Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria

Summary

The use of bacteria as live drug delivery agents is an emerging application of synthetic biology. Toward this end, efforts have gone to develop controlled interactions between mammalian cells and bacteria. This paper details the development of a mammalian cell invading device that allows bacteria, specifically Escherichia Coli, to invade mammalian cells upon environmental signals. E. Coli have been engineered to invade various types of cancer-derived cells such as HeLa, HepG2, and U2OS cell lines. The expression of the invading device is induced by various environmental signals. The fdhF promoter and the araBAD promoter are induced by hypoxia and arabinose, respectively. When bacterial cells reach a density of greater than 10^8 bacteria/ml or are in the presence of 0.02% arabinose, activation of the invasion device occurs. The actual gene that induces the invading is the inv gene from the organism Yersinia pseudotuberculosis, which when expressed in E. Coli, will bind tightly to beta-1 integrins on mammalian cells. The binding of beta-1 integrins on the surface of mammalian cells stimulates Rac-1 and induces uptake of the bacteria into the cell. The invasin device effectively couples environmental inputs with the invasion as the output. Tuning of the input to output strength was done using a combinatorial strategy of constructing ribosome binding libraries and genetic selection. The use of invasin can then be coupled to the delivery of therapeutic proteins and plasmids into the cell.

Results

Assays were done to test for the nature of invasin. The ability of invasin to invade mammalian cells were tested by putting the inv gene under the constitutive tet promoter and allowing the MC1061 E. coli strain to interact with HeLa cells. The readout for the assay was the fraction of bacteria recovered from lysis, which is ~8% for inv+ E. coli and negligible for inv- E. coli. To determine the role that P pili plays in the adhesion and uptake of E. coli to mammalian cells, a fim deficient strain (CAMC600) was tested, and the strain retained its invasion abilities. Similar experiments with other cancer cell lines such as HepG2 and U2OS show that invasin is capable of invading cancer cells from different origins. It should be noted, however, that the level of invasion probably depends on the amount of beta-1 integrins expressed and their accessibility. Also, invasin only acts on cancer cells that are actively expressing beta-1 integrins. These results are shown in the figure below. The white bars indicate percent bacteria recovered from lysis with the inv+ system. The grey bar indicates the percent recovered with the inv+ CAMC600 system. The black bar represents the inv­- system. An asterisk indicates no bacteria recovered.

The inducibility of the invasin system was then tested with arabinose induction. The araBAD promoter was fused to the inv gene, and the expression of invasin upon induction of arabinose was tested. It was noted that the basal expression of the inv gene due to a leaky promoter was enough to produce invasin and invade mammalian cells. Hence, an RBS library was made where the RBS sequence was randomized and the clones that did not invade cells without arabinose induction were chosen. In the figure below, the white bar represents without induction. The grey bar is induction with arabinose, and the black is anaerobic induction.

Invasin is further linked to hypoxia by switching the promoter to the fdhF promoter which results in the expression of the inv gene in only anaerobic conditions. The expression levels were tuned by an RBS library. It was shown that after 2hours in anaerobic conditions, about 10% of added bacteria were recovered, using the same gentimycin assay. The inv gene is further put under the control of luxR promoter, which is part of the quorum sensing genetic circuit that is activated upon increase in cell density. Under quorum control, the inv gene is only detectable under high cell densities.

Significance

The engineering of an invasin gene under various promoters allows it to invade mammalian cells selectively at high cell density, in anaerobic growth, or chemical induction. This scheme can be used to invade cancer cells of diverse origins. The engineered invasin can also be a target for protein engineering to alter its properties such that the expression of invasin results in binding but not invasion of cells. A very significant result of engineering an inv+ E. coli is that other devices can be added to the system to make a therapeutic bacterium. The inv+ system lays the groundwork for applications such as live vaccines and gene delivery vectors. In particular, invasive bacteria is being engineered with additional genetic devices such a self-lysis and drug delivery system that would allow bacteria to invade cancer cells and release drugs to kill the cell.

Critique

One problem with the system is that bacteria will only invade mammalian cells that are actively expressing beta-1 integrins, so that cancer cells that do not express that surface protein will not be detected by the bacteria. Moreover, cancer cells are not the only cells expressing beta-1 integrins as surface proteins, so a possibility of invasion of other cells is present. Another major problem is the anti-LPS response from the mammalian immune system. Modifications to the lipopolyssacharide structure of the E. coli will have to be done for the bacteria to survive in the body.

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