Danielle A. Garsin

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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

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

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Identification of Bacterial Virulence Factors by
Screening for Attenuated Mutants

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Screen mutants for those that dont kill nematodes
Test for mutants attenuated in the nematode in a
variety of mammalian models
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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).

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pTV1-OK temperature-sensitive carrier plasmid
for Tn917
repAts-pWVO1
Tn917
aph3 (kan)
pTV1-OK
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How Screen Carried Out
transformants
E. faecalis
Kan 28C
transposants
Erm 28C
Erm 48C
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.
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Mutants Attenuated in C. elegans
found
Homolog
Function(s)
DNA Repair
Sucrose Utilization
Quorum Sensing
Transcriptional Regulation
Biosynthesis
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Mouse Peritonitis Model
Inject bacteria
skin
peritoneal membrane
organs
Results in systematic bloodstream infection and
death.
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scrB Attenuated in Mouse Peritonitis Model
100
75
Survival
50
scrB wildtype

25
0
0
10
20
30
40
Time (hrs)
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Mutants Attenuated in C. elegans
found
Homolog
Function(s)
DNA Repair
Sucrose Utilization
Quorum Sensing
Transcriptional Regulation
Biosynthesis
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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.

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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

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How Library is Being Sequenced
Tn917
genomic E. faecalis DNA
PCR 1
TnP 1
ARB
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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
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Current Progress on Non-Essential Library
of OG1RF Transposants 14,282
Sequenced Thus Far 5,496
Trimmed Sequences 4,087
Sequences with Bit Score gt 60 2,669
Distinct Genes Hit 377
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Statistics on Current Progress
of Library Sequenced 35.9
Trimmed Sequences 74.4
Sequences with Bit Score gt 60 out of total sequenced 48.6
Sequences with Bit Score gt 60 out of total trimmed sequence 65.3
Distinct Genes Hit 377
of non-essential genes hit out of estimated total 16
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of Transposon Insertions over Genome (100 KB
bins)
1.5-1.6 MB 1894/2669 or 70 of Insertions are here
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of Transposon Insertions over Genome (10 KB
bins)
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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 5-dehydrogenase EF1561 11
tcaA EF1542 1
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Distribution of Insertion sites
31B11 5F1 15G12 25G6 29B8 4D8 8D9 3E1 10B10 29E1 2
2A5 28C12 28C11
1.35 1.36 Mb
28G12
6A5
30A5
3H1
7G12
29C3
0
2.8 Mb
S. aureus chromosome
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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 (gt
  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.

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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.

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Hits over 100 KB Regions
Hot Spot
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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?

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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)?

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Guarente Kenyon, Nature (2000)
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Longevity of daf-2 on E.coli and B. subtilis
100
E. coli
75
daf-2
Survival
wildtype
50
B. subtilis

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daf-2
wildtype
0
0
10
20
30
40
Time (days)
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Lifespan Extension for Longevity Mutants is not
as Dramatic on B. subtilis
150
E. coli
B. subtilis
100
Lifespan Extension Relative to wildtype
50
0
daf-2
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Will the difference in lifespan of C. elegans
longevity mutants disappear when grown on B.
subtilis? Partially
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Long-lived C. elegans Mutants Enhanced
Resistance to Pathogens Mutants (erp)? Will
enhanced lifespan mutants be resistant to
pathogen killing?
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Longevity Mutants Resistant to Killing by E.
faecalis
100
75
daf-2 age-1 wildtype
Survival
50
25
0
0
5
10
15
20
Time (days)
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Longevity Mutants Resistant to Killing by S.
aureus
daf-2 age-1 wildtype
100
75
Survival
50
25
C. Sifri J. Begun
0
0
50
100
150
Time (hours)
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Longevity Mutants Resistant to Killing by P.
aeruginosa
100
daf-2 age-1 wildtype
75
Survival
50
25
D.Kim J.Villaneuva
0
0
50
100
150
Time (hours)
37
Guarente Kenyon, Nature (2000)
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daf-16 Blocks daf-2 Pathogen Resistance
100
75
daf-16 daf-2 daf-2/16 wildtype
Survival on E. faecalis
50

25
0
0
5
10
15
20
Time (days)
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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.
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Glp Mutants are Resistant to Pathogens
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Germ-line Ablated Mutants Have More DAF-16
Localization to the Intestinal Cells
K. Lin et al. Nature Genetics. 2001. Vol. 28.
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glp-4/daf-2 Resistance to E. faecalis Killing
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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.

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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.
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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