• Part 1: Introduction
  • Part 2: Description
  • Part 3: Exemplary example:
                QEP&AQ
  • Part 4: Current delivery system
  • Part 5: Our solution
  • Secretion module
  • Adhesion module
  • Biosafety module
  • References

    • Part 3: Exemplary example: QEP&AQ

    Two representative small peptides, QEP (Gln-Glu-Pro) and AQ (Ala-Gln) have demonstrated significant health benefits when used as therapeutics or nutritional supplements.

    QEP(Gln-Glu-Pro)

    QEP is shown to mitigate glucose absorption in the intestine, by inhibiting the expression of SGLT1, a glucose cotransporter protein1.

    Fig10. Model depicting the presumed RS1-dependent mechanism for downregulation of SGLT1 by QEP at high D-glucose (adapted from Hermann et al. 2020)

    In contrast to traditional SGLT1 inhibitors, the small peptide has additional antidiabetic effects such as increasing insulin sensitivity and normalizing elevating fasting glucose,etc. Detailed comparisons betweuen QEP and traditional SGLT1 inhibitors are shown in the table below.

    AQ(Ala-Gln)

    AQ belongs to the family of glutamine supplement. After entering the body, it will be digested into alanine and glutamine. Compared to glutamine, it has increased solublility, stablility, and ease of absorbtion3.

    Fig2. Mechanisms of enteral and parenteral glutamine (GLN) supply.(Cruzat V et al.2018)

    As the most abundant free amino acid in the bloodstream, glutamine exhibits important functions. Some of them are listed below.

    There are Various formulations available for these prospective small peptides, but they each has their own shortcoming.

    Part 4: Current delivery system

    Current delivery systems include controlled-release microparticle depots, in-situ-forming polymer matrix, implants, etc11. Although some of them have already had clinical products launched in the market, they still possess shortcomings like invasive injection, unsustainable and non-inducible release. Invasive injection: Delivery methods like intravenous and subcutaneous routes require injection, which is invasive and brings pain and suffering to patients. Unsustainable release: Traditional delivery methods like oral administration are unsustainable. Patients have to take medicine frequently (for example, 3 times a day), which increases their burden. Non-inducible release: delivery systems like controlled-release microparticle depots release therapeutics slowly for up to 6 months11. However, drugs are released constitutively and not due to the microenvironment response, making the system more uncontrollable.

    Fig3. Disadvantages of current small peptide delivery systems

    Part 5: Our solution

    To create a non-invasive, sustainable, and inducible delivery system, we genetically engineer Escherichia coli into probiotic Intestide. Our proposed design encompasses three interconnected modules—Secretion, Adhesion, and QS (Quorum Sensing) safety—and involves two distinct strains of engineered bacteria: Peptide Producers and Controllers.

    Fig 4. MGenetic circuit of Intestide

    Secretion module:

    The Secretion module is developed for the peptide producer strain, facilitating the secretion of small peptides. This module features a recombinant transmembrane protein, Lpp'OmpA, which would be.integrated into the outer membrane of E. coli upong syntheses. By synthesizing a chimera protein consisting of this small peptide and transmembrane protein, we enabled their delivery into the surrounding environment12.

    Fig 5. Schematic view of the delivery system

    Adhesion module

    This module would be incorporated into both the peptide Producer and Controller strains, which consists of HSP60 and LAP adhesins that are capable of binding to each other. HSP60, also present as a membrane protein on intestinal cells, interacts with LAP through a β-barrel protein structure displayed on the outer membrane of E. coli. This arrangement results in the formation of a strain-layered matrix that securely adheres to the intestinal lining13,14.

    Fig6. The inspiration for the Adhesion module. HSP60, as a membrane protein on intestinal cells, can bind to LAP(blue dots on bacteria) present in the outer membrane of E. coli
    Fig7. The expected effect for the Adhesion module, which adheres to the engineering bacteria and intestine

    Biosafety module

    The Biosafety module comprises two distinct components within the peptide producer and controller strains. Based on a reciprocal activation principle, whereby one strain's product induces the activation of the other's gene promoter, this module regulates the population dynamics of the peptide producer and controller strains to ensure optimal small peptide production15. Furthermore, the effective functioning of the QS-safety mechanism is contingent upon the cohabitation of both bacterial strains and the formation of the adhesion matrix. The viability of these strains is primarily sustained within the low-oxygen environment of the intestine, effectively mitigating the risk of bacterial leakage16,17.

    Fig8. Shcematic view of QS system

    To sum up, our engineered E.coli will be delivered into the intestine, in which they will colonize and reproduce with the aid of the adhesion module. The secretion module displays small peptides on the surface of bacteria, which are cleaved by enterokinase in the intestine and conduct their tasks. To confirm the biosafety, peptide producer and controller regulate the number of each other through the QS system.

    Fig9. Schematic view of Intestide

    References


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    Otto, C. et al. Antidiabetic Effects of a Tripeptide That Decreases Abundance of Na+-D-glucose Cotransporter SGLT1 in the Brush-Border Membrane of the Small Intestine. ACS Omega (2020).

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    Ishida N, Saito M, Sato S, Tezuka Y, Sanbe A, Taira E, Hirose M. Mizagliflozin, a selective SGLT1 inhibitor, improves vascular cognitive impairment in a mouse model of small vessel disease. Pharmacol Res Perspect. 2021 Oct;9(5):e00869. doi: 10.1002/prp2.869.

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    Cruzat V, Macedo Rogero M, Noel Keane K, Curi R, Newsholme P. Glutamine: Metabolism and Immune Function, Supplementation and Clinical Translation. Nutrients. 2018 Oct 23;10(11):1564. doi: 10.3390/nu10111564.

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    Fläring UB, Rooyackers OE, Wernerman J, Hammarqvist F. Glutamine attenuates post-traumatic glutathione depletion in human muscle. Clin Sci (Lond). 2003 Mar;104(3):275-82. doi: 10.1042/CS20020198.

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    Cruzat VF, Rogero MM, Tirapegui J. Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise. Cell Biochem Funct. 2010 Jan;28(1):24-30. doi: 10.1002/cbf.1611.

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    J C Hall, K Heel, R McCauley, Glutamine, British Journal of Surgery, Volume 83, Issue 3, March 1996, Pages 305-312.

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    Mills EL, Kelly B, O'Neill LAJ. Mitochondria are the powerhouses of immunity. Nat Immunol. 2017 Apr 18;18(5):488-498. doi: 10.1038/ni.3704.

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    Vargason AM, Anselmo AC, Mitragotri S. The evolution of commercial drug delivery technologies. Nat Biomed Eng. 2021;5(9):951-967. doi:10.1038/s41551-021-00698-w

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    Nicchi, S. et al. Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems. Microb Cell Fact 20, 1-14 (2021).

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    Drolia, R. et al. Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nat Commun 11, 6344 (2020).

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