Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

David Piwnica-Worms

Abstract

Salmonella Typhimurium is a Gram-negative bacterial pathogen and a common cause of gastroenteritis in humans. The organism utilizes a multitude of well-studied virulence factors to invade and replicate in host intestinal epithelial cells and macrophages. Interestingly, Salmonella is also capable of localizing to tumors in in vivo model systems, and while the typical route of Salmonella infection and pathogenesis has been thoroughly investigated, the behavior of Salmonella in the tumor microenvironment has not. Therefore, to investigate Salmonella and host behavior during bacterial-neoplastic cell interactions, I utilized two high-throughput screens. In the first, I designed a bioluminescent transposon-reporter trap to identify specific Salmonella genes activated in the context of cancer cell co-culture conditions. Through this work, I identified five Salmonella genes reproducibly activated by co-culture with cancer cells, and further isolated the activating stimulus to low pH. Because low pH is a common characteristic of the tumor microenvironment, I also demonstrated the pH inducibility and reversibility of Salmonellagene activation in tumors ex vivo and in vivo. In a separate study, to better understand how host neoplastic cells respond to Salmonella, I investigated the ability of Salmonellato induce pro-inflammatory responses in HCT116 colon carcinoma cells, specifically, NF-κB activation. Then, I performed a high-throughput siRNA screen to identify novel host kinases and phosphatases involved in detection of Salmonellaand activation of NF-κB signaling. For this work, I used a reporter construct consisting of an IκBα-firefly luciferase fusion protein transcriptionally activated by NF-κB. The reporter permitted imaging of both degradation of the NF-κB negative inhibitor IκBα and its resynthesis, which is dependent on NF-κB activation, following stimulus with Salmonella. The host kinase, NME3, was identified in the screen as a specific modulator of NF-κB. Knockdown of NME3 prevents proper activation of NF-κB signaling pathways in HCT116 cells exposed to Salmonella, demonstrating the role of this kinase as a positive regulator of NF-κB pro-inflammatory signaling in colon carcinoma cells.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7DN433M

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