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Gram-Positive DegP Evaluation

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

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Gram-Positive DegP Evaluation

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  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. 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

  7. Cellular Fractionation Results… • 1-Control Cell Wall Fraction 2-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 8-Triton X-100 Total Cell Fraction

  8. 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

  9. 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

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

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