Presentation on theme: "Kelly Bachta, MD PhD Ramakrishnan Laboratory UW Internal Medicine Residency Research In Progress Towards an Understanding of the role of ESAT-6 in Mycobacterial."— Presentation transcript:
Kelly Bachta, MD PhD Ramakrishnan 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
ESAT6 Secretion System Flint J L et al. PNAS 2004;101:12598-12603 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 MockESAT6 50uM ESAT6 100uMESAT6 250uMESAT6 500uM ESAT6 20uM
ESAT6 induces pore formation ONLY in epithelial cells MockESAT6 50uM ESAT6 100uMESAT6 500uM ESAT6 250uM ESAT6 20uM
ESAT6 kills macrophages, but not epithelial cells MacrophagesEpithelial Cells
ESAT-6 has distinct actions on Macrophages and Epithelial Cells MacrophagesEpithelial Cells Pore FormationYes Cell DeathYesNo MMP9 inductionNoYes
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 collaborator Tiffany, James, Shane, Steven, Kevin, Russ, and Chen for support and office entertainment Klevit lab – for allowing me to use their equipment and protein purification expertise RamaLab for making me feel so welcome…