A novel pathway to sequester ASC at mitochondria-associated membranes dampens inflammasome activation during early Salmonella infection

• Main Author: Fang, Yuan
• Language: English
• Published: University of British Columbia 2012
• Online Access: http://hdl.handle.net/2429/43102

Salmonella enterica is an intracellular bacterial pathogen that injects effector proteins into host cells and induces rapid cell death. Mitochondria associated membranes (MAMs) are important contact sites between mitochondria and ER and selectively facilitate Ca²⁺ uptake from ER lumen into mitochondria. In the context of early Salmonella infection of THP-1 cells, a discontinuous sucrose gradient was used in combination with Stable Isotope Labeling by Amino acids in Cell culture (SILAC) to profile organelle proteomes. Protein profiles were generated for >800 mitochondria and ER proteins. I observed unique protein recruitments to MAMs during early Salmonella infection. The inflammasome adaptor protein, ASC is co-purified with MAM markers. SILAC immunoprecipitation (IP) experiments showed that ASC interacts directly with the VDAC/Stress-70 complex at MAMs, suggesting that ASC is specifically recruited to MAMs during Salmonella infection. SILAC IP experiments identified an interaction between Flightless-I and ASC. Flightless-I is an actin binding protein and also interacts with Salmonella flagellin (FliC). siRNA knockdown of Flightless-I and mitofusin 2 (a structural protein of MAMs) both leads to significant increase of IL-1β during Salmonella infection. Simultaneous knockdown of Flightless-I and mitofusin 2 does not have additive effect, suggesting that they are in the same pathway to dampen inflammasome activation. Actin isolation experiments showed that ASC is enriched at actin filaments during Salmonella infection. Therefore, I proposed a model that during early Salmonella infection, FliC interacts with Flightless-I that then interacts with and transports ASC to MAMs via actin filaments. This process specifically sequesters ASC at MAMs to dampen inflammasome activation in the cytosol. Experimental data from ΔfliCΔfljB Salmonella infection of THP-1 cells and inflammasome reconstitution in 293T cells supported this model. This study was the first characterization of MAM protein composition during bacterial infection. Furthermore, enrichment of ASC at MAMs was identified during early Salmonella infection, with Flightless-I interacting with and transporting ASC to MAMs via actin filaments at the presence of Salmonella flagellin. This novel pathway dampened the inflammasome activation during early Salmonella infection. This is the first report of the negative regulation of pyroptosis by Salmonella flagellin.