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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. Kelly Bachta , MD PhD Ramakrishnan Laboratory UW Internal Medicine Residency. Why is TB so scary? Drug Resistance.

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Kelly Bachta , MD PhD Ramakrishnan Laboratory UW Internal Medicine Residency

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  1. Research In Progress Towards an Understanding of the role of ESAT-6 in MycobacterialGranuloma Formation Kelly Bachta, MD PhD RamakrishnanLaboratory UW Internal Medicine Residency

  2. 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

  3. Pathogenesis of TB: Rethinking the role of the granuloma

  4. The Granuloma Promotes Bacterial Expansion Gran Dannenberg, AM. Immunobiology 1994; 191 (4-5) 461-73.

  5. ESAT6 Secretion System Rv3871 Rv3872 Rv3873 CFP-10 ESAT-6 Rv3876 Rv3877 Rv3878 Rv3879c Region of Difference (RD1) Hsu T et al. PNAS 2003;100:12420-12425 Flint J L et al. PNAS 2004;101:12598-12603

  6. Structure of ESAT-6 W-X-G motif Brodin P et al. J. Biol. Chem. 2005;280:33953-33959

  7. 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.

  8. ESAT6 induces MMP9 in human epithelial cells, but not macrophages Volkman, HE, et al. Science. 2010. Jan 22;327(5964):466-9.

  9. Summer Project: Does ESAT-6 induce MMP9 in epithelial cells through mediating cell death?

  10. Developing assays to detect pore formation and cell death • Cell Staining • stained with Lucifer Yellow • Visualized by light and fluorescence microscopy • LDH release assay • cell supernatants harvested and LDH measured (as a surrogate for cell lysis)

  11. ESAT6 induces cell death AND pore formation in macrophages Mock ESAT6 50uM ESAT6 20uM ESAT6 100uM ESAT6 250uM ESAT6 500uM

  12. ESAT6 induces pore formation ONLY in epithelial cells ESAT6 100uM ESAT6 500uM Mock ESAT6 50uM ESAT6 250uM ESAT6 20uM

  13. ESAT6 kills macrophages, but not epithelial cells Macrophages Epithelial Cells

  14. ESAT-6 has distinct actions on Macrophages and Epithelial Cells

  15. Summer Accomplishments… • 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

  16. Future Directions… • Is the pore-forming function of ESAT-6 required for MMP9 production in epithelial cells? • -Purification of non-pore forming ESAT6 mutants 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

  17. 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…

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