Team:UT Dallas/immunobot intro

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Introduction

Inflammatory bowel disease (IBD) is a group of increasingly prevalent disorders that afflicts parts of the human gastrointestinal tract and results in significant tissue damage. IBD manifests primarily as Crohn’s disease and ulcerative colitis. These disorders both have ambiguous disease etiology, which complicates a timely diagnosis. Generally considered autoimmune disorders, treatment often involves anti-inflammatory and immunosuppressive medications and surgical intervention. However, these have temporary success and mainly work to alleviate symptoms and maintain remission. Thus, IBD remains without a cure.

Wound healing involves a complex series of biochemical events that progress in distinct stages: inflammation, proliferation and remodeling. Tissue repair takes place during the proliferative and remodeling stages during which a balance of collagen production and degradation is maintained. However, repair of IBD-damaged tissue is particularly difficult since these are chronic wounds locked in a prolonged inflammatory state that can result in further damage as collagen degradation overwhelms production. In 2009, Stanford developed a two-component probiotic that regulates the activity of lymphocytes involved in inflammation. To build on their work, we developed a means to localize tissue repair following control of inflammation. As a human gut symbiont, we selected an E. coli chassis for its well-characterized machinery and the vast supply of E.coli-compatible tools available in the Registry.

"Immunobots" are composite sensor-taxis devices that interpret wound signals to identify tissue damage and migrate towards damage sites, such as those resulting from IBD. Immunobots can work with another device that facilitates tissue repair, which takes an important step towards achieving a more durable treatment for IBD.

References:

Baumgart, Daniel C C. et. al. (2007). Inflammatory bowel disease: clinical aspects and established and evolving therapies. The Lancet. 369(9573): 1641-57 (doi:10.1016/S0140- 6736(07)60751-X)

Midwood, K.S. et. al. (2004). Tissue repair and the dynamics of the extracellular matrix. The International Journal of Biochemistry & Cell Biology. 36 (6): 1031–1037. (doi:10.1016/ j.biocel.2003.12.003)

Stanford iGEM 2009 (https://2009.igem.org/Team:Stanford/)

Sensing Module

Fibroblasts play a critical role in the proliferative stage of wound healing. Fibroblast growth factors (FGFs) promote increased fibroblast cell differentiation and proliferation. Therefore FGFs can represent wound signals for our immunobot sensing module. FGFs work with Heparin sulfate-containing proteoglycans to induce the FGF receptor (FGFR). Once induced, FGFR dimerizes and triggers FGF signal transduction.

We wished to connect wound signaling inputs to movement in order to achieve localization at the site of tissue damage. This requires an intermediate that would link FGFR to chemotaxis, since the systems do not naturally share common machinery. Kolmar et. al. achieved a similar effect in E. Coli. Their system included a chimeric receptor consisting of a maltose-inducible component and ToxR transcription factor. Activated ToxR forms a homodimer that induces the CTX promoter. They cloned CTX upstream of bacterial chemotaxis genes that produce straight- line movement to achieve maltose-induced chemotaxis.

The dimerization properties of ToxR receptor made it particularly attractive for use in our system. Thus, we produced a chimeric receptor that includes a FGFR-ToxR fusion, which is to our knowledge, the first of its kind in the Registry. The activated complex induces CTX- controlled chemotaxis, hence completing the linkage between wound signals and movement.

Taxis Module

Bacterial chemotaxis in our chassis (E. coli DH5a) involves interactions of transmembrane receptor proteins known as methyl accepting chemotactic proteins (MCP). Each MCP within the cell uses the same intracellular signals to control movement towards different chemicals. These signals include the six Che proteins: CheA, CheB, CheR, CheW, CheY and CheZ.

Chemotaxis involves systematic interactions among the Che proteins to produce movement along gradients of inducers. Initially, a ligand binds to the extracellular portion of MCP, which and induces a three dimensional conformational change that extends throughout the cell. Once this happens, CheA also undergoes a conformational change leading to an increase in activity by autophosphorylation. At this point, CheY interacts with the activated phosphorylated CheA via diffusion and becomes phosphorylated itself by the newly activated CheA. CheY then forms a complex with the flagellar motor proteins (FLiM) and induces clockwise rotations of the flagella. This causes the cell to tumble in place with no net movement. Alternatively, CheZ, another chemotactic regulator protein, dephosphorylates CheY. This decreases the cellular concentration of CheY, so less phosphorylated CheY binds the FLiM protein. As a result, the flagella instead rotate counter clockwise, inducing straight line movement as the cell runs along gradients. CheW and CheR respectively methylate and demethylate the MCP receptor. The opposing actions of methylating and demethylating prevent double counting the chemical signals. These events are summarized in this video.

We wished to produce runs along wound signal gradients to demonstrate chemotactic activity toward tissue damage sites. This requires overexpressing CheZ, which will keep cellular concentrations of CheY low and favor straight line movement over tumbling. To achieve this, we fused the CTX promoter with CheZ. In addition, Silversmith et. al. reported a hyperactive mutant form of CheZ protein, CheZ*, which we also fused with the CTX promoter to further increase dephosphorylation of CheY. Wound signals will thus activate dimerization of our chimeric receptor, which then induces the CTX-controlled CheZ and CheZ* constructs producing straight line movement along wound signal gradients.

References

Plotnikov, A. et. al. (1999). Structural Basis for FGF Receptor Dimerization and Activation Cell. Cell. 98: 641–650.

Ornitz, D.M. et. al. (2001). Fibroblast growth factors. Genome Biol. 2 (3): Reviews 3005. (doi:10.1186/gb-2001-2-3-reviews3005)

Kolmar et. al. (1994). Dimerization of Bence Jones Proteins: Linking the Rate of Transcription from an Escherichia coli Promoter to the Association Constant of REIv. Biol. Chem. Hoppe- Seyler. 375: 61-70.

Silversmith, R et. al. (2007). Kinetic Characterization of Catalysis by the Chemotaxis Phosphatase CheZ: Modulation of Activity by the Phosphorylated CheY Substrate. Journal of Biological Chemistry. 283 (2): 756-65.