Engineered Curli-Expressing Probiotic Bacteria as a Platform for Therapeutics and Diagnostics
View/ Open
P Praveschotinunt Dissertation Final Draft.docx (67.42Mb)
Access Status
Full text of the requested work is not available in DASH at this time ("restricted access"). For more information on restricted deposits, see our FAQ.Author
Praveschotinunt, Pichet
Metadata
Show full item recordCitation
Praveschotinunt, Pichet. 2019. Engineered Curli-Expressing Probiotic Bacteria as a Platform for Therapeutics and Diagnostics. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.Abstract
Extracellular matrix-associated proteins play important roles in host-microbe interactions. We and others have identified curli amyloid fibers, which are major components of the extracellular matrix produced by Enterobacteriaceae during biofilm formation, to be a highly robust and versatile platform for microbial engineering. To exploit these curli amyloid fibers, we have developed a platform called “Biofilm Integrated Nanofiber Display” (BIND), in which we genetically engineered CsgA, a major subunit of curli fibers, to have novel properties either by adding functional domains or by modifying the CsgA sequence to influence the properties of curli fibers.In this work, we demonstrate the ability of the BIND platform to influence the host-microbe interaction in vitro, the amelioration of colitis in murine models using engineered probiotics, the formation of self-renewal living hydrogels, and the non-invasive tracking of engineered bacteria in vivo.
We used BIND to rationally design a bacterial strain that could interact with mucosal epithelium models. We engineered a commensal strain of E. coli to produce curli fibers fused to the trefoil factor family (TFF) peptides. These engineered microbes showed improved affinity for soluble mucins, Caco-2 intestinal cell lines, and goat intestinal explants. Some curli-TFF conjugates/fusions also enhanced cell migration of simulated Caco-2 wounds, which reflected their bioactivity comparable to their soluble TFF counterparts. Next, we engineered probiotic E. coli Nissle 1917 (EcN) to produce curli-TFFs in vitro and in vivo and confirmed that these engineered bacteria were non-pathogenic. Inoculation with the probiotic conferred improved protective effects against dextran sodium sulfate induced colitis in mouse models associated with barrier function enforcement and immunomodulation. We also found that engineered commensal E. coli expressing curli-TFFs could be fabricated into living hydrogels consisting of concentrated bacterial cultures. Such hydrogels had the ability to selectively interact with the gastrointestinal (GI) tract and to self-renew in vitro and in vivo. Finally, we demonstrated a method for tracking engineered microbes within the GI tract via non-standard amino acid incorporation into extracellular curli fibers followed by click chemistry-based labeling.
Overall, this work lays the foundation for a novel and versatile platform for creating engineered probiotics with programmable therapeutic and diagnostic functions.
Terms of Use
This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAACitable link to this page
http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029630
Collections
- FAS Theses and Dissertations [5370]
Contact administrator regarding this item (to report mistakes or request changes)