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 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 MDR – TB – resistant to at least 2 of the most common ABX (INH, RIF) XDR – TB – resistant to INH, RIF + any flouroquinolone and at least one of 3 second line drugs (amikacin, kanamycin, capreomycin) Traditionally have thought of bacterial targets for ABX, vaccines. Has not worked in the development of TB drugs or in TB vaccine. Switch gears and uncover host targets that could be targeted in order to halt infection.

Pathogenesis of TB: Rethinking the role of the granuloma

The Granuloma Promotes Bacterial Expansion Bacterial Expansion Factory: For every 1 macrophage that dies, >2 become subsequently infected. This process requires 2 key elements – Macrophage cell death and Macrophage recruitment Dannenberg, AM. Immunobiology 1994; 191 (4-5) 461-73.

Region of Difference (RD1) ESAT6 Secretion System Rv3871 Rv3872 Rv3873 CFP-10 ESAT-6 Rv3876 Rv3877 Rv3878 Rv3879c Region of Difference (RD1) This specialized secretion system mediates both functions… BCG vaccine developed in 1921 2/2 serial passages of M Tb on growth media. Been given to more people in the world than any other vaccine. While it doesn’t work well as a vaccine, it does demonstrate principles of pathogenesis and reveals that the ESAT-6 cluster of genes is required for pathogenesis. What is missing in BCG? The ESAT6 secretion system cluster. ESAT6 has homologues in Staph aureus and some Bacillus spp. Pore forming TOXIN…. Hsu T et al. PNAS 2003;100:12420-12425 Flint J L et al. PNAS 2004;101:12598-12603

Brodin P et al. J. Biol. Chem. 2005;280:33953-33959 Structure of ESAT-6 6kD secreted protein found in the supernatants of cultures of MTb Pore formation independent of CFP10 Forms a 1:1 heterodimeric complex with CFP-10 Forms ~4-4.5 nm pores in host-cell membranes/Mφ Mediates macrophage cell death Illicits MMP-9 production in epithelial cells W-X-G motif Brodin P et al. J. Biol. Chem. 2005;280:33953-33959

Volkman, HE, et al. Science. 2010. Jan 22;327(5964):466-9. Bacterial ESAT-6 induces MMP-9 expression in neighboring epithelial cells to induce granuloma formation ESAT-6 released from infected macrophages, in turn activates epithelial cells to secrete MMP9 (matrix metalloproteinase) which causes activation of chemokines/ECM degradation and serves to recruit other macrophages to ‘clean up apoptotic debri’. May also facilitate spread of infection 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 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) Lucifer Yellow a polar, fluorescent compound that is cell-membrane impermeable. Can only cross cell membranes that have been disrupted/have pores. LDH: and compared to fully lysed controls resulting in a % lysis

ESAT6 induces cell death AND pore formation in macrophages Mock ESAT6 50uM ESAT6 100uM ESAT6 250uM ESAT6 500uM ESAT6 20uM Lucifer Yellow staining of THP-1 cells treated with WT ESAT6

ESAT6 induces pore formation ONLY in epithelial cells Mock ESAT6 20uM ESAT6 50uM ESAT6 100uM ESAT6 250uM ESAT6 500uM Lucifer Yellow NHBE Cells treated with wild-type ESAT6 Top line: DIC – light microscopy Bottom Row: FITC labeling – fluorescence microscopy

ESAT6 kills macrophages, but not epithelial cells THP-1 cells are VERY sensitive to cell death by wild-type ESAT6 (75-90% lysis) More cytolysis with increasing [ESAT6] NHBE cells are not very sensitive to wild-type ESAT6 (20-30% lysis) Greater cell death when THP/macrophages are treated with ESAT6 than when NHBE epithelial cells are. Does this mean that ESAT6: cell interactions might be activating different pathways in different cell types?

ESAT-6 has distinct actions on Macrophages and Epithelial Cells Pore Formation Yes Cell Death No MMP9 induction

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

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

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…