Scientists at Harvard’s Wyss Institute for Biologically Inspired Engineering say they have developed a living material approach that uses a strain of genetically engineered E. coli Nissle gut bacteria as a locally acting probiotic. The engineered bacteria produce a network of nanofibers that directly binds to mucus to fill inflamed areas like a patch, shielding them from gut microbes and environmental factors.

This probiotic-based therapeutic strategy protected mice against the effects of colitis induced by a chemical agent and promoted mucosal healing as described in a study (“Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut”) in Nature Communications.

Inflammatory lesions destroy epithelial cells
Inflammatory lesions destroy epithelial cells that function as a barrier between the inside of the gut (lumen) and the rest of the body (left). This loss of barrier function leads to a feedback cycle of worsening inflammation fueled by bacteria and other particles crossing the barrier. PATCH is a bioactive material synthesized by engineered probiotic bacteria that helps to maintain gut barrier function even in the presence of inflammatory insults, thereby helping to keep bacteria and other particulates in the lumen and ameliorating the symptoms of inflammation (right). [Wyss Institute at Harvard University]
“Mucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae, and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer E. coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution,” the investigators wrote.

“We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo and is nonpathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.”

“With this ‘living therapeutics’ approach, we created multivalent biomaterials that are secreted by resident engineered bacteria on-site and attach to many mucus proteins at a time, firmly adhering to the viscous and otherwise moving mucus layer, which is a challenging thing to do,” explained Neel Joshi, PhD. “The ‘Probiotic Associated Therapeutic Curli Hybrids’ (PATCH) approach, as we named it, creates a biocompatible, mucoadhesive coating that functions as a stable, self-regenerating BAND-AID® and provides biological cues for mucosal healing.”

Joshi presently is a core faculty member of the Wyss Institute and associate professor at Harvard’s Paulson School of Engineering and Applied Sciences (SEAS) and will shortly be appointed as a professor at Northeastern University in Boston.

In previous work, Joshi’s group has demonstrated that self-regenerating bacterial hydrogels firmly attached to mucosal surfaces ex vivo, and, when orally given to mice, withstood the harsh pH and digestive conditions of the stomach and small intestine without affecting the health of the animals. To fabricate them, his team programmed a laboratory E. coli strain to synthesize and secrete a modified CsgA protein, which as part of E. coli’s “curli” system assembles into long nanofibers at the outer surface of the bacteria.

“To enable mucus adhesion, we fused CsgA to the mucus-binding domain of different human trefoil factors (TFFs), proteins that occur naturally in the intestinal mucosa and bind to mucins, the major mucus proteins present there. The secreted fusion proteins form a water-storing mesh with tunable hydrogel properties,” said co-author Anna Duraj-Thatte, PhD, a postdoctoral fellow working with Joshi. “This turned out to be a simple and robust strategy to produce self-renewing, mucoadhesive materials with long residence times in the mouse intestinal tract.”

In their new study, the team further built on these findings by introducing the machinery for producing one of the mucoadhesive hydrogels based on TFF3 into an E. coli Nissle strain that is a normal gut bacterium which can thrive in the colon and cecum sections of the intestinal tract affected by IBD, and is currently sold in many commercial probiotic formulations.

Histology Demonstrates PATCH Action in the Mouse Mucosa
These images show histological cross-sections of the colon from mice used as a disease model for colitis. The colons of injured mice (middle) lose the characteristic columnar cell structure of the healthy gut (left). When treated with PATCH, the mouse colons were able to maintain a healthy morphology even in the presence of inflammatory insults. [Wyss Institute at Harvard University]
“We found that the newly engineered Nissle bacteria, when given orally, also populated and resided in the intestinal tract, and that their curli fibers integrated with the intestinal mucus layer,” said first-author Pichet Praveschotinunt, who is a graduate student mentored by Joshi.

“When we induced colitis in the colons of mice by orally administering the chemical dextran sodium sulfate, animals that had received the PATCH-generating E. coli Nissle strain by daily rectal administration starting three days prior to chemical treatment, had significantly faster healing and lower inflammatory responses, which caused them to lose much less weight and recover faster compared to control animals. Their colon epithelial mucosa displayed a more normal morphology and lower numbers of infiltrating immune cells.”

Joshi and his team think that their approach could be developed as a companion therapy to existing anti-inflammatory, immuno-suppressant, and antibiotic therapies to help minimize patients’ exposure to the drugs and potentially provide protection against IBD relapses.

“This powerful and simple approach could potentially impact the lives of thousands of patients with IBD for whom there is no disease-specific cure available. It also is a testament to the creativity and vision of the Wyss Institute’s ‘Living Cellular Devices’ initiative that engineers living cells to perform key therapeutic and diagnostic tasks in our bodies,” said Wyss Institute founding director Donald Ingber, MD, PhD, who is also the Judah Folkman professor of vascular biology at Harvard Medical School, the Vascular Biology Program at Boston Children’s Hospital, and professor of bioengineering at SEAS.

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