Kelly Bachta , MD PhD Ramakrishnan Laboratory UW Internal Medicine Residency

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

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

The Granuloma Promotes Bacterial Expansion

Gran

Dannenberg, AM. Immunobiology 1994; 191 (4-5) 461-73.

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

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

Mock ESAT6 50uM ESAT6 100uM ESAT6 250uM ESAT6 500uMESAT6 20uM

ESAT6 induces pore formation ONLY in epithelial cells

Mock ESAT6 50uM ESAT6 100uM ESAT6 500uMESAT6 250uMESAT6 20uM

ESAT6 kills macrophages, but not epithelial cells

Macrophages Epithelial Cells

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

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 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…