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
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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
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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
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Pathogenesis of TB:
Rethinking the role of the
granuloma
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The Granuloma Promotes Bacterial Expansion
Gran
Dannenberg, AM. Immunobiology 1994; 191 (4-5) 461-73.
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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
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Structure of ESAT-6
Brodin P et al. J. Biol. Chem. 2005;280:33953-33959
W-X-G motif
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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.
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ESAT6 induces MMP9 in human epithelial cells, but not macrophages
Volkman, HE, et al. Science. 2010. Jan 22;327(5964):466-9.
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Summer Project:Does ESAT-6 induce MMP9 in epithelial cells
through mediating cell death?
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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
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ESAT6 induces cell death AND pore formation in macrophages
Mock ESAT6 50uM ESAT6 100uM ESAT6 250uM ESAT6 500uMESAT6 20uM
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ESAT6 induces pore formation ONLY in epithelial cells
Mock ESAT6 50uM ESAT6 100uM ESAT6 500uMESAT6 250uMESAT6 20uM
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ESAT6 kills macrophages, but not epithelial cells
Macrophages Epithelial Cells
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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
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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…
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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
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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…