) 2000 1750 1500 1.5-1.6 MB 1894/2669 or 70% of Insertions are here 1250 1000 750 500 250 0 Genome Divided into 100 KB Bins # of Transposon Insertions over Genome (10 KB bins) 1000 750 500 250 0 Genome Divided into 10 KB Bins Genome Location of Previously Identified Mutants Gene Homolog Location # Hits in Library photolyase EF1598 159 recQ EF1545 0 scrR EF1603 92 scrB EF1604 107 sacU EF1570 0 oppA EF1513 0 dipeptidase EF1157 0 cynR? lysR? EF1302 1 pai1 EF1590 5 Shikimate 5dehydrogenase EF1561 11 tcaA EF1542 1 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 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. 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. Hits over 100 KB Regions 75 25 Hot Spot 50 0 Pathogenicity Island Vancomycin Resistance Plasmid Integration Phage Insertion Part II of Group Meeting 5/28/03 • Understanding the relationships between germline, 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? 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)? Guarente & Kenyon, Nature (2000) Longevity of daf-2 on E.coli and B. subtilis 100 % Survival E. coli 75 daf-2 wildtype 50 B. subtilis 25 daf-2 wildtype 0 0 10 20 Time (days) 30 40 Lifespan Extension for Longevity Mutants is not as Dramatic on B. subtilis % Lifespan Extension Relative to wildtype 150 E. coli B. subtilis 100 50 0 daf-2 age-1 Will the difference in lifespan of C. elegans longevity mutants disappear when grown on B. subtilis? Partially Long-lived C. elegans Mutants = Enhanced Resistance to Pathogens Mutants (erp)? Will enhanced lifespan mutants be resistant to pathogen killing? Longevity Mutants Resistant to Killing by E. faecalis 100 % Survival 75 daf-2 age-1 wildtype 50 25 0 0 5 10 Time (days) 15 20 Longevity Mutants Resistant to Killing by S. aureus daf-2 age-1 wildtype % Survival 100 75 50 25 C. Sifri & J. Begun 0 0 50 100 Time (hours) 150 Longevity Mutants Resistant to Killing by P. aeruginosa 100 daf-2 age-1 wildtype % Survival 75 50 25 D.Kim & J.Villaneuva 0 0 50 100 Time (hours) 150 Guarente & Kenyon, Nature (2000) % Survival on E. faecalis daf-16 Blocks daf-2 Pathogen Resistance 100 75 daf-16 daf-2 daf-2/16 wildtype 50 25 0 0 5 10 Time (days) 15 20 Glp Mutants have Germ-Line Proliferation Defects 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. Adapted from Austin and Kimble. Cell. 1987. Recently it has been shown that both mutants increase lifespan by about 30%. Glp Mutants are Resistant to Pathogens wildtype glp-1 glp-4 100 50 0 0.0 2.5 5.0 7.5 Time (Days) 10.0 12.5 Germ-line Ablated Mutants Have More DAF-16 Localization to the Intestinal Cells K. Lin et al. Nature Genetics. 2001. Vol. 28. glp-4/daf-2 Resistance to E. faecalis Killing wildtype glp-4 daf-2 daf-2/glp-4 100 50 0 0 10 20 30 40 Time (Days) 50 60 70 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. Insulin-Growth Factor (daf-2 homolog) Mutant Mice Have a Longer Lifespan Will They Also be More Resistant to Pathogens????? M. Holzenberger et al. Nature. 2003. Vol. 421. Acknowledgements Jas Villaneuva Jake Begun Dennis Kim Costi Sifri Jonathan Urbach Dan Lee Nikki Liberati Terry Moy Fred Ausubel postdoctoral funding: Irvington Institute for Immunological Research