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CMC (arttificial adhesion protein)

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• Name: CMC
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Usage and Biology

The adhesion factor CMC is an engineered artificial glucan-binding protein designed to simulate the interaction between glucan-binding proteins and glucan substrates. It can recruit intestinal probiotics, enabling the stable attachment of engineered bacteria to the intestinal tract, thereby alleviating intestinal inflammation and modulating the gut microenvironment more effectively. CMC consists of two copies of CBM_CipC linked by the fluorescent protein mCherry sequence. The N-terminally attached outer membrane protein A (OmpA) signal peptide facilitates the translocation of the protein across the inner membrane, localizing it to the outer membrane surface and conferring surface display capability.

Figure1:The Design of CMC

Design of the Adhesion Factor CMC

The CBMcipc domain can serve as a "bridge" between intestinal microorganisms and the gut.

Mucus secreted by the surface of intestinal epithelial cells acts as a protective barrier, with its main component being the highly glycosylated glycoprotein MUC2, which contains various glycan structures such as Core1, Core2, and Core3. The beneficial properties of probiotics are related to their production of extracellular polysaccharides (EPS), with most probiotic surfaces exposing glucans. Lactobacillus, a genus within the phylum Firmicutes, plays roles in balancing the gut microbiota, promoting digestion and nutrient absorption, and alleviating intestinal inflammation and infections. Its cell wall is primarily composed of peptidoglycan and polysaccharides, such as glucansucrase (GS) produced by Lactobacillus, which synthesizes EPSs like α-glucans through glucosyl transfer. Unlike probiotics, Salmonella, a common foodborne pathogen, utilizes various virulence factors to overcome colonization resistance and induce intestinal inflammation. As Gram-negative bacteria, their cell walls contain lipopolysaccharides, which are endotoxins and include the O antigen.

Therefore, we designed the Clostridium cellulolyticum CipC (CBM_CipC) domain with the goal of "binding glucans." CBMcipc is derived from the scaffold protein CipC of Clostridium cellulolyticum and contains a type III cellulose-binding domain (CBD), a hydrophilic domain, and two hydrophobic domains. The CBD domain confers glucan-binding ability to CBM_CipC. This adhesion factor can serve as a "bridge" between intestinal microorganisms and the gut, recruiting probiotics and enabling the stable attachment of engineered bacteria in the intestinal tract.

Mathematical Modeling to Simulate the Adhesion Effects of Three Adhesion Factors with Different CBM_CipC Copy Numbers

The copy number of CBM_CipC may affect its ability to bind glucans. Therefore, proteins with different copy numbers, CM, CMC, and CMCC, were designed. These proteins consist of the fluorescent protein mCherry and one copy of CBM_CipC for CM, mCherry and two copies of CBM_CipC for CMC, and mCherry and three copies of CBM_CipC for CMCC.

To simulate the changes in probiotic and pathogenic bacteria numbers in the intestines of individuals with enteritis after introducing different engineered bacteria (EcM, EcCM, EcCMC, EcCMCC), we developed a mathematical model. The health status index is represented by the number of probiotics minus the number of pathogens within cells. Negative values are shown in blue, and positive values in red, with deeper colors indicating higher quantities of the respective bacteria.

In the results, we observe that CM (Figure 2a) is the least effective, with pathogens predominantly occupying the space. In contrast, the differences between CMC (Figure 2b) and CMCC (Figure 2c) are not significant, but a closer look reveals that CMC reaches a healthy state more quickly, with probiotics dominating faster. Therefore, we selected CMC as the adhesion factor for this experiment.

Figure2a Figure2b Figure2c

Functional Validation of the Adhesion Factor CMC

FITC-labeled glucan (FITC-glucan) was used to stain the cells for flow cytometry and confocal microscopy. After incubating the cells with FITC-glucan for 10 minutes, EcM cells showed almost no FITC fluorescence, while EcCM, EcCMC, and EcCMCC cells exhibited significant FITC fluorescence. Among the three strains containing CBMcipc, EcCMC cells displayed the highest FITC fluorescence (Fig. 3b, c), indicating 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, whereas EcCMC cells showed a bilateral distribution of mCherry (Fig. 3d). This suggests that CMC exhibits stronger surface display capabilities. Since the cell surface display of binding proteins is necessary for extracellular glucan binding, this characteristic of EcCMC likely contributes to its enhanced glucan-binding ability.

Fig. 3. Design and characterization of the synthetic bacteria used for glucan binding. (Based on the preliminary research findings from the laboratory)

An oral gavage experiment was conducted on mice using EcCM, EcCMC, and EcCMCC mixed with Lactobacillus plantarum (probiotic) to evaluate the adhesion rate, defined as the ratio of beneficial bacteria in the gut to the administered beneficial bacteria after 12 hours. The results showed that EcCMC had the highest adhesion rate (Fig. 5). When the probiotic was replaced with Salmonella enterica serovar Typhimurium (pathogen), the adhesion rates were much lower (Fig. 4), demonstrating the adhesion factor's preference for binding with probiotics.

Fig.4 The adhesion rate of exogenous yeast was detected by intragastric administration.

Conclusion

In summary, we ingeniously leveraged the mechanism of glucan-binding proteins to design the artificial glucan-binding protein CMC. This bio-inspired approach ensures both effectiveness and biocompatibility, providing a novel solution for the stable adhesion of engineered bacteria. Through extensive literature review, we selected the type III cellulose-binding domain (CBD) from the scaffold protein CipC of *Clostridium cellulovorans* due to its superior glucan-binding capability. This choice not only enhanced binding efficiency but also maintained stability in the complex gut environment.

The successful design of the CMC protein was a result of thorough literature research and systematic experimental validation by our team. By designing CBMcipc proteins with different copy numbers (M, CM, CMC, CMCC), we precisely regulated binding strength and stability to optimize protein performance and identify optimal binding conditions. The incorporation of the mCherry fluorescent protein allowed for direct observation of binding results through fluorescence intensity, enhancing the clarity and accuracy of experiments. Using the outer membrane protein A (OmpA) signal peptide, we localized the protein to the cell outer membrane, ensuring full exposure and functionality of the binding domain, thus improving the efficiency of protein display.

Our design not only increased the adhesion of engineered bacteria but also facilitated the recruitment and stabilization of probiotics in the gut, achieving dual functionality. This binding domain can serve as a modular component in our plasmid, offering wide adaptability and flexibility. Through these innovative designs, our CMC protein demonstrated significant novelty and practicality both theoretically and practically, making it an excellent foundational component.

References

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.

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.

5 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

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

7 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

8 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.

9 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.

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

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