450690 Non-Standard Amino Acid Labeling of the Extracellular Matrix of Probiotics for In Vivo Imaging of the Gut

Thursday, November 17, 2016: 8:48 AM
Golden Gate 7 (Hilton San Francisco Union Square)
Noémie-Manuelle Dorval Courchesne1, Pichet Praveschotinunt1,2, Anna Duraj-Thatte1,2, Alexis J. Rovner3, George M. Church1,3 and Neel Joshi1,2, (1)Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, (2)School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, (3)Department of Genetics, Harvard Medical School, Boston, MA

Current methods for diagnosing and observing the healing or progression of diseases of the gastrointestinal tract are often invasive and do not allow for easy time monitoring. Alternative approaches would be beneficial if they allowed for continuous monitoring of the patient’s condition non-invasively and for the use of molecular signatures or changes in the local gut environment to diagnose diseases. Probiotics are tools of choice for colonizing the gut, promoting health and even delivering therapeutics. By engineering probiotics to produce a detectable signal, they could also be used to image and probe diseases. In order to accomplish specific probing of probiotics, bio-orthogonal functional groups can be incorporated in proteins produced by probiotic bacteria via genetic engineering and use of non-standard amino acids (NSAA). Of particular interest are non-standard residues with side-chains containing azides, alkynes or other moieties that can participate in simple and efficient chemical reactions for labeling proteins with dyes, peptides or nanoparticles. Combining probiotics and NSAA could lead to the development of non-invasive methods for in vivoimaging in the gut.

Here we present the engineering of a probiotic strain, Escherichia coli Nissle 1917 (ECN), to produce curli nanofibers containing NSAA that can participate in click chemistry reactions. Curli nanofibers are part of the extracellular matrix of ECN, and contribute to the bacterial adhesion in the gut and to biofilm formation. In vitro experiments demonstrate that ECN can effectively incorporate NSAA into proteins without recoding of the ECN genome, and only with minimal effects on the fitness of the bacteria. Subsequent dye binding via click chemistry allows for the selective labeling of curli nanofibers containing NSAA. During in vivo experiments, after colonization of the gut and expression of the curli nanofibers in the presence of NSAA, fluorescent labels can be used to detect the presence over time and the spatial distribution of ECN bacteria in the gastrointestinal tract. At a longer term, our technology could be optimized for triggered expression of NSAA-containing curli nanofibers at specific locations in the gut where disease markers are produced.

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