After selecting the target gene, we need a suitable "switch" to make it express at the right place and time. According to the characteristic that the concentration of ROS in the intestine of patients with enteritis is often higher than that in other parts of the body, this "switch" should be able to increase the expression of target genes in response to ROS, and hardly enhance the expression in the absence of ROS environment.

ROS Promotor

ROS, or reactive oxygen species, are molecules commonly found in organisms undergoing aerobic respiration. Under normal circumstances, it does not threaten the health of the organism. Typically, a single electron is initially transferred to oxygen to form a superoxide (O2-), which is then catalyzed by superoxide dismutase to become a hydroperoxide (H₂O₂). Superoxide is less likely to cross the membrane, which limits its range of influence. However, H₂O₂can easily spread to other sites. When the human body is in a state of disease, ROS production is greatly increased, and H2O2 which is of high diffusion will spread from the lesion to other healthy parts, causing oxidative damage to the Escherichia coli in this place [1].

Fig1. ROS are highly reactive and quickly transition between species.

(Picture resource: Brieger K, Schiavone S, Miller FJ Jr, Krause KH. Reactive oxygen species: from health to disease. Swiss Med Wkly. 2012 Aug 17;142:w13659. doi: 10.4414/smw.2012.13659. PMID: 22903797.)

To counter this adverse effect, organisms have evolved relative clearance systems. The well-known ways in which Escherichia coli eliminate H₂O₂ are glutathione reduction, the thioredoxin system, and the gludoxin system. The genes that respond to ROS and encode related enzymes differs within organisms. In E. coli, the key regulators of adaptive responses to hydrogen peroxide and superoxide anions are OxyR and SoxR and SoxS. Among them, OxyR direct oxidation is the mechanism by which Escherichia coli sense hydrogen peroxide and induce the production of OxyR regulon. OxyR proteins are homologous to the LysR-NodD family of bacterial regulatory proteins and bind to the promoter of the oxyR regulatory gene. OxyR protein can activate the transcription of oxyR regulatory genes in vitro [2]. As shown in Fig2, there are multiple cysteine residues on the reduced OxyR protein, containing many sulfhydryl groups. These groups can be oxidized by H2O2 to form disulfide bonds. Under the action of glutathione and gludoxin, OxyR can be regenerated in reduced form [3]. This suggests that oxidation of the OxyR protein brings about conformational changes [2]. The changes are mainly due to the intramolecular disulfide bond Cys199-Cys208[3]. The activated OxyR binds upstream of the promoters −35 sequence of the target genes such as dps，katG and gorA in the form of tetramers [3][4]. Transcription is stimulated by direct interaction with the carboxyl terminal domain of the alpha subunit of RNA polymerase [4]. In addition, both states of OxyR is capable to bind to the oxyR promoter as repressors that limit the amount of regulatory protein synthesis [3]. OxyR regulates at least eight genes, including gorA encoding glutathione, dps encoding non-specific DNA-binding protein, katG encoding hydroperoxidase I and ahpCF encoding alkyl hydroperoxide reductase [5]. When the expression of OxyR regulators increased, the expression of these genes also augmented. Therefore, we conducted comparative analysis in the genes of katG, dps, gorA and ahpCF to select the final promoter base sequence.

Fig2. The oxyR regulon

(Picture resource: Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001 Mar;19(3):109-14. doi: 10.1016/s0167-7799(00)01542-0. PMID: 11179804.)

In a series of related studies, we founded that CARMEN MICHAÂN et al. used wild-type Escherichia coli in a culture medium containing 10μM H₂O₂ and measured gene expression of katG，dps，gorA and ahpCF over a period of time [5]. As shown in Fig3, we can find that katG has a rapid response to reactive oxygen stress and the longest duration among the genes to be selected. Therefore, it is the first choice as the promoter of engineered bacteria.

Fig3.In vivo transcription of the Escherichia coli oxyR regulon

Wild-type Escherichia coli from an overnight culture in M9 minimal medium were diluted in fresh medium and incubated at 37°C with shaking at 150 rpm. At an OD600 of 0.2, H₂O₂ was added to half of the culture, to make a final 10 M solution, and the rest was used as a control. Samples were collected immediately (1min) after the addition of H₂O₂ and at 5, 10, 15, and 20 min of exposure. Samples were frozen with liquid nitrogen, and total RNA was purified. The fluorescence signal of each PCR product was compared to that of gapA. Data are from an average of eight MPCR amplifications. Values from treated samples were divided by those from the corresponding control. All genes were analyzed, but only those genes for which statistically significant (P＜0.05) increases were observed at a given time are represented. Error bars were estimated from the corresponding SEM. (Picture resource: Michán, C., Manchado, M., Dorado, G., & Pueyo, C. (1999). In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress. Journal of bacteriology, 181(9), 2759–2764. https://doi.org/10.1128/JB.181.9.2759-2764.1999)

There is an experimental analysis of reactive oxygen stress response of katG gene conducted separately. we could see that with the multiplication of bacteria (the curve indicated the change of OD600 value), the expression of katG gradually increased at first, and then showed a trend of decline after the number of bacteria was saturated. The activity of peroxidase, a gene expression product, had the same trend as the gene, but remained stable instead of decreasing later. This phenomenon further indicates that the expression of katG is related to H₂O₂ concentration. When H₂O₂ increased, the expression of katG went up. While the reaction between peroxidase and H2O2 reaches equilibrium, the expression of katG will gradually decrease. This mechanism can reduce the extra energy expenditure of cells. Therefore, the promoter of katG is suitable for the design of engineered bacteria.

In this paper, the changes of katG gene expression due to the increase of H₂O₂ caused by aerobic respiration during bacterial proliferation were analyzed by the RNA content expressed by katG. Escherichia coli cultured overnight in LB broth were diluted in fresh medium and incubated at 37°C and shaken at 150 rpm. Samples are collected at regular intervals and quickly cooled to 0°C. The culture growth is monitored by measuring OD600 for a specified time and is measured in the usual RNA purification and measurement manner.There is an experimental analysis of reactive oxygen stress response of katG gene conducted separately. With the multiplication of bacteria (the curve indicated the change of OD600 value), the expression of katG gradually increased at first, and then showed a trend of decline after the number of bacteria was saturated. The initial upregulation was the result of increased H2O2 output by aerobic metabolism under favorable conditions. The activity of peroxidase, a gene expression product, had the same trend as the gene, but remained stable instead of decreasing later. This phenomenon further indicates that the expression of katG is related to H₂O₂ concentration. When H2O2 increased, the expression of katG went up. While the reaction between peroxidase and H₂O₂ reaches equilibrium, the expression of katG will gradually decrease. This mechanism can reduce the extra energy expenditure of cells. Therefore, the promoter of katG is suitable for the design of engineered bacteria [5].

Fig4.Peroxidase activity during course of growth of wild-type bacteria in nutrient LB medium.

Activity data (darkly shaded bars) are from an average of four independent determinations. Error bars represent SEM. The katG expression data are shown in light bars. Statistical significance (P＜0.05), for comparisons with minimal values at 1.5 h of outgrowth, is marked with asterisks. Bacterial growth, monitored as OD600, is indicated by the line. (Picture resource: Michán, C., Manchado, M., Dorado, G., & Pueyo, C. (1999). In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress. Journal of bacteriology, 181(9), 2759–2764.https://doi.org/10.1128/JB.181.9.2759-2764.1999)

[1] Brieger K, Schiavone S, Miller FJ Jr, Krause KH. Reactive oxygen species: from health to disease. Swiss Med Wkly. 2012 Aug 17;142:w13659. doi: 10.4414/smw.2012.13659. PMID: 22903797.

[2] Storz G, Tartaglia LA, Ames BN. The OxyR regulon. Antonie Van Leeuwenhoek. 1990 Oct;58(3):157-61. doi: 10.1007/BF00548927. PMID: 2256675.

[3] Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001 Mar;19(3):109-14. doi: 10.1016/s0167-7799(00)01542-0. PMID: 11179804.

[4] Tao K. In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli. FEBS Lett. 1999 Aug 20;457(1):90-2. doi: 10.1016/s0014-5793(99)01013-3. PMID: 10486570.

[5] Michán C, Manchado M, Dorado G, Pueyo C. In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress. J Bacteriol. 1999 May;181(9):2759-64. doi: 10.1128/JB.181.9.2759-2764.1999. PMID: 10217765; PMCID: PMC93716.

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Ideas Of Anti-inflammatory Factors

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How to determine effective drugs?

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As mentioned before, the engineered bacteria we designed have three important elements: reactive oxygen species initiation factors, adhesion factors, and anti-inflammatory factors. Among them, anti-inflammatory factors are the key to the therapeutic effect of engineered bacteria. How to determine effective and easy-to-express anti-inflammatory drug molecules is an important issue we have to face.

In the early stages of the project, we considered a variety of drugs that may have anti-inflammatory effects, such as Short chain fatty acids (SCFAs), DL-endopeptidase, IL-10, etc（We will display these ideas in the idea library）. However, taking into account factors such as the absorption capacity of the pharmaceutical molecules, their anti-inflammatory capabilities and the size of the introduced gene, we believe that peptide-based drugs should be chosen. Peptides are small in molecular weight, easily absorbed, have a small gene length to save vector space, they are ideal pharmaceutical molecules.

Peptides with anti-inflammatory effects are currently less applied, and we have screened two peptides with anti-inflammatory effects through extensive literature review, namely melittin (MEL) and Indolicidin (Indo). Among them, melittin is the main active substance of bee venom, which has long been used in traditional Chinese medicine to treat inflammatory diseases, and modern researchers have also confirmed the anti-inflammatory effects of melittin. This has inspired us to apply melittin in the treatment of IBD. Below are the introductions to these two drugs.

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Basic information on melittin:

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Melittin is an amphipathic, water-soluble peptide consisting of 26 amino acid residues and is the principal active component of natural bee venom. The N-terminal region contains four positively charged residues, while the C-terminal region contains two positively charged residues. Due to the uneven distribution of polar and non-polar amino acid residues, melittin exhibits both hydrophilic and lipophilic amphipathic properties. The first 20 amino acids (from the N-terminus) are hydrophobic, whereas the subsequent 6 amino acids are hydrophilic. The complete sequence is as follows:

NH2-Gly-ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Gly-Leu-Pro-Ala-Leu-ile-Ser-Trp-ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH2

Reference: Ceremuga M, Stela M, Janik E, Gorniak L, Synowiec E, Sliwinski T, Sitarek P, Saluk-Bijak J, Bijak M. Melittin-A Natural Peptide from Bee Venom Which Induces Apoptosis in Human Leukaemia Cells. Biomolecules. 2020 Feb 6;10(2):247.

Each melittin chain exhibits a helix-hinge-helix motif or bent rod structure. Amino acid residues 3-10 form an α-helix, while residues 12-14 create a hinge region that connects to another α-helix containing residues 15-24. This unique structure, along with its amphipathic characteristics, enables melittin to penetrate cell membranes and interact with cellular substructures at the molecular level.

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Action principle of melittin:

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Effect of melittin on cell membrane:

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Reference: Zhang HQ, Sun C, Xu N, Liu W. The current landscape of the antimicrobial peptide melittin and its therapeutic potential. Front Immunol. 2024 Jan 22;15:

Melittin monomers can attach to the membrane surface and spontaneously integrate into natural or artificial phospholipid bilayers, thereby reducing the rigidity between the polar and non-polar regions and decreasing the permeability barrier. It has been proposed that melittin-induced membrane permeability may result from the formation of toroidal pores or fissures within the membrane.

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Anti-inflammatory effect of melittin:

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Reference: Liu Minchen & Du Ruofei, Chinese Journal of Modern Applied Pharmacy, 2023

In terms of anti-inflammatory effects, melittin plays a crucial role in regulating inflammatory signaling pathways. It inhibits the activity of the NF-κB pathway, reducing the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Melittin can suppress the phosphorylation of IKK and IκB, thereby blocking the nuclear translocation of NF-κB and interfering with inflammatory signal transduction. Additionally, melittin affects the MAPK signaling pathway by inhibiting the phosphorylation of JNK and p38, thus regulating the production of pro-inflammatory cytokines and reducing the activity of inflammatory cells and the secretion of inflammatory mediators. Furthermore, melittin interferes with the JAK/STAT pathway, inhibiting the activity of STAT transcription factors decreasing the secretion of inflammatory cytokines, which helps alleviate inflammatory diseases. Melittin also reduces inflammation by inhibiting Akt phosphorylation, which interferes with the expression of inflammatory proteins and suppresses the production of inflammatory mediators such as COX-2, iNOS, and cPLA2. Additionally, melittin promotes the recruitment of immune cells to the site of inflammation and induces T-cell apoptosis, thereby modulating the immune response and further alleviating inflammation.

Current experiments have demonstrated that melittin can be used to treat various inflammatory diseases, such as arthritis, skin inflammation, ulcerative colitis, and other inflammatory conditions.

table 1: The inflammatory diseases can be treated by melittin.

Reference:Liu Minchen &Du Ruofei, Chinese Journal of Modern Applied Pharmacy, 2023

This project focuses on the treatment of IBD, with particular attention to ulcerative colitis (UC), a form of IBD characterized by persistent inflammation primarily affecting the superficial layers of the rectum and colon.

Interviews with clinicians reveal that conventional therapies for UC often fail to achieve satisfactory efficacy and have notable side effects. Therefore, more effective treatments for UC are needed. We think melittin may be a good choice for treatment, our and other experiments have proved this.

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Therapeutic effect of melittin on ulcerative colitis:

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Ahmedy et al. (2020) evaluated the anti-inflammatory effects of melittin through oral administration in an acetic acid-induced colitis mouse model. Melittin treatment ameliorated acetic acid-triggered decreases of body weight and an increase of colon mass index in colitis model mice （Figure 5）. Compared to untreated colitis model mice, the melittin-treated group showed significantly reduced levels of tumor necrosis factor (TNF-α) and IL-6 (Figure 6). Melittin amended changes induced by acetic acid in the upstream inflammatory signaling pathways in colon such as NF-κB, Toll-like receptor 4 (TLR4), p38 MAPK, TNF receptor-associated factor 6 (TRAF6) (Figure 7). Melittin also reversed acetic acid-induced alterations in PGE2 and enzymes involved in its synthesis pathway in colon inculding secretory phospholipase A2 (sPLA2), and cyclooxygenase-2 (COX-2) (Figure 8). Furthermore, Melittin attenuated acetic acid-induced oxidative stress (Figure 9) and protected colon mucosa and submucosa against acetic acid-induced damage (Figure 10).

5-10Refernce:Ahmedy OA, Ibrahim SM, Salem HH, Kandil EA. Antiulcerogenic effect of melittin via mitigating TLR4/TRAF6 mediated NF-κB and p38MAPK pathways in acetic acid-induced ulcerative colitis in mice. Chem Biol Interact. 2020 Nov 1;331:109276.

Melittin inverted changes induced by acetic acid in body weight and colon mass index. a Change in body weight from day 0 to day 5 induced by acetic acid and melittin administration. b Effect of acetic acid and melittin on colon mass index in mice. Each bar with vertical line represents the mean ± S.E.M. of 10 mice per group; * significantly different from control group at p < 0.05, ** significantly different from control group at p < 0.001, *** significantly different from control group at p < 0.0001, significantly different from acetic acid group at p < 0.05, significantly different from acetic acid group at p < 0.0001 using One-Way ANOVA followed by Tukey multiple comparisons test.####

Melittin mitigated acetic acid-induced alterations in TNF-α and IL-6 levels in colon. Each bar with vertical line represents the mean ± S.E.M. of 6 mice per group; *** significantly different from control group at p < 0.0001, significantly different from acetic acid group at p < 0.0001 using One-Way ANOVA followed by Tukey multiple comparisons test.###

Melittin amended changes induced by acetic acid in the upstream inflammatory signaling pathways in colon. a Western blot analysis for the protein expression levels of TLR4, TRAF6 and p38 MAPK using β-actin as loading control (n = 3) b NF-κB level as measured by ELISA (n = 6). Each bar with vertical line represents the mean ± S.E.M.; * significantly different from control group at p < 0.05, ** significantly different from control group at p < 0.001, *** significantly different from control group at p < 0.0001, significantly different from acetic acid group at p < 0.0001 using One-Way ANOVA followed by Tukey multiple comparisons test.###

Melittin reversed acetic acid-induced alterations in PGE2 and enzymes involved in its synthesis pathway in colon. a PGE2 level as estimated by ELISA (n = 6). b Western blot analysis for the protein expression level of sPLA2 using β-actin as loading control (n = 3). c COX-2 level as estimated by ELISA (n = 6). Each bar with vertical line represents the mean ± S.E.M.; * significantly different from control group at p < 0.05, ** significantly different from control group at p < 0.001, *** significantly different from control group at p < 0.0001, significantly different from acetic group at p < 0.001 using One-Way ANOVA followed by Tukey multiple comparisons test.###

Melittin attenuated acetic acid-induced oxidative stress. Each bar with vertical line represents the mean ± S.E.M. of 6 mice per group; *** significantly different from control group at p < 0.0001, significantly different from acetic acid group at p < 0.05, significantly different from acetic acid group at p < 0.001, significantly different from acetic acid group at p < 0.0001 using One-Way ANOVA followed by Tukey multiple comparisons test.######

Melittin protected colon mucosa and submucosa against acetic acid-induced damage. (A) H & E staining of histological sections in the colon of different experimental groups. Control mice as well as those receiving melittin alone showed normal histological structures of colon wall with apparent intact mucosal lining including glandular elements and lining enterocytes, normal submucosa as well as outer muscular coat with minimal inflammatory cells infiltrates and normal vasculatures. However, mouse received acetic acid showed focal areas of erosion and damaged lining mucosa including glandular elements (arrows) with abundant intraluminal necrotic depresses and marked congestion of mucosal as well as submucosal blood vessels (dotted arrows), moderate submucosal edema and inflammatory cells infiltrates (star). On the other hand, mouse treated with melittin showed apparent intact intestinal mucosa, intact submucosal and outer muscular layers with occasional focal mononuclear inflammatory cells aggregates (H & E X 200 and X 50). (B) Histological scoring of colon mucosal damage. Results are expressed as median and range of 3 mice per group; ** significantly different from control group at p < 0.001, # significantly different from acetic acid group at p < 0.05, using Kruskal–Wallis one-way ANOVA followed by Dunn's multiple comparisons test.

Additionally, melittin reduced oxidative stress in colitis model mice. Atieh Yaghoubi et al. (2022) evaluated the anti-inflammatory properties of melittin in a dextran sulfate sodium (DSS)-induced colitis model.(Figure 11) They also analyzed the synergistic effects of melittin and sulfasalazine (SSZ) to assess whether combination therapy could improve clinical symptoms in UC model mice. The results indicated that both melittin alone and in combination with sulfasalazine significantly alleviated pathological damage in the colon tissues of UC model mice.(Figure 12-15) Melittin treatment exhibited antioxidant effects, suggesting that melittin could be a potential alternative therapy for UC intervention.

11-15Reference:Yaghoubi A, Amel Jamehdar S, Reza Akbari Eidgahi M, Ghazvini K. Evaluation of the therapeutic effect of melittin peptide on the ulcerative colitis mouse model. Int Immunopharmacol. 2022 Jul;108:108810. 

Melittin peptide improve the clinical symptoms of DSS-induced colitis in mice. (A, B, and D) Therapy with melittin peptide effect on colon weight and colon shortening that induced by DSS; (C) Evaluation of the colon weight to length ratio in a different group in comparison to the colitis group. [(*p < 0.05; ***p < 0.001 compare to the control group); (#p < 0.05; ##p < 0.01; ### p < 0.001 compare to the colitis group)]. (DSS=dextran sulfate sodium; SSZ=Sulfasalazine).

Melittin peptide ameliorated the disease activity index. (A and B) Therapy with melittin peptide (2.4 mg/kg/day) alone or along with SSZ (100 mg/kg/day) was measured in a different group in comparison to colitis group. [(**p < 0.01; ***p < 0.001 compare to the control group); (##p < 0.01; ### p < 0.001 compare to the colitis group); (@@p < 0.01; @@@p < 0.001 compare to the Melittin group); (++p < 0.01; +++p < 0.001 compare to the Sulfasalazine group); ($p < 0.05; $$p < 0.01; $$$p < 0.001 compare to the Melittin in combination with sulfasalazine group)].

Melittin peptide attenuated edema and inflammation of colon tissue induced by DSS in colitis mice. Evaluation of the effect of the therapy with melittin peptide alone or along with SSZ on (A) inflammation score; (B) Mucosal damage score; (C) and crypt loss in different groups. [(*p < 0.05; **p < 0.01; ***p < 0.001 compare to the control group); (##p < 0.01; ### p < 0.001 compare to the colitis group); (+p < 0.05; ++p < 0.01 compare to the Sulfasalazine group)].

Melittin peptide effect on histological damage to colon tissue in DSS-induced colitis mice. (A) Pathological damage ranges were measured in the different groups; (B) Evaluation of histological score of the colon in different treatment groups; (C) Submucosal edema and infiltration of the inflammatory cell in the histological result of different groups. [(*p < 0.05; **p < 0.01; ***p < 0.001 compare to the control group); (### p < 0.001 compare to the colitis group); (+p < 0.05; ++p < 0.01 compare to the Sulfasalazine group)].

Melittin effect on oxidative stress markers in DSS-induced colitis mice. The outcomes of melittin and melittin along with SSZ (A) MDA; (B) SOD activity; (C) catalase enzyme activity; and (D) total thiol of the serum in comparison to the colitis group. [(***p < 0.001 compare to the control group); (#p < 0.05; ### p < 0.001 compare to the colitis group); (@@p < 0.01; @@@p < 0.001 compare to the Melittin group); (+++p < 0.001 compare to the Sulfasalazine group)] (DSS=dextran sulfate sodium; SSZ=Sulfasalazine; MDA=malonyl dialdehyde; SOD=Superoxide dismutase).

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Artificial modification of melittin

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To further enhance the performance of melittin, we conducted in-depth research and precise modifications. In the modification process, we employed an innovative approach by connecting two melittin monomers with a linker to form a unique hairpin structure. This modification resulted in multiple performance improvements. The specific amino acid and polypeptide sequences are shown in Figure 16.

The artificial modification significantly reduced the cytotoxicity of melittin. By introducing this structure, we effectively decreased the peptide's toxicity to normal cells, greatly enhancing its application safety. This improvement allows melittin to be used in a wider range of scenarios, particularly in treatments requiring prolonged contact with biological tissues. Additionally, the modified melittin shows a marked increase in immunostimulatory capacity (building on previous laboratory work). This feature enhances its potential in regulating immune and inflammatory responses. The enhanced immunostimulatory ability not only helps activate the immune system more effectively but may also facilitate appropriate inflammation modulation, offering new possibilities for treating IBD.

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Conclusion

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Therefore, due to its anti-inflammatory and antioxidant properties, we have selected melittin as one of the drug molecules for this project.

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Indolicidin

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Indolicidin, as a naturally occurring compound with significant bioactivity, is gradually revealing its potential applications in the treatment of colitis. Studies indicate that due to its unique anti-inflammatory and immunomodulatory properties, indolicidin can act through various mechanisms, particularly excelling in modulating the gut microbiota and enhancing intestinal barrier function. The synergistic effects of these functions enable indolicidin to effectively alleviate symptoms of colitis, thereby improving patients' quality of life.

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Basic information:

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Indolicidin, discovered by Selsted et al. (1992), is a natural peptide belonging to the cathelicidin family isolated from bovine neutrophils. This peptide consists of 13 amino acid residues, nearly half of which are hydrophobic, including 5 tryptophan residues. These tryptophan residues are responsible for insertion and distribution within biological membranes, leading to the peptide's hemolytic activity (Subbalakshmi et al., 1996). The physicochemical properties are shown in Table 2.

Table 2: Sequence and physicochemical properties of indolicidin

Reference:Batista Araujo J, Sastre de Souza G, Lorenzon EN. Indolicidin revisited: biological activity, potential applications and perspectives of an antimicrobial peptide not yet fully explored. World J Microbiol Biotechnol. 2022 Jan 12;38(3):39.

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Action principle:

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Representation of indolicidin's mechanism of action after transient membrane perturbation,intracellular potential targets are proteins and nucleic acids (a), indolicidin-LPS interactionrelated to anti-sepsis and anti-biofilm activity (b) and the indolicidin anion carrier mechanismis thought to explain hemolytic activity(c).

(a)The bacteriostatic effect of indolicotin: Indolicidin has a broad range of biological targets, including both Gram-positive and Gram-negative bacteria (Vergis et al., 2020). Additionally, Indolicidin can disrupt established biofilms and inhibit biofilm formation. At the molecular level, although Indolicidin can insert into the plasma membrane, it does not directly cause membrane rupture. Instead, it enters the cytoplasm, binds to DNA, and inhibits the synthesis of proteins and DNA.

(b)Indolicidin neutralizes the pro-inflammatory effects of LPS: Lipopolysaccharide (LPS) is considered an endotoxin and a major outer membrane component of almost all Gram-negative bacteria, acting as a potent immune system stimulant (Cohen 2002). LPS activates inflammatory cells, such as monocytes and macrophages, leading to the release of cytokines (e.g., TNF-α, IL-1β, and IL-6) and nitric oxide, thereby triggering an inflammatory cascade (Nan et al., 2009a; Park et al., 2009). Indolicidin can bind and neutralize LPS, making it a potential anti-inflammatory agent.

(c)The indolicidin anion carrier mechanismis thought to explain hemolytic activity.

Reference:Batista Araujo J, Sastre de Souza G, Lorenzon EN. Indolicidin revisited: biological activity, potential applications and perspectives of an antimicrobial peptide not yet fully explored. World J Microbiol Biotechnol. 2022 Jan 12;38(3):39.

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Conclusion:

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Melittin and indolicidin, as novel peptide-based drugs, exhibit significant anti-inflammatory potential. Both inhibit inflammatory responses through different mechanisms, improving gut health. Our project focuses on these two anti-inflammatory agents, aiming to fully utilize their properties and enhance clinical efficacy and safety, providing new therapeutic options for IBD patients.

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Anti-inflammatory Factors

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Peptide cell experiment

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Next, we will use the cell model constructed above for preliminary drug screening experiments. Thaw macrophages and culture them in wells on a 24 well plate after two passages. Wait for the cells to grow full again and perform the same starvation treatment on the cells. After infection, different concentrations of Mel and Indo were applied to the experimental group, and the cells were collected after four hours of cultivation. Compared with the control group treated only with PBS solution, the experimental group cells showed varying degrees of decreased levels of reactive oxygen species（ROS）, decreased levels of some inflammatory factors, and increased expression of anti-inflammatory factor IL-10.

Use flow cytometry to detect cellular ROS levels. As shown in Figure 1A, the experimental groups treated with 5, 10, and 15 ppm Mel and 5 and 15 ppm Indo showed a significant decrease in the level of ROS in cells. However, the experimental results of applying different concentrations of peptides to uninfected macrophages showed that Mel and Indo did not reduce the level of ROS in healthy cells (Figure 1B). The above results indicate that appropriate concentrations of Mel and Indo can significantly reduce the levels of ROS in inflammatory cells.

The expression levels and changes of four inflammatory factors in cells were detected by RT-qPCR technology, and the results are shown in Figure 2A. Only concentrations of 5 and 10 ppm in the Indo group showed significant decrease in IL-1 β. In the Mel group, a concentration of 15ppm of Mel significantly upregulated all four inflammatory factors, while a concentration of 10ppm significantly downregulated the expression levels of IL-1 β and IL-6. We speculate that both peptides at appropriate concentrations can downregulate the expression of some inflammatory factors. Excessive concentrations may lead to peptides acting as antigenic substances to stimulate macrophages to generate inflammatory response, resulting in a significant increase in the expression level of inflammatory factors.

In addition, we also conducted cell counts and intracellular cell counts after infection. As shown in Figure 2B, there was no significant change in the number of cells after experimental treatment in all groups. Except for a significant decrease in the number of intracellular bacteria in cells treated with Mel at concentrations of 5 and 10 ppm, there was no significant change in the number of intracellular bacteria in the other groups.

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In vivo peptide experiments

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The above experiments have demonstrated that Mel and Indo at appropriate concentrations have certain anti-inflammatory effects in an in vitro cellular inflammation model. Next, we will further validate the anti-inflammatory effects of these two drugs in mice.

Firstly, a mouse intestinal inflammation model was constructed. A batch of female Balb/cJNju-Foxn1nu/Nju Hfh11nu mice were ordered and experimental procedures were carried out according to the table and all fluids were administered into the mice by gavage. The concentration of Salmonella is , Mel and Indo is 10ppm. The intestinal specimens taken were from the cecum to the colon.

We measured the colon length of each group of mice to characterize the level of intestinal inflammation. The results showed that both Mel and Indo could significantly alleviate the shortened colon length in mice caused by intestinal inflammation, and Mel had better alleviating effect (Figure 3A).

Take some colon tissue for microscopic observation. After dehydration, embedding, and sectioning of colon tissue, HE staining was performed. The results are shown in Figure 3B. It can be seen that compared with the PBS group, the S.Tm group had damaged intestinal epithelial cells and disrupted intestinal mucosal integrity. However, the symptoms of the treatment group were significantly relieved, and the Mel group had better effects. Some tissue sections were processed for immunofluorescence staining. The results are shown in Figure 3C. It can be seen that the IL-6 fluorescence signal intensity of the tissues treated with Salmonella by gavage was significantly higher than that of the PBS group, indicating more severe intestinal inflammation. However, the IL-6 fluorescence intensity of the Mel and Indo groups was significantly lower than that of the S.Tm group, and both Mel and Indo had certain anti-inflammatory effects.

Cut off a portion of colon tissue, completely dissociate it, stain immune factors and immune cell marker proteins, and then measure their expression levels using flow cytometry. As shown in Figure 3D, Mel can significantly reduce the levels of IFN-γ, IL-6, TNF-α, F4/80, and Ly6G in the colon tissue of mice with enteritis, while Indo can only reduce the levels of IFN-γ, TNF-α, and F4/80

Take a portion of colon tissue, extract cellular RNA, and perform RT qPCR to detect the expression levels of various inflammatory factors inside the cells. The results are shown in Figure 3E. Compared with the control group, the expression levels of IL-6, IL-1 β, and IL-8 in the intestinal tissue after Mel treatment were significantly reduced, while after Indo treatment, only IL-6 expression levels were significantly reduced, and other inflammatory factors showed no significant changes or significant increases. In addition, compared to the positive control group, there was no significant change in IL-10 levels in the Mel group, while the Indo group showed a significant increase.

Through in vitro and in vivo experiments with the two peptides mentioned above, we found that compared to Indo, Mel exhibited better anti-inflammatory performance in all the indicators we tested. Therefore, we selected Mel for further experimental research.

Figure 3E

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Adhesion Factor CMC

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The key to the efficient functioning of FMK is to make the engineered bacteria FMK stay in the intestine longer and express anti-inflammatory factors in response. Therefore, the team simulated the binding between the glucan binding protein and the glucan substrate, and designed the artificial glucan binding protein CMC.

Glucan is an extremely important polysaccharide produced by a large number of bacteria and fungi. At the same time, the surface of intestinal epithelial cells secretes mucus, which plays a protective role as a barrier. The main component of mucus is the highly glycosylated glycoprotein MUC2, which has various polysaccharide structures such as Core1, Core2 and Core3 [1]. At the same time, the beneficial properties of probiotics are related to the extracellular polysaccharide (EPS) they produce [2], and the surface of most probiotics can be exposed to glucan. Lactobacillus in Firmicutes has various functions, such as regulating the balance of intestinal microbial community, promoting food digestion and nutrient absorption, alleviating intestinal inflammation and infection, etc. Its cell wall is mainly composed of peptidoglycan and polysaccharide. For example, Lactobacillus can produce glucan sucrase (GS), and synthesize EPSs through glucose-transfer glycogen [3], such as α-glucan [4]. Salmonella is a common food-borne pathogen that uses a variety of virulence factors to overcome colonization resistance and induce intestinal inflammation [5], all of which are Gram-negative bacteria. Lipopolysaccharide, an important component of cell wall, is an endotoxin and contains O antigen [6]. S.typhimurium is a zoonotic pathogen that causes a large proportion of non-typhoid Salmonella (NTS) infections, accounting for a quarter of infections, second only to Salmonella enteritidis serotype in incidence [7]. Therefore, we thought of combining this characteristic of glucan as the core idea for designing synthetic proteins, so that the adhesion factors we designed can serve as a "bridge" between intestinal microorganisms and the intestines, recruit intestinal probiotics, and achieve stable attachment of engineered bacteria to the intestines. This idea inspired us to design CBMcipc domain, which is derived from the scaffold protein CipC of Clostridium cellulolyticum and contains a group III cellulose binding domain (CBD), a hydrophilic domain and two hydrophobic domains. The CBD domain confers CBMcipc with the ability to bind to glucan[8].

In order to select the best glucan-binding protein, fluorescent protein mCherry sequence was introduced in the experiment, and proteins M, CM, CMC and CMCC with different copy numbers of Clostridium cellulolyticum CipC (CBMcipc) were designed. By introducing pET-28a plasmid carrying corresponding genes (namely M, CM, CMC, CMC, CMCC) into Escherichia coli BL21, the synthetic Escherichia coli strains EcM, EcCM, EcCMC, EcCMCC were constructed. The proteins M, CM, CMC and CMCC are composed of mCherry, mCherry and one copy of CBMcipc, mCherry and two copies of CBMcipc, mCherry and three copies of CBMcipc (Fig1a). The N-terminal connected outer membrane protein A (OmpA) signal peptide helps the protein to pass through the intima and locate on the outer membrane surface [8], thus giving the protein surface display ability.

To verify the above properties, cells were stained with FITC-labeled glucan for flow cytometry and confocal microscopy. After incubation with FITC-glucan for 10 minutes, EcM cells showed almost no FITC fluorescence, while EcCM, EcCMC and EcCMCC cells showed obvious FITC fluorescence. Among the three CBMcipc containing strains, EcCMC cells showed the highest FITC fluorescence (Fig 1b, c), indicating that EcCMC had the highest glucan-binding capacity.

Confocal microscopy images and fluorescence quantification further revealed that EcM, EcCM, and EcCMCC cells had a whole-cell distribution of mCherry fluorescence, while EcCMC cells had a bilateral distribution of mCherry, indicating that CMC exhibited a stronger surface display ability(Fig1d). Since the cell-surface display of the binding protein is necessary for it to bind to dextran outside the cell, this property of EcCMC may help enhance its dextran binding ability.

Fig1. Design and characterization of the synthetic bacteria used for glucan binding[7]. (Based on the preliminary research findings from the laboratory)

Gavage experiments with EcCM, EcCMC, EcCMCC mixed with Lactobacillus plantarum (probiotics) were done on mice to assess the ratio of the number of beneficial intestinal bacteria to the amount of beneficial bacteria in the gavage after 12 hours of gavage (adherence rate) and the results showed that EcCMC had the highest adherence rate (Fig2). Whereas, after replacing the probiotics with Salmonella enterica serovar Typhimurium subsp. enterica (pathogenic bacteria), the adhesion rate was lower in all cases, which reflects the preference of adhesion factors for binding to probiotics.

Fig2 The adhesion rate of exogenous yeast was detected by intragastric administration.

Based on the above characteristics, CMC was selected as a adhesion factor in the experiment, hoping to achieve the colonization of engineered bacteria in the intestine and the recruitment of intestinal probiotics through its expression in the intestine, so as to better alleviate intestinal inflammation and regulate the intestinal microenvironment.

Reference:

[1].NIE Shuo, WEN Zhengshun. Secretion, Structure, Synthesis Regulation of Intestinal Mucin 2 and Its Role in Development of Intestinal Diseases. Chinese Journal of Animal Nutrition, 2020, 32(6): 2521-2532.

[2]. Pourjafar, Hadi et al. “Functional and health-promoting properties of probiotics' exopolysaccharides; isolation, characterization, and applications in the food industry.” Critical reviews in food science and nutrition vol. 63,26 (2023): 8194-8225. doi:10.1080/10408398.2022.2047883

[3].Yu, Liansheng et al. “Glucansucrase Produced by Lactic Acid Bacteria: Structure, Properties, and Applications.” Fermentation (2022): n. pag.

[4].Chen, Ziwei et al. “Lactic acid bacteria-derived α-glucans: From enzymatic synthesis to miscellaneous applications.” Biotechnology advances vol. 47 (2021): 107708. doi:10.1016/j.biotechadv.2021.107708

[5].Whitfield, Chris et al. “Lipopolysaccharide O-antigens-bacterial glycans made to measure.” The Journal of biological chemistry vol. 295,31 (2020): 10593-10609. doi:10.1074/jbc.REV120.009402

[6].Park, Jeong Soon et al. “Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane.” The FASEB Journal 26 (2012): 219 - 228.

[7]. Yin, Hongda et al. “Synthetic physical contact-remodeled rhizosphere microbiome for enhanced phytoremediation.” Journal of hazardous materials vol. 433 (2022): 128828. doi:10.1016/j.jhazmat.2022.128828

idea library

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DL-endopeptidase

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IBD, as an inflammatory disease, is considered to be caused by the interaction of multiple factors, among which immune imbalance is a key factor. The gut, as a major digestive organ directly connected to the outside world, has a complex microbial ecology and naturally faces a complex immune environment. To maintain intestinal immune balance, there is abundant information exchange between the gut and the microbiota to regulate immune activity, which is often achieved through chemical signals, such as the specific recognition of receptors and signal molecules (ligands).

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The NOD2 receptor receives MDP signals to regulate immune activity.

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The nucleotide oligomerization domain 2 (NOD2) receptor is a classical pattern-recognition receptor sensing specific microbial peptidoglycan fragments, including muramyl dipeptide (MDP), and N-acetylglucosamine (NAG)-MDP. Activation of NOD2 is involved in a lot of physiological processes, including immune training, epithelial regeneration, insulin resistance, vaccination response, and promoting the effects of anti-PD-L1 immunotherapy. Research shows that loss-of-function mutations in NOD2 are the strongest risk factor for CD.

Figure 1: The NOD2 receptor can accept the signal of muramyl dipeptide (MDP) for regulation, and by modulating pro-inflammatory cytokines, anti-inflammatory factors, antimicrobial peptides, adaptive immunity, and autophagy, it can promote intestinal homeostasis and enhance resistance to the invasion of pathogens that can cause infectious colitis, thereby maintaining intestinal homeostasis by strengthening the epithelial barrier function.

Reference: Khor B, Gardet A, Xavier RJ (2011).Genetics and pathogenesis of inflammatory bowel disease. Nature, 474.

https://doi.org/10.1038/nature.2011.10209

In addition to mutations in the NOD2 receptor gene itself, a low level of MDP signaling may also lead to insufficient NOD2 functional response, and the loss of NOD2 function may lead to Crohn's disease (CD). Moreover, increasing the level of MDP signaling can also partially compensate for the reduced function of the NOD2 receptor caused by gene mutations, thereby alleviating CD.

Figure 2: NOD2 mutation leading to CD mechanism diagram (upper figure) and reduction of DL-peptidase levels causing CD mechanism diagram (lower figure).

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H (2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe, 30.

https://doi.org/10.1016/j.chom.2022.08.002.

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DL-endopeptidase can increase the levels of MDP.

The main component of bacterial cell walls is peptidoglycan, and peptidoglycan hydrolases (PGHs) are the main enzymes responsible for cleaving peptidoglycan chains to release fragments. D-L endopeptidase is a type of PGH that can specifically hydrolyze special sites on bacterial peptidoglycan and produce muramyl dipeptide (MDP) molecular structures. Since some DL-endopeptidases are secreted enzymes, they could interact with other bacteria and enable them to release NOD2 ligands. We speculate that the DL-endopeptidase gene can be introduced into a gene expression vector, and the engineered bacteria can be used to express and secrete it, thereby increasing the level of MDP.

Figure 3: A schematic diagram of the catalytic sites of the 8 PGHs, where ⑤ shows the site where DL-peptidase catalyzes the production of MDP.

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H (2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe, 30.

https://doi.org/10.1016/j.chom.2022.08.002.

Figure 4: A schematic diagram of the principle of DL-peptidase regulating intestinal homeostasis.

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H (2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe, 30.

https://doi.org/10.1016/j.chom.2022.08.002.

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Experimental validation of the anti-inflammatory bowel disease (IBD) effects of DL-endopeptidase.

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To investigate whether DL-endopeptidase deficiency could edispose mice to colitis and the role of NOD2 signaling in this process, researchers collected feces from healthy controls and CD patients, divided them into healthy, high-endopeptidase, or low-DL-endopeptidase activity groups (indicated as healthy, DL-endoHigh, or DL-endoLow), and transferred the fecal microbiota to antibiotic-treated wild-type (WT) mice or their Nod2 knockout littermates (Nod2−/−) individually. After 10 days of fecal microbiota transplantation, colitis was induced in mice by adding 3% dextran sodium sulfate (DSS). To assess the efficiency of microbiota transplantation, researchers collected feces from all the donors and recipient mice before the DSS challenge.

Figure 5:Mice that received fecal microbiota from CD patients with low DL-endopeptidase activity were more predisposed to colitis.

(A) Schematic outline of DL-endopeptidase activity stratification and the fecal microbiota transplantation experiments.

(B) First two axes of a principal coordinate analysis (PCoA) of weighted UniFrac distances from donors in health, \( \mathrm{DL-endo^{High}} \), or \( \mathrm{DL-endo^{Low}} \)groups. Group differences were tested by pairwise PERMANOVA. n = 3 for each group.

(C) Selected DL-endopeptidase gene-encoding microbiota from Firmicutes decreased in \( \mathrm{DL-endo^{High}} \)or \( \mathrm{DL-endo^{Low}} \)groups, compared with the healthy controls, as revealed by t statistics of linear mixed-effects analysis: DL-endopeptidase group (healthy,\( \mathrm{DL-endo^{High}} \) , or \( \mathrm{DL-endo^{Low}} \) and NOD2 gene status (WT, Nod2−/−) as a fixed effect and donor ID as a random effect. Healthy and WT were used as references.

(D) NOD2 activity in HEK-Blue-NOD2 cells after incubation with the fecal supernatant of humans (n = 3/group), or mice (n = 9–12/group) that received human fecal microbiota from indicated groups. The values are relative ratios normalized to \( \mathrm{DL-endo^{Low}} \)group, valued as 1.

(E–H) Colitis severity of mice in different groups: body weight change (E), colon length (F), and intestinal barrier permeability, indicated by FITC-dextran permeability (G). Histology manifestation on hematoxylin-eosin (H&E) staining, scale bars represent 100 μm, and histology inflammation score (H).

(I–O) The transcriptional levels of Tnfa (I), Il-6 (J), Ccl2 (K), Cxcl1 (L), Col1a1 (M), Ripk2 (N), and Atg16l1 (O) in colonic tissue of mice (n = 8–11/group).

Each dot indicates one sample. Data are shown as mean ± SD. Statistical comparison was performed by two-way ANOVA followed by Bonferroni post hoc test (E), one-way ANOVA followed by Bonferroni post hoc test (D and F–O), or Student’s t test (N and O). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant.

Reference: Gao J, Zhao X, Hu S, Huang Z, Hu M, Jin S, Lu B, Sun K, Wang Z, Fu J, Weersma RK, He X, Zhou H (2022). Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway. Cell Host Microbe, 30.

https://doi.org/10.1016/j.chom.2022.08.002.

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Discussion about this idea

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DL-endopeptidase, an enzyme secreted by probiotics in the gut, can activate the NOD2 receptor by catalyzing the production of MDP signals to participate in intestinal immune regulation, maintain intestinal homeostasis, and alleviate Crohn's disease (CD). Since DL-endopeptidase acts on the cell walls of gut bacteria and does not directly act on the human body, it can be effective without being absorbed by the human body, making it suitable for the expression of engineered bacteria in the gut. DL-endopeptidase relies on the human immune system to maintain intestinal homeostasis, has fewer side effects, and is safer. However, due to its dependence on the NOD2 receptor pathway, it may be difficult to achieve therapeutic effects for IBD caused by NOD2 receptor gene mutations. In addition, because the number of engineered bacteria introduced into the patient's gut is limited, and DL-endopeptidase does not directly exert a therapeutic effect, it may be challenging for the engineered bacteria to express enough DL-endopeptidase to achieve the desired therapeutic effect. Therefore, we choose to first use peptides with direct therapeutic effects as the drug molecules for this project, and then consider introducing the DL-endopeptidase gene to enhance its efficacy or combine it with IBD typing to create differentiated products in subsequent product improvements.

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miRNA

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Inflammatory bowel disease (IBD) is believed to be caused by the dysregulation and persistent inflammatory response of genetically susceptible individuals to intestinal microbes, and epigenetic factors play an important role in its pathogenesis.

microRNA (miRNA), as endogenous small non-coding RNA, can recognize target mRNA through sequence complementation, inhibit target mRNA, and mediate post-transcriptional regulation of gene expression. Current studies have shown that mirnas exhibit a high degree of specificity in different types of IBD, so mirnas can be used as biomarkers and therapeutic targets after determining the functional consequences of miRNA dysregulation. Based on the above ideas, we propose the application of mirNa-based logic circuits in the treatment of IBD.

We selected specifically malregulated mirnas and protein responsive mrnas in IBD as control switches to create a logic circuit to regulate the expression of anti-inflammatory factors or adhesion factors associated with IBD therapy by detecting miRNA input. As shown in Fig2, in OFF state (absence of input miRNAs), L7Ae protein represses translation of the output gene-coding mRNAs by interacting with the kink-turn motif (Kt). In ON state (presence of input miRNAs), the L7Ae translation is repressed by the miRNAs, which leads to output translation.

At present, experiments have verified that miRNA-based circuits can control cell death signals through logical operation. As shown in Fig3a, a 2-input (miR-21 AND miR-302a) AND circuit was designed in the experiment, with EGFP as output. The AND circuit expresses the output only in the presence of two miRNAs. This experiment not only proves that miRNA-based logic loops can actually be used for expression regulation, but also provides ideas for designing AND, OR, NAND, NOR logic loops in response to multiple mirnas (Fig3b).

This miRNA-based logical loop treatment of IBD has higher biosafety. At the same time, due to the highly specific characteristics of miRNA in different IBD subtypes, miRNA can more sensitive to distinguish between different IBDs to achieve accurate and sensitive treatment. However, this scheme still has many problems, such as the difficulty of engineering bacteria to detect miRNA changes in the intestinal environment in time, the difficulty of the experiment, and the high cost, so the team did not use this idea. In the future, we will continue to pay attention to the research progress of miRNA as a therapeutic target, and look forward to a more optimized experimental program.

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SCFAs

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Short-chain fatty acids (SCFAs) are primarily produced through the metabolism of carbohydrates, particularly propionate and butyrate. Their health effects include inhibiting histone deacetylase (HDAC) activity, enhancing the expression of anti-inflammatory factors, strengthening mucosal barrier function, and reducing inflammation. Research indicates that SCFAs can improve the integrity of intestinal cells, influence the homeostasis and function of different cell types, and promote the expression of tight junction proteins such as claudin and occludin. Further clinical studies suggest the need for sustained SCFA production to achieve anti-inflammatory effects, which can be delivered via oral or enema routes, or by utilizing specific live bacteria to enhance their production. Therefore, due to the plasticity of yeast fatty acid synthase (FAS) structures, we aim to use engineered yeast colonization in the gut to provide more SCFAs for the treatment of inflammatory bowel disease (IBD). SCFAs offer a potential direction for this project.

Short-chain fatty acids (SCFAs) are fatty acids produced by gut microbiota through the fermentation of undigested carbohydrates, typically containing fewer than six carbon atoms. The main SCFAs include acetate, propionate, and butyrate. Propionate and butyrate are primarily derived from the metabolism of carbohydrates during glycolysis. The synthesis process of short-chain fatty acids is shown in Figure 1.

Figure 1: Acetate is the most abundant SCFA in the gut, produced from pyruvate via acetyl-CoA. It can also be converted into butyrate through the action of acetyl-CoA:butyryl-CoA transferase (BCoAT). Butyrate is synthesized by the condensation of two acetyl-CoA molecules, which are then reduced to butyryl-CoA and subsequently converted into butyrate. Butyrate kinase and butyryl-CoA:acetate CoA-transferase are the two main enzymes involved in butyrate production.

Reference:Parada Venegas, D., De la Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra, G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

Short-chain fatty acids (SCFAs) play a crucial role in protecting the intestinal mucosa and exerting anti-inflammatory effects. They inhibit histone deacetylase (HDAC) activity, enhance the expression of anti-inflammatory factors, and strengthen the intestinal mucosal barrier function. Additionally, SCFAs can reduce systemic inflammatory responses. The specific role of SCFAs is shown in Figure 2

Figure 2: Health effects of SCFAs

In a healthy intestinal mucosa, SCFAs can increase the expression of anti-inflammatory factors and inhibit pro-inflammatory factor expression, while enhancing mucosal barrier function and reducing inflammation. In inflamed mucosa, the reduction of SCFAs leads to an increase in pro-inflammatory factors, a decrease in anti-inflammatory factors, compromised mucosal integrity, and abnormal cell autophagy. Figure 3 illustrates the mechanisms of SCFAs.

Figure 3:

SCFAs in healthy (A) and inflamed (B) colonic mucosa. In healthy mucosa, (1) bacterial fermentation of dietary fiber (DF) by SCFAs-producing bacteria (e.g., F. prausnitzii), increases luminal content of butyrate (green), propionate (blue) and acetate (purple) (2), forming a gradient along the crypt. In lamina propria (LP) macrophages under acute inflammatory stimulus (4), butyrate inhibits histone deacetylases (HDACs) thus; NF-κB-induced pro-inflammatory mediators (e.g. TNF-α, IL-6, IL-12 and iNOS) expression whereas increases anti-inflammatory mediators (e.g., IL-10). In colonocytes (5), butyrate is β-oxidized to Acetyl-CoA and constitutes the main source of energy by entering the TCA cycle. Alternatively, butyrate initiates signaling pathway activation (or repression) by GPCRs and/or directly inhibits HDACs, thus activating (e.g., HIF-1, STAT3 and SP1) or repressing (e.g., NF-κB) transcription factors (TFs), increasing epithelial barrier function, antimicrobial peptides (AMPs) production, cell proliferation and decreasing inflammation.

In inflamed mucosa as IBD, (1) a decreased fermentation of DF by low levels of SCFAs-producing bacteria (e.g., F. prausnitzii) (2), reduces SCFAs luminal content (3). In LP inflammatory macrophages (4), butyrate-GPCRs activation and -HDACs inhibition are downregulated, thus, there is uncontrolled NF-κB-induced pro-inflammatory mediators’ expression (e.g. TNF-α, IL-6, IL-12 and iNOS) and decrease of anti-inflammatory mediators (e.g., IL-10), although it appears that the inflammation increasesthe GPCRs and transporters expression. In inflamed colonocytes (5), butyrate uptake and oxidation are decreased and GPCRs and transporters are also downregulated. This contributes to decreased epithelial barrier integrity, AMPs production, cell proliferation and increased inflammation.

Reference:Parada Venegas, D., De la Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra, G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

Table 1 describes the role and mechanism of different types of SCFAs in different mouse models.

Table 1: lmpact of SCFAs on intestinal homeostasis.

Reference:Parada Venegas, D., De la Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra, G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

In current clinical treatments, butyrate or SCFA mixtures are typically administered orally or via enema for IBD. Studies have shown that continuous production and delivery of SCFAs to the mucosa are necessary for anti-inflammatory effects. However, long-term medication and enemas can be burdensome for patients. Therefore, we aim to directly introduce live bacteria that produce SCFAs into the mucosa for continuous production and delivery. This approach requires pretreatment to enrich or restore SCFA-producing bacteria.

The plasticity of fungal fatty acid synthase (FAS) supports this concept. Fatty acid synthesis is carried out by FAS in a cyclical, repetitive manner through seven distinct reaction steps, with each cycle extending the carbon chain by two atoms. Each step is completed by an independent enzyme module within the FAS. The acyl carrier protein (ACP) acts as a carrier, transporting substrates and intermediates between different enzyme modules, facilitating their transfer and reaction. We aim to incorporate additional enzyme modules into yeast FAS to alter its activity, reducing the cycles to synthesize more short-chain fatty acids.

Figure 4: The yeast fatty acid biosynthesis cycle is depicted. ACP, acyl carrier protein (purple); AT, acetyltransferase (blue); MPT, malonyl-palmitoyl-transferase (yellow);KS, ketosynthase (red); KR, ketoreductase (orange); DH, dehydratase (light green); ER, enoyl reductase (green); FMN, flavin mononucleotide.

Reference:Singh, K., Bunzel, G., Graf, B., Yip, K. M., Neumann-Schaal, M., Stark, H., & Chari, A. (2023). Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural snapshots. Cell, 186(23), 5054–5067.e16. https://doi.org/10.1016/j.cell.2023.10.009

Figure 5: Visualized chemical details in the 1.9 A˚ structure. One of the six asymmetric units of FAS (indicated in cyan, top left) is extracted and depicted in the center. Individual enzymatic domains are colored following the same scheme as in (A). The locations of zooms (i–v) in the asymmetric unit are indicated. (i) FMN bound in the ER active site. The density indicates a 22 kink in the isoalloxazine ring system, consistent with its reductive role in FA biosynthesis. (ii) FMN recognition by ER active site amino acids and their accompanying water network. (iii) NADP+ bound in the ER active site cleft with its nicotinamide group stacked between the FMN isoalloxazine and His740, a solvent network aids in recognition of the ADP moiety. (iv) Recognition of NADP+ by amino acids and solvent in the KR active site cavity. (v) Malonyl-CoA was found in a post-hydrolysis state in the MPT active site. CoA interacts with many MPT active sites residues, while the malonyl group is covalently attached to Ser1808 and interacts with Arg1834.

Reference:Singh, K., Bunzel, G., Graf, B., Yip, K. M., Neumann-Schaal, M., Stark, H., & Chari, A. (2023). Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural snapshots. Cell, 186(23), 5054–5067.e16. https://doi.org/10.1016/j.cell.2023.10.009

This study has detailed the synthesis process, showing that the \(\gamma\) subunit of yFAS can be used as a carrier to integrate other exogenous globular proteins (about 150 kDa per dome or 300 kDa per FAS) into the FAS dome. This discovery offers the potential to regulate the fatty acid profile of yFAS products. Based on this, researchers reconstructed the FAS and conducted validation experiments. The results are shown in Figure 6.

Figure 6:

(A) SDS-PAGE of peak gradient fractions of FAS reconstituted with various proteins. From left to right, these are: FAS (buffer addition only), g subunit (wild type), g-Tes V. harveyi, Tes V. harveyi (V. harveyi TesA alone), g-1xUnaG, or g-2xUnaG. The respective proteins reconstituted with FAS are denoted with red asterisks.

(B) Efficiency of reconstitution can be monitored by additional fluorescence intensity. Shown are three density gradients after sedimentation (g-1xUnaG left, g-2xUnaG middle, FAS alone right). Note that both g-1xUnaG and g-2xUnaG increase the intrinsic FMN fluorescence of FAS alone.

(C) Relative abundance of free acyl-CoAs in the absence of protein (no protein; left), Dg-FAS (FAS; 2nd from left), FAS bound to the g subunit (+ g subunit, middle), FAS bound to g subunit-TesA fusion protein (+ g-Tes V. harveyi; 2nd from right), and Dg-FAS subsequently treated with TesA (+ Tes V. harveyi; right) as measured by LC/MS. Note that C12-CoA and C14-CoA are absent when FAS is bound to g subunit-TesA fusion protein; C16-CoA is markedly reduced, whereas the relative abundance of C8-CoA and C10-CoA is increased (2nd from right). This can be attributed to thioesterase activity, as a Dg-FAS reaction subsequently treated with TesA shows a similar product profile (right) and argues that C12, C14, and C16 are present as free fatty acids and therefore not detected in LC/MS. All measurements were performed in triplicate (n = 3), and the error bars for the relative abundance of the individual species are indicated.

Reference:Singh, K., Bunzel, G., Graf, B., Yip, K. M., Neumann-Schaal, M., Stark, H., & Chari, A. (2023). Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural snapshots. Cell, 186(23), 5054–5067.e16. https://doi.org/10.1016/j.cell.2023.10.009

The experimental results indicate that the recombination directly modulates the configuration of FAS products, demonstrating that TesA can alter the product profile by integrating into the γ subunit. We can attempt to introduce enzyme modules for short-chain fatty acid synthesis to enhance the production of SCFAs in yeast. This improved yeast could continuously deliver SCFAs to the intestine, aiming to treat IBD and alleviate the discomfort associated with long-term medication and enemas in traditional therapies.

However, yeast, due to its larger size and more complex biological structure, significantly increases the workload in genetic engineering. In contrast, bacteria, while simpler in certain biochemical pathways, offer advantages as experimental models due to their ease of manipulation and cost-effectiveness. Therefore, after comprehensive evaluation, we have decided to use bacteria as the primary experimental subject for this study. Additionally, the aforementioned research results were mainly achieved in yeast and have not yet been validated in bacterial models. Furthermore, the redesign of yeast fatty acid synthase is technically more challenging than in bacteria, adding to the complexity and uncertainty of the experiments.

Considering the urgency of the project, budget constraints, and experimental feasibility, we ultimately decided to use engineered bacteria to explore new strategies for treating inflammatory bowel disease (IBD). This choice is based not only on the convenience of genetic manipulation in bacterial models but also on their potential advantages in clinical applications. Through this strategy, we hope to overcome the limitations of existing treatments and provide more effective therapeutic options for IBD patients.