Transcript PowerPoint - Oregon State University

Kate Bateman
Mentors: Dr. Dennis Hruby
…… Tove’ Bolken
Department of Microbiology
-Gram+ bacterial pathogens:
ANTIBIOTIC RESISTANCE
- Staphylococcus aureus:
HOSPITAL-ACQUIRED INFECTIONS
95% penicillin resistant
50% methicillin resistant
Vancomycin resistance on the rise
-Anti-infective compounds
-Block tissue attachment
-Keep bacteria from hiding from the immune system
-Work on antibiotic-resistant strains
-Less likely to lead to resistance
-Compounds don’t kill bacteria Less selective pressure
S. aureus
adhering to host
tissue
S. aureus
infecting wound
tissue
-Highly conserved
in G+ bacteria
-High Throughput
Screening (HTS)
assays
-Test potential drug
candidates
-Bacteria growth inhibition assays
Incubate strains in the
presence of the inhibiting
compound, or a control
Measure optical density of
each treatment
S. aureus: Compound 6959776
S. aureus: Compound 6054217
0.9
0.9
0.8
0.8
0.6
tetracycline
0.5
0.4
2.5 uM
5 uM
0.3
50 uM
0.2
100 uM
Control
0.6
tetracycline
0.5
2.5 uM
0.4
0.3
5 uM
50 uM
0.2
100 uM
0.1
0.1
0
0
0
1
2
3
4
5
0
6
0.8
0.7
0.6
0.5
Control
tetracycline
0.4
2.5 uM
0.3
5 uM
10 uM
0.2
0.1
0
1
2
3
Time (hours)
2
3
4
5
6
S. aureus (G+) has the
sortase enzyme.
S. aureus: Compound 7570470
0
1
Time (hours)
Time (hours)
OD 610
0.7
Control
OD 610
OD 610
0.7
4
5
6
Growth slightly inhibited
as concentration of
compound increases
E. coli: Compound 6959776
1
1
0.9
0.9
0.8
Control
0.8
0.7
0.6
tetracycline
0.7
0.6
0.5
chloramphenicol
5 uM
0.4
25 uM
0.3
100 uM
0.3
0.2
200 uM
0.2
0.1
Control
tetracycline
chloramphenicol
0.5
5 uM
0.4
25 uM
100 uM
0.1
0
0
0
5
10
15
20
25
0
Time (hours)
0.9
0.8
Control
0.7
tetracycline
0.6
0.5
chloramphenicol
1.25 uM
0.4
5 uM
0.3
25 uM
0.2
50 uM
0.1
0
5
10
15
Time (hours)
10
15
20
25
E. coli (G-) does not have
the sortase enzyme
1
0
5
Time (hours)
E. coli: Compound 7570470
OD 610
OD 610
OD 610
E. coli: Compound 6054217
20
25
Growth not inhibited at
higher concentrations of
compound
-Surface protein assays
Western blot for protein A
Protein A: Surface factor that
inhibits phagocytes from
engulfing the bacterium
-Grow S. aureus in presence or
absence of sortase-inhibiting
compounds
-Control (no compounds)
-6054217
-6959776
-7570470
-Berberine chloride (known
inhibitor of sortase)
-Harvest by centrifugation, retain
supernatant
-Resuspend cells in Tris-HCl and EDTA
-Lyse cells with lysostaphin and add
DNase
-Dilute 10X and add gel loading dye
with SDS to cell lysate and to
supernatant, boil samples
-Run samples (and marker) on an
SDS polyacrylamide gel
Protein A:
50 kDa when cut at LPXTGX
56.7 kDa when uncut
-Place gel in a transfer cassette
with a PVDF membrane
-Develop with antibodies
Antibody development
Antibody development
Antibody development
Antibody development
Antibody development
Every treatment looks the same in
Intensity and Size
- Harvest cells during exponential
phase of growth
- Do an assay for another surface
protein (fibrinogen clumping assay)
-Surface protein assays
Fibrinogen assay
Clumping factor/fibrinogen binding
proteins anchored in cell wall:
-Recognize and bind to fibrinogen in blood, forming
clumps.
-Promote attachment to blood clots and traumatized tissue
-Grow S. aureus in presence or
absence of sortase-inhibiting
compounds
-Control (no compounds)
-6054217
-6959776
-7570470
-Berberine chloride (known
inhibitor of sortase)
-Harvest by centrifugation, retain cells
-Resuspend cells in solution containing
fibrinogen
-Measure optical density over time
O.D. Decreases  Fibrinogen clumping  Proteins present  Sortase Active
O.D. Constant  Fibrinogen not clumping  Proteins not present  Sortase Inactive
-Compounds do not inhibit growth of S.
aureus
-Assay for protein A needs to be more
refined
-Fibrinogen assay shows promise
-Compounds do not inhibit growth of S.
aureus
-Assay for protein A needs to be more
refined
-Fibrinogen assay shows promise
*Several new compounds identified by HTS
at Siga
- Howard Hughes Medical Institute
- Dr. Hruby
- Tove’ Bolken
- Everyone in the Hruby lab
- Kevin Ahern