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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