Transcript Powerpoint - Oregon State University

Gram-Positive DegP Evaluation
Dr. Dennis Hruby
Jaimie Smith
Dept. of Microbiology,
Oregon State University
What is DegP?
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DegP is a serine protease that is found in the
periplasm of all Gram-negative bacteria, and is
highly conserved among these bacteria
DegP functions to degrade misfolded proteins that
accumulate in the periplasm when the organism is
under stress (high temperatures, oxidative
conditions, etc.)
DegP has been proven vital to the full virulence of
the bacteria
DegP has recently been found in several types of
Gram-positive bacteria, but little is known about it
Why Study DegP?
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When non-functional proteins aggregate in a
bacteria cell, it weakens the cell
Since DegP is essential for the full virulence of a
bacteria, removing it will make the cell more
susceptible to damage from stressful conditions
It also leaves the cell more susceptible to antibiotics
By studying DegP researchers are aiming to develop
an antibiotic or vaccine that is non-species specific
and will work against all bacteria
My Project…
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Questions to be
answered are:
Where is DegP located
in Gram-positive
bacteria? (there is no
periplasm)
Is DegP overexpressed in the same
conditions as Gramnegative bacteria?
Study Design…
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Study species are E. coli (Gram-negative), S. aureus
(Gram-positive), and S. pyogenes (Gram-positive)
Supernatant, soluble, and insoluble fractions of each
species will be analyzed using Western Blot
technique to determine, if any, the cross-reactivity of
each of the DegP antiseras
Cellular fractions (cell wall, cytoplasmic, and
membrane bound fractions) will be analyzed using
Western Blot technique to determine the location of
DegP in the Gram-positive species
E. coli Antisera S. pyogenes
Antisera
S. aureus
Antisera
Supernatant
EC
X
Soluble EC
X
X
X
Insoluble EC
X
X
X
DegP EC
X
Supernatant
SP
Soluble SP
Insoluble SP
X
DegP SP
X
X
X
Supernanant
SA
X
X
X
Soluble SA
X
X
X
Insoluble SA
X
X
X
X
X
X
X
DegP1 SA
DegP2 SA
X
What Does This Tell Us?
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Since the DegP antiseras show cross
reactivity with the DegPs from other species,
both Gram-negative and Gram-positive, it
can be inferred that DegP is highly
conserved in Gram-positive bacteria as it is
in Gram-negative bacteria, and possibly even
highly conserved between the two types
Cellular Fractionation Results…
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1-Control Cell Wall Fraction
3-Control Membrane Bound Fraction
5-Trypsin Cytoplasmic Fraction
7-Triton X-100 Cytoplasmic/Membrane
2-Control Cytoplasmic Fraction
4-Trypsin Cell Wall Fraction
6-Trypsin Membrane Bound Fraction
8-Triton X-100 Total Cell Fraction
What Does This Mean?
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This data shows that DegP is present in S.
aureus as a membrane bound protein
Further blots using E. coli antisera as the
primary antibody will be run to determine if it
is DegP1 or DegP2 because E. coli antisera
only shows cross reactivity with S. aureus
DegP2
Future Research…
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Cellular fractionation procedures will be
followed with S. pyogenes as it was with S.
aureus to determine the location
Develop a protocol to analyze the conditions
under which DegP is over-expressed in S.
pyogenes and S. aureus
Many thanks to…
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The Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Dennis Hruby, Tove Bolken, the Hruby
Lab, and SIGA Technologies