by Justin Wang, Sameen Belal, and Suyasha Shrestha
The ability of bacteria to sense their surroundings allows them to be useful mediums through which scientists can deliver drugs and vaccines to specific targeted cells. This is a novel technique in synthetic biology that can be especially effective against cancer cells. In the article Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria, researchers made use of this special capability of bacteria by regulating the environment that these bacteria live in. The researchers isolated the inv gene in Yersinia pseudotuberculosis and expressed it in E. Coli in order to help them bind to cancer derived cells, specifically HeLa, HepG2 and U20S cells. Furthermore, the researchers made use of various techniques in order to synthetically link the bacteria to environmental factors such as cell density and inadequate supplies of oxygen, and even placed the gene under the control of an operon in order to selectively induce its effects.
Invasin is a long, rigid protein that extends from the surface of bacteria that allows for the penetration of mammalian cells. Bacteria accomplish this by interacting with surface carbohydrate molecules (such as B1 integrins) that bind to invasin. To express invasin, the inv gene was inserted into E. coli plasmids and approximately 8% of the bacteria were recovered with invasiveness to HeLa cells, a commonly used human cell line. Next, Anderson et al examined other potential hosts for invasin binding, specifically the human cancer cell lines U2OS and HepG2. Despite varying efficiency levels due to different levels of B1 integrin expression or accessibility, the inv+ were able to invade cancer cells with multiple origins and demonstrated specificity to only B1 integrin expression.
One way to mediate the bacteria’s ability to respond to environmental stimuli is to place it under the control of an operon that can regulate when the targeted gene should be expressed. The invasin gene was placed under the control of the araBAD operon, a gene that codes for enzymes needed for catabolism of arabinose in E. coli. The araBAD operon is involved in the production of the transcription factor AraC, which acts as its repressor. The researchers inserted araBAD and AraC into plasmids along with the invasin gene. They found that the invasive phenotypes were present and that transcription of the araBAD promoter led to invasin synthesis. After positively selecting for only plasmids able to invade cells, it was found that invasin was able to invade only in the presence of arabinose, the activator of araBAD. These results suggest that invasion can be controlled environmentally through operons.
In order for E. coli to be successful as therapeutic bacteria, it must be able to invade only tumor cells. The low oxygen, or hypoxic, environment of tumor cells can help E. coli distinguish tumor cells from other cells. The scientists used the fdhF promoter, a gene that is strongly induced in hypoxic environments, to create a system in which bacteria will infect only tumor cells. Invasin was placed under control of fdhF and cells were grown in both aerobic and anaerobic conditions. It was found that only cells in the anaerobic chamber were invasive, meaning that the promoter successfully restricted invasion.
Invasion can also be restricted by cell-density. The high cell density of cells acts as another cue for invasin. Invasin was placed under the control of the lux genetic circuit, a circuit the enables Vibrio fischeri to make cellular products based on its own cell density. Bacteria with invasin were grown to various cell densities and their abilities to invade HeLa cells were measured. It was found that under control of the genetic circuit, the bacteria were only invasive at high cell densities.
By designing a bacteria delivery system with the ability to invade cancer cells with specificity, future applications involve genetically modifying invasin to bind to new targets. Subsequent genetic logic circuits and more complicated regulatory systems could allow invasin to be more accurate in environmental sensing. However, specific invasion of cancer cells is only one component of bacterial treatment delivery. A cytotoxic response would be needed to cause destruction in the tumor after membrane penetration via invasin. Other applications could involve a release of therapeutic proteins for treatment or even antigen modification to facilitate cell-recognition. Nevertheless, Anderson et al demonstrate that therapeutic functions can be programmed into bacteria by genetic modulation.
Anderson, J.C., Clarke E.J., Arkin, A.P., and Voigt, C.A. (2005). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355, 619-27.