Transcript Modeling Gram-positive pathogen/host interactions using C

Exploring Relationships Between
Longevity, Pathogenicity and Food
Source
and
Future Directions with
E. faecalis/C. elegans model
Presented by
Danielle A. Garsin
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
Outline
• Studying C. elegans mutants and relationship between
pathogenicity and longevity.
– C. elegans lifespan on E. coli vs. B. subtilis
– C. elegans lifespan mutations resistance to pathogens
• Preliminary and future studies of Enterococcus
mutants identified in screen.
–
–
–
–
Non-polar deletions, killing in C. elegans and mouse
Biofilm assay for Enterococcus
Macrophage assay for Enterococcus
Pilot RT-PCR
Is E. coli a completely nonpathogenic food source?
Nematode food (E. coli strain OP50) Can Kill
% Survival
100
75
50
BHI
NG
25
0
0
50
100
Time (hrs)
150
C. elegans Live 30-50% Longer on
B. subtilis than E. coli (25°C)
% C. elegans alive
100
N2 (E. coli)
N2 (B. subtilis)
75
50
25
0
0
10
20
Time (days)
30
C. elegans Live 2x Longer on
B. subtilis than E. coli (27°C)
% C. elegans alive
100
75
50
B. subtilis
E. coli
25
0
0
5
10
Time (days)
15
20
Esp-3 has a Shortened Lifespan
on E. coli, but not B. subtilis
% C. elegans alive
100
esp-3 (E. coli)
N2 (E. coli)
esp-3 (B. subtilis)
N2 (B. subtilis)
75
50
25
0
0
10
20
Time (days)
30
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)?
Lifespan Extension for Longevity
Mutants is not as Dramatic on B. subtilis
% Lifespan Extension
Relative to N2
150
OP50
PY79
100
50
0
age-1
daf-2
% Lifespan Extension on
PY79 relative to OP50
Lifespan Extension for Longevity
Mutants is not as Dramatic on B. subtilis
60
50
40
30
20
10
0
N2
age-1
daf-2
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
N2
50
25
0
0
5
10
Time (days)
15
20
Longevity Mutants Resistant
to Killing by S. aureus
daf-2
age-1
N2
% Survival
100
75
50
25
0
0
50
100
Time (hours)
150
Longevity Mutants Resistant
to Killing by P. aeruginosa
100
daf-2
age-1
N2
% Survival
75
50
25
0
0
50
100
Time (hours)
150
Conclusions
• C. elegans live longer on B. subtilis than they do
on E. coli.
• At least one enhanced susceptibility to
pathogens mutant (esp-3) is sensitive to E. coli
but not B. subtilis on NG medium plus FUDR
• Two C. elegans mutants with enhanced longevity
have less dramatic increases in lifespan on B.
subtilis suggesting part of the phenotype may be
due to resistance to pathogens
• These two longevity mutants are indeed more
resistant to pathogens
Outline
• Studying C. elegans mutants and relationship between
pathogenicity and longevity.
– C. elegans lifespan on E. coli vs. B. subtilis
– C. elegans lifespan mutations resistance to pathogens
• Preliminary and future studies of Enterococcus
mutants identified in screen.
–
–
–
–
Non-polar deletions, killing in C. elegans and mouse
Biofilm assay for Enterococcus
Macrophage assay for Enterococcus
Pilot RT-PCR
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.
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?
Transposon Insertions vs. Non-polar Deletions
Operon
scrB
scrR
Transposon insertion
sc
Tn917
rB
scrR
Non-polar (in-frame) deletion
s B scrR
C. elegans Killing with Non-Polar scrB Deletion
% Survival
100
75
wildtype
50
25
Transposon
insertion
Non-polar
deletion
0
0
50
100
Time (hours)
150
200
Possible Function of Sucrose
Utilization in Pathogenesis
Sucrose used to make glucan and fructan,
part of the extracellular polysaccharide matrix
which helps some bacteria stick to surfaces.
Biofilm Attachment Assay
0.18
0.16
0.14
OD 600
0.12
0.1
0.08
0.06
0.04
0.02
0
wildtype
scrB
fsrB
Preliminary data suggests that scrB may
be attenutated in the rat endocarditis
model
Markus Hufnagel, Johannes Huebner
Channing Laboratory
Possible Function of DNA Repair
Enzymes in Pathogenesis
Macrophage
ROS
DNA enzymes could be needed to repair damage
from reactive oxygen species (ROS).
Isolation of Human Macrophages for Assay
• Collect 150 mls of human blood from healthy
donor.
• Run over Histopaque 1077 gradient (Sigma) to
isolate mononuclear cells
• Incubate in culture in donor’s serum for a week to
allow macrophage differentiation.
Antibiotics
Intracellular Survival
Expected Outcome of Macrophage Assay
Wildtype
Mutant
Time
Analyzing Gene Expression of New
Virulence Factors by RT-PCR
OD = 0.262
RNA dilution*
1
0.1
0.01
OD = 1.688
1
0.1
0.01
scrB
*RNA prepped from approximately equal number of cfu’s
Differential Gene Expression of
Internalized Bacteria?
2. Wash
nematodes
1. Feed nematodes E. faecalis
3. Disrupt nematodes and isolate RNA from internalized
bacteria. Compare gene expression to bacteria on lawn.
Use specific primers to look at gene of interest or arbitrary
primers and gene chip to compare global differences.
More Details on C. elegans Disruption and
Bacterial RNA Isolation
• Disrupt washed nematodes with a tissue-tearor
(BioSpec). Low speed centrifugation to spin down
tissue.
• Isolate bacteria by high speed centrifugation,
wash.
• Isolate total RNA using Mini-BeadBeater-8
(Biospec) following Gilmore lab protocol*
*(www.enterococcus.ouhsc.edu/rna_Isolationl.asp)
Future Plans
• Current E. faecalis mutants: Test in other model
systems. Continue analysis.
• E. faecalis screen (help Terry): Screen nonredundant transposon-insertion library of all nonessential E. faecalis genes.
• Host Factors: Finish analysis of lifespan
mutants.
Acknowledgements
MGH
Costi Sifri
Jake Begun
Terry Mylonakis
Jas Villaneuva
Sylvia Lee
Dennis Kim
Steve Calderwood
Gary Ruvkun
Fred Ausubel
Harvard Medical School
Markus Hufnagel
Johannes Huebner
postdoctoral funding:
Irvington Institute for Immunological Research