kelly bachta , md phd ramakrishnan laboratory uw internal medicine residency
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Research In Progress. Towards an Understanding of the role of ESAT-6 in Mycobacterial Granuloma Formation. Kelly Bachta , MD PhD Ramakrishnan Laboratory UW Internal Medicine Residency. Why is TB so scary? Drug Resistance. - PowerPoint PPT PresentationTRANSCRIPT
Kelly Bachta, MD PhDRamakrishnan Laboratory
UW Internal Medicine Residency
Research In Progress
Towards an Understanding of the role of ESAT-6 in Mycobacterial Granuloma
Formation
Why is TB so scary?Drug Resistance
Current drugs are effective, but often require therapy courses of 6-12 months
MDR – TB XDR – TB For new drug development, new molecular
targets need to be identified Host targets
Pathogenesis of TB:
Rethinking the role of the
granuloma
The Granuloma Promotes Bacterial Expansion
Gran
Dannenberg, AM. Immunobiology 1994; 191 (4-5) 461-73.
ESAT6 Secretion System
Flint J L et al. PNAS 2004;101:12598-12603
Rv3871
Rv3872
Rv3873
CFP-10
ESAT-6
Rv3876
Rv3877
Rv3878
Rv3879
c
Region of Difference (RD1)
Hsu T et al. PNAS 2003;100:12420-12425
Structure of ESAT-6
Brodin P et al. J. Biol. Chem. 2005;280:33953-33959
W-X-G motif
Bacterial ESAT-6 induces MMP-9 expression in neighboring epithelial cells to induce granuloma
formation
Volkman, HE, et al. Science. 2010. Jan 22;327(5964):466-9.
ESAT6 induces MMP9 in human epithelial cells, but not macrophages
Volkman, HE, et al. Science. 2010. Jan 22;327(5964):466-9.
Summer Project:Does ESAT-6 induce MMP9 in epithelial cells
through mediating cell death?
Developing assays to detect pore formation and cell death
LDH release assay
•cell supernatants harvested and LDH measured (as a surrogate for cell lysis)
Cell Staining
•stained with Lucifer Yellow•Visualized by light and fluorescence microscopy
ESAT6 induces cell death AND pore formation in macrophages
Mock ESAT6 50uM ESAT6 100uM ESAT6 250uM ESAT6 500uMESAT6 20uM
ESAT6 induces pore formation ONLY in epithelial cells
Mock ESAT6 50uM ESAT6 100uM ESAT6 500uMESAT6 250uMESAT6 20uM
ESAT6 kills macrophages, but not epithelial cells
Macrophages Epithelial Cells
ESAT-6 has distinct actions on Macrophages and Epithelial Cells
Macrophages Epithelial Cells
Pore Formation Yes Yes
Cell Death Yes No
MMP9 induction No Yes
Purification of ESAT6 for assay development
Optimization of both LDH release assay and Lucifer Yellow microscopy to allow for study of interactions between ESAT-6 and host model cell lines
ESAT-6 induces cell death AND pore formation in macrophages, but only pore formation in epithelial cells
Summer Accomplishments…
2) Do other pore-forming proteins induce MMP-9 production in epithelial cells?
-Application of staphylococcal α-toxin (a pore forming toxin) to cell models
Future Directions…
1) Is the pore-forming function of ESAT-6 required for MMP9 production in epithelial cells?
-Purification of non-pore forming ESAT6 mutants
Thanks…Special thanks to
University of Washington Internal Medicine Residency
Lalli Frances Chu, post-doc and primary collaboratorTiffany, James, Shane, Steven, Kevin, Russ, and
Chen for support and office entertainmentKlevit lab – for allowing me to use their equipment
and protein purification expertise
RamaLab for making me feel so welcome…