1. Gram-Positive DegP Evaluation
Dr. Dennis Hruby
Jaimie Smith
Dept. of Microbiology, Oregon State University

2. What is DegP?
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

3. Why Study DegP?
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

4. My Project… Questions to be answered are:
Where is DegP located in Gram-positive bacteria? (there is no periplasm)
Is DegP over-expressed in the same conditions as Gram-negative bacteria?

5. Study Design…
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

6. E. coli Antisera
S. pyogenes Antisera
S. aureus Antisera
Supernatant EC X
Soluble EC
Insoluble EC
DegP EC
Supernatant SP
Soluble SP
Insoluble SP
DegP SP
Supernanant SA
Soluble SA
Insoluble SA
DegP1 SA
DegP2 SA

7. What Does This Tell Us?
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

8. Cellular Fractionation Results…
1-Control Cell Wall Fraction Control Cytoplasmic Fraction
3-Control Membrane Bound Fraction 4-Trypsin Cell Wall Fraction
5-Trypsin Cytoplasmic Fraction 6-Trypsin Membrane Bound Fraction
7-Triton X-100 Cytoplasmic/Membrane Triton X-100 Total Cell Fraction

9. What Does This Mean?
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

10. Future Research…
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

11. Many thanks to… The Howard Hughes Medical Institute Dr. Kevin Ahern
Dr. Dennis Hruby, Tove Bolken, the Hruby Lab, and SIGA Technologies