Studies on the structure and stimulating effect on Caco-2 cells of flagellin and its truncated proteins of Escherichia coli Nissle 1917

doi:10.21203/rs.3.rs-3247156/v1

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Studies on the structure and stimulating effect on Caco-2 cells of flagellin and its truncated proteins of Escherichia coli Nissle 1917

https://doi.org/10.21203/rs.3.rs-3247156/v1

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This study aimed to investigate the structural characteristics and their effects on stimulating Caco-2 cells of Escherichia coli Nissle 1917 flagellin (FliC_EcN) and its truncated proteins, FliC_△174−506 (D2-D3 domain deleted) and FliC_△274−406 (D3 domain deleted). The experiment predicted the tertiary structure of FliC_EcN by Alphofold2, analyzed the structural characteristics of FliC_EcN, FliC_△174−506 and FliC_△274−406 by surface-enhanced Raman spectroscopy (SERS) and circular dichroism (CD), and detected the secretion levels of IL-6 (interleukin-6), IL-10 (interleukin-10) and TNF-α (tumor necrosis factor-α) after FliC_EcN, FliC_△174−506 and FliC_△274−406 stimulated Caco-2 cells for 6 and 12 h, respectively. The results showed that the NH3-ends and COOH-ends of FliC_EcN were highly conserved, mainly composed of α-helix; the middle domains were highly variable, mainly composed of β-sheet and random coil. The Raman peaks of FliC_△174−506 and FliC_△274−406 generally maintained the main chain peaks of FliC_EcN, while the side chain and amino acid peaks were absent to varying degrees. The composition of the secondary structure of FliC_△174−506 and FliC_△274−406 was altered. FliC_EcN, FliC_△174−506 and FliC_△274−406 stimulated Caco-2 cells to secrete cytokines IL-10, IL-6 and TNF-α differently. The complete FliC_EcN structure could stimulate more secretion of IL-10; the FliC_△174−506 group had higher secretion of IL-6; and the FliC_△274−406 group had higher secretion of TNF-α. In conclusion, deletion of different domains of the hypervariable region of FliC_EcN affects its SERS and CD spectrum and stimulates Caco-2 cells to secrete cytokines.

Escherichia coli Nissle 1917

Flagellin

Truncated flagellin

Surface-enhanced Raman spectroscopy

Circular dichroism

Biological activity

Successfully predicted the tertiary structure of FliC_EcN; The characteristic spectrum and secondary structure of the truncated proteins were changed; Truncated proteins affected the secretion of cytokines by Caco-2 cells.

Flagellin, as the structural protein of bacterial flagella, is the structural basis for bacterial movement, adhesion, and invasion (Il Kim, et al. 2018). Flagellin is also an important immunologic adjuvant, which can trigger MyD88-dependent and MyD88-independent pathways to send signals and activate nuclear factors NF-κB by recognizing TLR5 ( Toll like receptor 5) and NOD like receptor, inducing the production of various cytokines and chemokines, activating the body's innate immune system, and enhancing the body's adaptive immunity to the target antigen (Song, et al. 2017). Flagellin adjuvants are widely used in the research of vaccines against influenza, cancer, AIDS, newcastle disease, COVID-19, and other diseases (Puth, et al. 2022; Barkhordari, et al. 2021; Jiang, et al. 2021; Pulendran, et al. 2021; Hinkula, et al. 2019).

Due to differences in the structure of flagellin from different bacterial species, flagellin may contain 2–4 different structural domains (D0, D1, D2, and D3). The D0 and D1 domains exist inside the flagella, mainly composed of α-helix, assembled to form a lumen of flagella filaments, with a relatively stable structure and relatively conserved among bacterial species, is the main region for flagellin recognition of TLR5 (Il Kim, et al. 2018). The D2 and D3 domains are displayed outside the flagellum, mainly interacting with the external environment; they are highly variable regions (HVR) of flagellin, and mainly determine the antigenicity of flagellin (Song, et al. 2017).. Studies have shown different perspectives on the impact of HVR of flagellin from different bacteria on their adjuvant effects. EBM found that the absence HVR of the Salmonella flagellin strongly weakened the inherent antigenicity without affecting TLR5-dependent immunostimulatory activity (Biedma, et al. 2019); however, Wangkahart E found that the immune stimulation ability was lost after the deletion of HVR of Brucella flagellin (Wangkahart, et al. 2019); besides, C ô t é - Cyr M found that the flagellin of Bacillus subtilis without HVR also has a good TLR5 activation effect (Côté-Cyr, et al. 2022).

Flagellin, the structural protein on the flagellum surface of Escherichia coli Nissle 1917 (FliC_EcN), is a new immunologic adjuvant with structural and functional diversity. Yang Y found that the HVR of FliC_EcN can be used to exhibit exogenous peptides and assemble them into flagella to achieve surface display of exogenous peptides (Yang, et al. 2016). Steimle confirmed that FliC_EcN with a complete flagella protein structure can alleviate colitis in mice and has better adjuvant effects (Steimle, et al. 2019). However, Deng Y-J found that FliC_△174−506, which lacks amino acids at positions 174–506 of the HVR of FliC_EcN, can more effectively stimulate the secretion of inflammatory factor IL-8 (interleukin-8) and has an adjuvant effect comparable to that of full-length FliC_EcN (Deng. 2021). In summary, the function of FliC_EcN is still in the preliminary research stage, and its structure has not yet been elucidated.

The structure of a protein determines its biological function, making it of great significance to analyze the structural characteristics of FliC_EcN for the study of its biological function. In recent years, surface-enhanced Raman spectroscopy (SERS) and circular dichroism (CD) have become cutting-edge and potential analytical techniques in the field of life science, and are very good tools for measuring protein structure. SERS uses laser emission wavelengths ranging from ultraviolet to near-infrared regions, has high spatial resolution, can be analyzed in aqueous solution, is easy to operate, and is non-destructive to the sample (Yan, et al. 2021). CD can be used to determine protein conformation in solution state, with a fast and simple method that is sensitive to conformational changes (Sreerama, et al. 2004). At the same time, the development of various artificial intelligences such as AlphaFold2 provides another way for the research of protein structure and function (Nene, et al. 2022; Goulet, et al. 2022).

Our laboratory successfully expressed and purified FliC_EcN and two truncated proteins, FliC_△174−506 (D2-D3 domain deleted) and FliC_△274−406 (D3 domain deleted), through prokaryotic expression and NTA column purification in the early stage. To study their structural and functional characteristics more comprehensively, this study first predicted and analyzed the structure of FliC_EcN using Alphofold2, then analyzed the structural characteristics of FliC_EcN, FliC_△174−506 and FliC_△274−406 using SERS and CD, and finally stimulated Caco-2 cells with FliC_EcN, FliC_△174−506 and FliC_△274−406 respectively, and detected the secretion of IL-6 (interleukin 6), IL-10 (interleukin 10), and TNF- α (tumor necrosis factor-α).

2.1 Structural prediction and analysis of FliC_EcN

Obtain the amino acid sequence of FliC_EcN through the NCBI website. Use AlphaFold2 to predict the tertiary structure of FliC_EcN, SAVESv6.0 to select the best tertiary structure of FliC_EcN, and PyMOL to analyze the structural characteristics and the interactions between its HVR of FliC_EcN.

2.2 SERS analysis of FliC_EcN and its truncated proteins

2.2.1 Ag@ICNPs (silver nanoparticles coated with silica) preparation

Prepare silver sol nanoparticles with citrate ion according to the method of Lee (Lee, et al. 2014), add trisodium citrate (1%, 6 mL) into silver nitrate solution (0.034 g, 200 mL) with slight boiling (93 ℃), and wait for 20 min to obtain emerald green silver sol. Centrifuge 10 mL of silver sol (5500 rpm, 10 min, 15 ° C) and remove the supernatant. Take 25 µL silver sol and 25 µL of potassium iodide solution (1 mmol/L) and mix at room temperature for 3 h. Mix the mixture with 25 µL FliC_EcN, FliC_△174−506 and FliC_△274−406 separately, then add another 10 µL of Ca²⁺ (0.01 mol/L CaCl2) in each system and mix thoroughly for SERS testing.

2.2.2 Sample testing

FliC_EcN, FliC_△174−506 and FliC_△274−406 were frozen in the laboratory of the Institute of Animal Epidemics at Guizhou University. The SERS spectra of the three proteins were performed on a WITec alpha300R confocal Raman microscope (WITec GmbH) with a working wavelength of 633 nm, a grating of 600 g/mm, a scanning time of 20 seconds, and a laser power of 15 mW. The detection data was analyzed using WITec Project Plus, Origin 2019, and PeakFit v4.12.

2.3 CD analysis of FliC_EcN and its truncated proteins

FliC_EcN, FliC_△174−506 and FliC_△274−406 were sent to Microspectral Testing Technology Group Co. (Shanghai, China) for CD testing.

2.4 Biological activity detection of FliC_EcN and its truncated proteins

Caco-2 cells were cultured in Minimum Essential Medium (Gibco, Shanghai, China), supplemented with 20% Fetal Bovine Serum (Gibco, Shanghai, China). The cells were seeded in a 24-well microtiter plate (guaranteed to have a density of 1 × 10⁵/well) at 37°C in 5% CO₂. Then, FliC_EcN, FliC_△174−506, and FliC_△274−406 were added,༕µg/m༬respectively, to stimulate mature Caco-2 cells for 6 and 12 h. Finally, the levels of IL-6, IL-10, and TNF-α induced by flagellins in the supernatants were collected and sent to Seville Biotechnology Co. (Wuhan, China) for testing of IL-6, IL-10, and TNF-α. All experiments were repeated at least three times.

3.1 Structural prediction and analysis of FliC_EcN

The amino acid registration number of FliC_EcN was AID79011. The 3D structure of FliC_EcN showed that the N/C ends of FliC_EcN were highly conserved, mainly composed of α-helix, while the middle domains were highly variable, mainly composed of β-sheet and random coil (Fig. 1A). The flexibility of the α-helix and β-sheet that constitute the FliC_EcN structure was relatively small, while a few amino acids at the N/C end and random coil in the HVR of FliC_EcN had greater flexibility (Fig. 1B). According to the comprehensive score of SAVESv6.0, the overall quality of the best FliC_EcN model was 98.69, and the Ramachandran plot showed that 92.90% of the residues were clustered in the maximum allowable area (Fig. 1C).

Analyze the polar contacts and hydrogen bond interactions within a distance of 3.2 Å between the D1, D2, and D3 domains of FliC_EcN. The results showed that the interaction between the D1 and D2 domains was mainly between individual amino acid residues. Aspartic acid (Asp) at position 110 of FliC_EcN was linked to Serine at position 178 (Ser) and Asparagine (Asn) at position 176 with two hydrogen bonds, and Phenylalanine (Phe) at position 173 of FliC_EcN was linked to Asparagine (Asn) at position 174 with one hydrogen bond. The Aspartic acid (Asp) at position 506 of FliC_EcN was connected to Lysine (Lys) at position 509, Leucine (Leu) at position 508, and Proline (Pro) at position 507 with three hydrogen bonds (Fig. 2A). There were complex interactions between the D2 and D3 domains of FliC_EcN. The D2 and D3 domains at the N-end of FliC_EcN were connected with two pairs of reverse-parallel β-sheets, the D2 and D3 domains at the C-end were connected with a pair of reverse-parallel β-sheets, and the main interaction force formed was hydrogen bonds (Fig. 2B).

2.2 The SERS analysis of FliC_EcN and its truncated proteins

The characteristic spectrum of FliC_EcN included characteristic amino acid peaks and main and side chain peaks, without disulfide bonds. The main characteristic amino acid spectrum of FliC_EcN included serine (Ser) at 725 cm^− 1, L-tyrosine (Tyr) at 830 cm^− 1, and phenylalanine (Phe) at 1 004 cm^− 1. The side chain spectrum of FliC_EcN included hydrogen bonding sites of Tyr at 850 cm^− 1 and the Phe-ring breathing at 1 006 cm^− 1, etc. The main chain spectrum of the amide Ⅰ region of FliC_EcN included the α-helix located in 1 650-1 660 cm^− 1, the β-sheet located in 1 610-1 640 cm^− 1, the β-turn located in 1 660-1 700 cm^− 1, and the random coil located in 1 640-1 650 cm^− 1 (Fig. 3A).

The characteristic Raman peaks of FliC_△174−506 and FliC_△274−406 generally maintained the main chain Raman peaks, while the side chain and characteristic amino acid characteristic peaks were missing to varying degrees. Only a few side chain and characteristic amino acid characteristic peaks of FliC_△174−506 were absent (Fig. 3B), while most side chain peaks, including serine (Ser), tyrosine (Tyr), and phenylalanine (Phe) of FliC_△274−406, as well as the hydrogen bonding sites of Tyr and the Phe-ring breathing, were absent (Fig. 3C).

2.3 CD analysis of FliC_EcN and its truncated proteins

The CD analysis of FliC_EcN and its truncated proteins showed a characteristic positive peak of the α-helix at 192 nm, and characteristic negative peaks of α-helix at 208 nm and 222 nm. A negative peak at 217–218 nm and 216 nm, and a positive peak at 195–198 nm belonged to the characteristic peaks of β-sheet. A negative peak near 198 nm belonged to the characteristic peak of random coil. The CD spectrum of FliC_△174−506 was similar to that of FliC_EcN, and the CD spectrum of FliC_△174−506 was similar to that of FliC_EcN, while FliC_△274−406 exhibited negative peaks near the wavelength of 192 nm, which belonged to the positive peak of the α-helix. The remaining characteristic bands of FliC_△274−406 were similar to those of FliC_EcN and FliC_△174−506 (Fig. 4).

The composition of the secondary structure showed that the α-helix and β-sheet were the main secondary structures of the three proteins. Compared to FliC_EcN, the composition of the secondary structure of FliC_△174−506 and FliC_△274−406 had changed (Table 1).

Table 1

Secondary structure composition of FliC_EcN and its truncated proteins
Component	FliC_EcN	FliC_△174-506	FliC_△274-406
α-helix	44.4	54.5	47.6
β-sheet	23.4	24.5	32.4
β-Turn	13.5	11.7	12.2
randomcoil	19.0	10.9	8.0
Total	100.3	100.6	100.2

2.4 Biological activity detection of FliC_EcN and its truncated protein

After stimulating Caco-2 cells for 6 h, compared to the control group, all three protein groups increased the secretion of IL-6, IL-10, and TNF-α to varying degrees. The secretion level of IL-6 in the FliC_△174−506 group was higher than that in the FliC_△274−406 group and the FliC_EcN group, but the difference was not significant; there was no significant difference in the secretion level of IL-10 among the three protein groups; the secretion level of TNF-α in the FliC_△274−406 group was higher than that in the FliC_△174−506 group and the FliC_EcN group, but the difference was not significant. After stimulating Caco-2 cells for 12 h, compared with the control group, all three protein groups increased the secretion of IL-6, IL-10, and TNF-α to varying degrees. The secretion level of IL-6 in the FliC_△174−506 group was higher than that in the FliC_△274−406 group and the FliC_EcN group, but the difference was not significant; the secretion level of IL-10 in the FliC_EcN group was significantly higher than that in the FliC_△274−406 group (P < 0.001) and the FliC_△174−506 group (P < 0.05); the secretion level of TNF-α in the FliC_△274−406 group was higher than that in the FliC_△174−506 group and the FliC_EcN group, but the difference was not significant ( Fig. 5).

Further analysis of the secretion of various cytokines at 6 and 12 hours showed that, as the stimulation time increased, the secretion levels of IL-6 in all three experimental groups showed a decreasing trend, but the difference was not significant; the secretion of IL-10 in the FliC_EcN group and FliC_△174−506 group showed a upward trend, but the difference was not significant; the secretion of IL-10 in the FliC_△274−406 group showed a downward trend and the difference was not significant; the secretion levels of TNF-α in all three experimental groups showed a upward trend, but the difference was not significant ( Fig. 6).

Flagellin, as a immunologic adjuvant, is widely used in the research of vaccines for bacteria, viruses, parasites and other diseases (Chen, et al. 2022; Bruxelle, et al. 2017). However, considering that flagellin has dual properties of antigenicity and immune activation, there are still some limitations in its clinical applications. On the one hand, flagellin is an important virulence factor in some pathogenic bacteria, which may cause inflammatory diseases such as intestinal inflammation and lung damage (Bergman, et al. 2005; Gonzalez, et al. 2005; Ivison, et al. 2010). On the other hand, flagellin will induce the anti-flagellin antibodies produced by the body, which may neutralize its triggered immune response ability, thereby affecting the adjuvant effect (Grimes, et al. 2016; Malapaka, et al. 2006). Therefore, further research on the structure and biological functions of flagellin is of great significance for the development of flagellin adjuvants.

In the past, there have been more studies on the structure of flagellin from Salmonella and other bacteria. Studies have shown that the flagellin is mainly composed of four domains: D0, D1, D2, and D3. The D0 and D1 domains located at the N/C ends of flagellin are mainly composed of highly conserved α-helix and are the main area for exerting adjuvant functions; the D2 and D3 domains located in the HVR of flagellin are highly variable, which mainly determines the antigenicity, and can reduce its antigenicity by deleting these domains (Liu, et al. 2010; Khim, et al. 2021). The results of structural prediction and analysis of FliC_EcN are consistent with previous research. It showed that α-helix mainly constituted the N/C ends of FliC_EcN, which was less flexibility and relatively conservative; the β-sheet and random coil mainly constituted the HVR of FliC_EcN, which were flexible and relatively non-conservative. We further analyzed the interactions between the HVR of FliC_EcN, and the results showed that the D1-D2 domain of FliC_EcN was mainly connected by hydrogen bonds between individual amino acid residues, and the D2-D3 domain was mainly connected by complex interactions, including multi-parallel and reverse parallel β-sheet, and the main interaction force is hydrogen bonds. Studies have shown that β-sheet has more stable residues than other secondary structure and that a single amino acid replacement in a β-sheet is more likely to damage the stability of the protein structure (Kulandaisamy, et al. 2018). Therefore, the lack of D2 and D3 domains in FliC_EcN may affect its structural stability.

In order to explore the impact of the absence of HVR in FliC_EcN on its structure, SERS and CD were used to analyze the structural characteristics of FliC_EcN, FliC_△174−506 and FliC_△274−406. It showed that FliC_△174−506 and FliC_△274−406 maintained the main chain peak positions of each secondary structure, and the characteristic amino acid and side chain peaks of FliC_△174−506 and FliC_△274−406 altered in varying degrees. Only a few side chains and characteristic amino acid peaks of FliC_△174−506 were absent, while most of the characteristic amino acid and side chain peaks of FliC_△274−406 were absent. Combining the with the results analysis of previous findings that FliC_△274−406 lacking the D3 domain of FliC_EcN destroyed the β-sheet structure connected by complex hydrogen bonds between the D2-D3 domain. At the same time, the CD results of the three proteins showed that the CD spectra of FliC_△174−506 was similar to FliC_EcN; while FliC_△274−406 exhibited negative peaks near the wavelength of 192 nm, which belonged to the positive peak of the α-helix. Therefore, the absence of the HVR of FliC_EcN, especially the D3 domain, could cause its correct conformity to be destroyed and affect its spectral characteristics.

We further analyzed the differences in biological functions of flagellin and its truncated proteins by stimulating Caco-2 cells. Studies have shown that flagellin can activate NF-κB signaling pathway and other pathways to promote the body's expression of inflammatory factors IL-6 and TNF-α (Barkhordari, et al. 2021). IL-10 can also play the role of immunosuppressive or immune stimulation at the same time, which is a key anti-inflammatory medium (Shouval, et al. 2014). On this basis, this study stimulated Caco-2 cells with FliC_EcN, FliC_△174−506 and FliC_△274−406 for 6 and 12 h respectively, and then detected the secretion of IL-6, IL-10 and TNF-α. Results showed that FliC_EcN, FliC_△174−506 and FliC_△274−406 stimulated Caco-2 cells to secrete cytokines IL-10, IL-6 and TNF-α differently.The complete FliC_EcN structure could stimulate more secretion of IL-10; the FliC_△174−506 group had higher secretion of IL-6; and the FliC_△274−406 group had higher secretion of TNF-α. It can be seen that FliC_EcN's HVR participated in the production of various cytokines. Therefore, we have reason to further speculate that FliC_EcN's HVR may also affect its adjuvant effect.

In summary, this study combined various technologies such as bioinformatics, spectral analysis, and immune detection to comprehensively analyze FliC_EcN and its truncated proteins, indicating that the loss of different domains in the HVR of FliC_EcN can alter its spectral characteristics and stimulate the secretion of cytokines by Caco-2 cells. This study provides data to support for further research on the adjuvant effects of FliC_EcN and its truncated proteins.

Funding

The project was supported by the National Natural Science Foundation of China (Grant No. 32102703), Guangdong Basic and Applied Basic Research Foundation (2019A1515111186),Guizhou Provincial Science and Technology Projects (Qian Ke He Basic-ZK[2023] General 105), the Plan of Science and Technology of Guizhou Province (Qian Ke He LH Zi [2017] No.7268), the project of Introducing Talents in Guizhou University (GDRJHZ[2016]77), the Support Plan Project of Science and Technology of Guizhou Province in 2021 (Qian Ke He Support [2021] General 164) .

Declaration of competing interest

The authors declare there are no competing interests.

Acknowledgments

The authors would like to be very thankful to the College for Veterinary Medicine, Guizhou University, China for supporting and helping to complete this research work timely. At the same time, we thank all the contributors who provided financial support for this project.

Data availability

The datasets generated in the present study are available from the authors upon request.

*Author Contributions

Conceptualization: Ying Yang, Ming Wen, Guilan Wen; Data curation:Bingming Ou; Formal analysis: Shuang Li; Funding acquisition: Ying Yang, Ming Wen; Methodology: Shuang Li, Bingming Ou; Project administration: Ying Yang; Supervision: Ying Yang, Ming Wen, Guilan Wen; Validation: Shuang Li; Writing - original draft: Shuang Li; Writing - review & editing: Shuang Li, Ying Yang.

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Studies on the structure and stimulating effect on Caco-2 cells of flagellin and its truncated proteins of Escherichia coli Nissle 1917

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Material and methods

2.1 Structural prediction and analysis of FliC_EcN

2.2 SERS analysis of FliC_EcN and its truncated proteins

2.2.1 Ag@ICNPs (silver nanoparticles coated with silica) preparation

2.2.2 Sample testing

2.3 CD analysis of FliC_EcN and its truncated proteins

2.4 Biological activity detection of FliC_EcN and its truncated proteins

3. Results

3.1 Structural prediction and analysis of FliC_EcN

2.2 The SERS analysis of FliC_EcN and its truncated proteins

2.3 CD analysis of FliC_EcN and its truncated proteins

2.4 Biological activity detection of FliC_EcN and its truncated protein

4. Discussion

Declarations

Funding

Declaration of competing interest

Acknowledgments

Data availability

References

Additional Declarations

Status:

Version 1

Studies on the structure and stimulating effect on Caco-2 cells of flagellin and its truncated proteins of Escherichia coli Nissle 1917

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Material and methods

2.1 Structural prediction and analysis of FliCEcN

2.2 SERS analysis of FliCEcN and its truncated proteins

2.2.1 Ag@ICNPs (silver nanoparticles coated with silica) preparation

2.2.2 Sample testing

2.3 CD analysis of FliCEcN and its truncated proteins

2.4 Biological activity detection of FliCEcN and its truncated proteins

3. Results

3.1 Structural prediction and analysis of FliCEcN

2.2 The SERS analysis of FliCEcN and its truncated proteins

2.3 CD analysis of FliCEcN and its truncated proteins

2.4 Biological activity detection of FliCEcN and its truncated protein

4. Discussion

Declarations

Funding

Declaration of competing interest

Acknowledgments

Data availability

References

Additional Declarations

Status:

Version 1

2.1 Structural prediction and analysis of FliC_EcN

2.2 SERS analysis of FliC_EcN and its truncated proteins

2.3 CD analysis of FliC_EcN and its truncated proteins

2.4 Biological activity detection of FliC_EcN and its truncated proteins

3.1 Structural prediction and analysis of FliC_EcN

2.2 The SERS analysis of FliC_EcN and its truncated proteins

2.3 CD analysis of FliC_EcN and its truncated proteins

2.4 Biological activity detection of FliC_EcN and its truncated protein