Nitrate-responsive genetically engineered probiotics locally release TNF-α nanobodies against inflammatory bowel disease.

Escherichia coli Nissle 1917 was successfully engineered to express TNF-α nanobodies (NbTNF-α) with confirmed strong anti-inflammation activity.

• Expression of NbTNF-α from E. coli was first confirmed in this work.
• Strong anti-inflammation activity of NbTNF-α expressed from E. coli was demonstrated.
• The nanobodies target TNF-α, a key cytokine in inflammatory bowel disease pathogenesis.

A NarX/L sensing system was tailored to detect nitrate as a biomarker of intestinal inflammation for triggering payload release.

• Nitrate is highly produced in the intestinal route during inflammatory bowel disease.
• The NarX/L sensing system was used to create a nitrate-responsive release mechanism.
• This system enabled the 'producing and storing first, then responding and releasing' mode proposed by the authors.

Two distinct release systems were designed and separately recombined into EcN, designated E5 and E7.

• E5 incorporated the Hly system, described as a protein complex secretion machine.
• E7 incorporated a φX174E-based lysis-release system.
• Both systems were designed to release NbTNF-α in response to nitrate stimulation.

E5 and E7 exhibited similar in vitro kinetics of NbTNF-α secretion.

• Both engineered strains showed comparable secretion profiles in vitro despite using fundamentally different release mechanisms.
• The Hly secretion system (E5) and the φX174E lysis-release system (E7) produced equivalent release kinetics.
• This similarity in kinetics held across both a continuous secretion mechanism and a lysis-based burst release mechanism.

Oral administration of both E5 and E7 achieved high anti-ulcerative colitis effects in vivo.

• Anti-ulcerative colitis effects were characterized by inflammation attenuation and recovery of intestinal mucosal barriers.
• Levels of NbTNF-α expressed by both E5 and E7 in the intestinal tract were similar.
• The therapeutic effects were achieved through local release of nanobodies in the intestinal environment.

The study introduces a novel 'producing and storing first, then responding and releasing' mode for genetically engineered probiotics.

• This release mode is distinguished from conventional continuous secretion approaches used in engineered probiotics.
• The impact of the release mode of bacterial payloads had previously remained unknown.
• The approach leverages pathological environmental cues (nitrate) to trigger payload release specifically at sites of inflammation.