Presentation on theme: "Research In Progress Towards an Understanding of the role of ESAT-6 in Mycobacterial Granuloma Formation Kelly Bachta, MD PhD Ramakrishnan Laboratory UW."— Presentation transcript:
1Research In ProgressTowards an Understanding of the role of ESAT-6 in Mycobacterial Granuloma FormationKelly Bachta, MD PhDRamakrishnan LaboratoryUW Internal Medicine Residency
2Why is TB so scary? Drug Resistance Current drugs are effective, but often require therapy courses of 6-12 monthsMDR – TBXDR – TBFor new drug development, new molecular targets need to be identifiedHost targetsMDR – 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.
3Pathogenesis of TB: Rethinking the role of the granuloma
4The 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 recruitmentDannenberg, AM. Immunobiology 1994; 191 (4-5)
5Region of Difference (RD1) ESAT6 Secretion SystemRv3871Rv3872Rv3873CFP-10ESAT-6Rv3876Rv3877Rv3878Rv3879cRegion 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:
6Brodin P et al. J. Biol. Chem. 2005;280:33953-33959 Structure of ESAT-66kD secreted protein found in the supernatants of cultures of MTbPore formation independent of CFP10Forms a 1:1 heterodimeric complex with CFP-10Forms ~4-4.5 nm pores in host-cell membranes/MφMediates macrophage cell deathIllicits MMP-9 production in epithelial cellsW-X-G motifBrodin P et al. J. Biol. Chem. 2005;280:
7Volkman, 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 formationESAT-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 infectionVolkman, HE, et al. Science Jan 22;327(5964):466-9.
8ESAT6 induces MMP9 in human epithelial cells, but not macrophages Volkman, HE, et al. Science Jan 22;327(5964):466-9.
9Summer Project:Does ESAT-6 induce MMP9 in epithelial cells through mediating cell death?
10Developing assays to detect pore formation and cell death Cell Stainingstained with Lucifer YellowVisualized by light and fluorescence microscopyLDH release assaycell 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
11ESAT6 induces cell death AND pore formation in macrophages MockESAT6 50uMESAT6 100uMESAT6 250uMESAT6 500uMESAT6 20uMLucifer Yellow staining of THP-1 cells treated with WT ESAT6
13ESAT6 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?
14ESAT-6 has distinct actions on Macrophages and Epithelial Cells Pore FormationYesCell DeathNoMMP9 induction
15Summer Accomplishments… Purification of ESAT6 for assay developmentOptimization of both LDH release assay and Lucifer Yellow microscopy to allow for study of interactions between ESAT-6 and host model cell linesESAT-6 induces cell death AND pore formation in macrophages, but only pore formation in epithelial cells
16Future Directions…Is the pore-forming function of ESAT-6 required for MMP9 production in epithelial cells?-Purification of non-pore forming ESAT6 mutants2) Do other pore-forming proteins induce MMP-9 production in epithelial cells?-Application of staphylococcal α-toxin (a pore forming toxin) to cell models
17Thanks… Special thanks to University of Washington Internal Medicine ResidencyLalliFrances 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 expertiseRamaLab for making me feel so welcome…