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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemical Engineering

Year Degree Awarded

Spring 2014

First Advisor

Neil Forbes

Subject Categories

Biochemical and Biomolecular Engineering

Abstract

Engineered Salmonella possess unique capabilities that make them ideal drug delivery vectors for tumors. Targeted bacterial delivery of anticancer proteins has the ability to overcome therapeutic resistance in tumors that limits the efficacy of chemotherapeutics. In my doctoral research, I identified a protein-drug that can be expressed by bacteria and rapidly kills cancer cells. I also created a density dependent switch that initiates gene expression in tumors and prevents expression in healthy tissue. Combining these two systems has created a potent anti-cancer system that targets tumors with minimal toxicity.

I cloned genes for five potential anti-cancer proteins into Salmonella. Supernatant from cultures was applied to MCF-7 mammary carcinoma cells to identify proteins that 1) were expressed, 2) secreted, and 3) rapidly killed cancer cells. Of the investigated proteins, α-hemolysin from Staphylococcus aureus (SAH) was the most promising because it secreted, caused trauma to cellular membrane, and induced oncosis in 18 minutes. After exposure for six hours, SAH decreased cell viability by 90%. The maximum death rate induced by SAH was a 7.1% reduction in cell viability per minute.

Due to systemic toxicity, bacteria that constitutively express anti-cancer drugs would not be an alternative to standard chemotherapy. To overcome this, I engineered Salmonella to initiate gene expression using the lux quorum sensing system. Quorum sensing (QS) allows bacteria to change gene expression based on differences in population density. Because salmonella preferentially accumulate in tumors, a QS genetic circuit could create an expression switch that only initiates in bacterial colonies in tumors. QS Salmonella only expressed GFP in high-density colonies in vivo. Gene expression of colonies was also dependent upon the radial distance of neighboring bacteria. At densities above 5x1010 cfu/g, 84% of the colonies whose neighbors were at an average radial distance less than 103µm expressed GFP, whereas no colonies expressed GFP when their neighbors were at radial distances greater than 108µm. A mathematical model correctly predicted GFP expression in 93% of 84,213 QS colonies based on density and radial distance from adjacent Salmonella. QS Salmonella will allow for targeted bacterial drug delivery to tumors while minimizing systemic toxicity.

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