1. 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

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
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.

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

4. 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)

5. 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 /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:
Flint J L et al. PNAS 2004;101:

6. 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:

7. 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 Jan 22;327(5964):466-9.

8. ESAT6 induces MMP9 in human epithelial cells, but not macrophages
Volkman, HE, et al. Science 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)
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

11. 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

12. 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

13. 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?

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

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…