Part:BBa_K5332000

ROS promoter->anti-inflammatory factor->adhesion factor

Our part has three special components, the ROS promoter, the anti-inflammatory factor, and the adhesion factor. By virtue of the ROS promoter, it can achieve spatio-temporal specific expression, and its action factors will be expressed only in the environment of high inflammation level; by virtue of the anti-inflammatory factor melittin, it can achieve the reduction of inflammation level in the intestine; by virtue of the adhesion factor, it can be colonised in the intestine and adhere to the beneficial bacteria to improve the microbial environment of the intestinal tract. This plasmid as a whole can be used to alleviate intestinal inflammation, and the promoter portion can be used as a switch for expression in environments with high ROS levels.

Usage and Biology

The NKU-China team has designed a gene element that can express anti-inflammatory factor Melittin and adhesion factor CMC under high reactive oxygen species conditions for the treatment of intestinal inflammation. The promoter that controls the transcription of this element is taken from the target gene katG of OxyR protein, which can initiate downstream gene expression under high conditions and achieve spatially specific expression. In order to reduce the cytotoxicity of the anti-inflammatory factor Melittin, we used a linker to connect two Melittin monomers, resulting in the formation of hairpin structures in the final expression product, which produced more beneficial effects. The adhesion factor CMC was independently designed by our team, and its core structural domain CBMcipc is derived from the cellulose solubilizing Clostridium scaffold protein CipC, which has strong glucan binding ability. The N-terminal connected outer membrane protein A (OmpA) signal peptide helps the protein locate on the outer membrane surface. Figure 1 shows the detailed design of the entire component.

The Design of three subparts

Reactive Oxygen Species Promoter

Reactive oxygen species (ROS) molecules are common molecules in aerobic respiration organisms, and under normal circumstances, they do not cause serious damage to the organism itself. However, in certain specific environments, the occurrence of diseases such as IBD in the human body can lead to abnormal accumulation of ROS, which have high diffusion and can spread from the lesion to other healthy areas, causing further damage to the cells in this area. In order to cope with this adverse effect, organisms have evolved a system for clearing reactive oxygen species, among which OxyR is one of the key regulatory factors that can regulate the expression of downstream target genes including katG, gdps, gorA, and ahpCF. After comparing and analyzing the promoter sequences of these four genes and reading relevant literature, we selected the promoter sequence of katG gene for subsequent experiments.

Details can be found in BBa_K5332001

Anti-inflammatory Factor Di-melittin

Melittin is the main active substance of bee venom. Bee venom, as a traditional Chinese medicine, has been used by Chinese people to treat inflammatory diseases for a long time. Some researchers have verified the anti-inflammatory effect of Melittin, but its high cytotoxicity has reduced its use range. In order to reduce the cytotoxicity of Melittin, we adopted an innovative approach of linking two melittin monomer molecules together through a linker to form a unique hairpin structure. This not only effectively reduces toxicity and improves safety, but also significantly enhances Melittin's ability in immune stimulation. This not only helps to activate the body's immune system more effectively, but may also promote moderate regulation of inflammation in some cases, providing new possibilities for the treatment of IBD.

Details can be found in BBa_K5332002

Adhesion Factor CMC

The main component of mucus secreted on the surface of intestinal epithelial cells is the highly glycosylated glycoprotein MUC2, which has various polysaccharide structures. Glucan is an extremely important polysaccharide that can be produced by a large number of bacteria and fungi. Meanwhile, the beneficial properties of probiotics are related to the extracellular polysaccharides (EPS) they produce, and most probiotic surfaces can expose glucan. Based on this, we have designed a protein that can act as a "bridge" between gut microbiota and the intestine, helping engineered bacteria adhere to the surface of the intestine while recruiting gut probiotics to play multiple roles.

We designed a CBMcipc domain derived from the cellulose solubilizing Clostridium scaffold protein CipC, which includes a group III cellulose binding domain (CBD), a hydrophilic domain, and two hydrophobic domains. The CBD domain endows CBMcipc with the ability to bind to glucan. After introducing the fluorescent protein mCherry sequence, proteins M, CM, CMC, and CMCC with different CBMcipc copy numbers were designed. After a series of experimental verifications, it was found that CMC has stronger surface display ability and glucan binding ability.

Details can be found in BBa_K5332003

Test

We cloned the element into pET-28a (+) and transformed it into the vector cell EcN to obtain the engineered bacterium FMK (iEraser). To verify whether FMK can exert its expected effect in vivo, we conducted the experiment according to the procedure shown in Table 1, where all liquids were administered into mice by gavage, and the concentrations of Salmonella and FMK were resuspended in PBS at . The intestinal specimens taken were from the cecum to colon.

Table1:Mouse Experimental Procedure

Relief of the shortened colon length in mice caused by IBD

After three days of oral administration of Salmonella, the mice became the colitis model constructed by the team. The colon length of mice with IBD was significantly shortened. After measuring the colon length of all mice, we found that FMK could significantly alleviate the shortened colon length caused by intestinal inflammation. (Figure 2)

Figure2a Figure2b

Anti-inflammatory effects shown in microscopic examination results

Take some colon tissue for microscopic observation. After dehydration, embedding, and sectioning of colon tissue, HE staining was performed, and the results are shown in Figure 3A. Compared with the PBS group, the S.Tm group showed damage to intestinal epithelial cells and disrupted intestinal mucosal integrity, while the symptoms of colitis in the mouse model treated with FMK were significantly alleviated. Some tissue sections were processed for immunofluorescence staining as shown in Figure 3B. It can be seen that the IL-6 fluorescence signal intensity of tissues treated with Salmonella by gavage was significantly higher than that of PBS group, while the IL-6 fluorescence intensity of tissues treated with engineered bacteria FMK was significantly lower than that of the S.Tm group, indicating that the synthetic bacteria FMK has a significant anti-inflammatory effect. Figure3a Figure3b

Immune cell aggregation are reduced

Take a portion of colon tissue, completely dissociate it, stain immune factors and immune cell marker proteins, and then use flow cytometry to measure their expression levels. As shown in Figure 4, after oral administration of FMK, the levels of IL-6, IFN-γ, F4/80, and Lys6G in the colon tissue of mice with enteritis significantly decreased. Due to the fact that F4/80 and Lys6G are the marker proteins of immune cells, the decrease in their levels indicates that the synthetic strain FMK can significantly reduce the aggregation of immune cells near colitis tissue, thereby alleviating colitis in mice.

Figure4

Downregulation of pro-inflammatory factors and elevation of anti-inflammatory factors

After extracting RNA from some colon tissue cells, RT-qPCR was performed to detect the expression levels of various inflammatory factors in the cells. The results are shown in Figure 5. Compared with the control group, the levels of pro-inflammatory factors IL-6, IL-1 β, and IL-8 in the intestinal tissue treated with FMK were significantly reduced, while the levels of anti-inflammatory factors TGF-β and IL-10 were significantly increased.

Figure5

Tenoristic enrichment of beneficial bacteria

To verify the effect of the engineered bacterium FMK on the gut microbiota of mice, we extracted total DNA from colon tissues of the experimental group and positive control group, sequenced 16s RNAv3+v4-b, and constructed a sequencing library after certain data processing. Perform inter group ANOVA analysis at the Phylum level, as shown in Figure 6a. At the phylum level, the engineered bacterium FMK significantly increased the abundance of Firmicutes and Verrucomicrobota in the mouse gut, while reducing the abundance of Proteobacteria and Bacteroidota. Previous studies have shown a decrease in the number of Firmicutes and an enrichment of Proteobacteria in the intestinal tract of IBD. However, this phenomenon was improved after the use of FMK. Therefore, FMK has a significant function in improving the structure of intestinal microbiota, promoting the enrichment of beneficial bacteria, and reducing the number of harmful bacteria. The heatmap analysis of sample species composition and relative abundance, as well as the sample community distribution map of species evolutionary tree, are shown in Figures 6b and 6c.

Figure6a Figure6b Figure6c

Increase diversity of gut microbiota shown in mathematical modeling results

The Shannon diversity index dilution curve (Figure 7a) was drawn using Mothur software and R language tools based on the sequencing volume of each sample at different sequencing depths. It can be seen that when the curve tends to flatten, except for the BCK1 group, the Shannon index in the group using engineered bacteria FMK is higher than that in the IBD mouse group, indicating that FMK has the function of enriching intestinal microbial diversity. Similar conclusions can also be drawn based on the sample grade abundance curve (Figure 7b).

Figure7a Figure7b

References

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.

2 Storz G, Tartaglia LA, Ames BN. The OxyR regulon. Antonie Van Leeuwenhoek. 1990 Oct;58(3):157-61.

3 Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001 Mar;19(3):109-14.

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.

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.

6 Zhou, Q., Zeng, J., & Liu, Z. (2023). Research Progress in the Treatment of Inflammatory Diseases with Melittin. Chinese Journal of Modern Applied Pharmacy, 40(9), 1270-1277.

7 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:

8 Memariani, H., & Memariani, M. (2020). Anti-fungal properties and mechanisms of melittin. Applied microbiology and biotechnology, 104(15), 6513–6526.

9 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

10 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.

11 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.

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

13 Chen, Ziwei et al. “Lactic acid bacteria-derived α-glucans: From enzymatic synthesis to miscellaneous applications.” Biotechnology advances vol. 47 (2021): 107708.

14 Fabrega A., Vila J. (2013). Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin. Microbiol. Rev. 26 308–341. 10.1128/CMR.00066-12

15 Whitfield, Chris et al. “Lipopolysaccharide O-antigens-bacterial glycans made to measure.” The Journal of biological chemistry vol. 295,31 (2020): 10593-10609.

16 Branchu, Priscilla et al. “Genome Variation and Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Pathovariants.” Infection and immunity vol. 86,8 e00079-18. 23 Jul. 2018

17 Pages, S., Gal, L., Belaich, A., Gaudin, C., Tardif, C., Belaich, J.P., 1997. Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation. J. Bacteriol. 179, 2810–2816.

18 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.

19 Yin, Hongda et al. “Synthetic physical contact-remodeled rhizosphere microbiome for enhanced phytoremediation.” Journal of hazardous materials vol. 433 (2022): 128828.

Information

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 182
  Illegal PstI site found at 1056
  Illegal PstI site found at 1259
  
• 12
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  Illegal EcoRI site found at 182
  Illegal NheI site found at 2030
  Illegal PstI site found at 1056
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• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 182
  Illegal BglII site found at 253
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 182
  Illegal PstI site found at 1056
  Illegal PstI site found at 1259
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 182
  Illegal PstI site found at 1056
  Illegal PstI site found at 1259
  Illegal NgoMIV site found at 400
  Illegal NgoMIV site found at 967
  Illegal NgoMIV site found at 2392
  Illegal NgoMIV site found at 2396
  Illegal AgeI site found at 881
  Illegal AgeI site found at 1982
  
• 1000
  COMPATIBLE WITH RFC[1000]