SUSTech-CHINA

Proof of Concept

Introduction

To eliminate the biofilm formed by pathogenic microbes, we expected to construct an engineered bacteria that can express and secrete two proteins, HMGB1 and PslG. HMGB1 can act on the DNA of the biofilm, and PslG can decompose the polysaccharides in it. To validate the efficacy of this system, we first purified these proteins and treated the biofilm with proteins to test their efficacy. Then, the side effects were examined by cytotoxicity test and inflammatory response validation. At the same time, we also selected the quorum sensing system as the sensor and tested its sensitivity. Finally, we selected Lysis E7 for the release of these proteins outside the cell and verified its role.

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Figure 1. Overall design of engineered bacteria

Section 1: Quantitative Analysis by Crystal Violet Staining

Crystal violet can be utilized to quantitatively analyze the amount of biofilm. By treating with different proteins, we demonstrated that these proteins could reduce biofilm formation. For one thing, when these proteins are present in the environment, they can inhibit biofilm formation. For another, in terms of a mature biofilm, these proteins are also able to disintegrate its structure within a certain period of time.

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Figure 2. Time course of PslG，FL，AB，A，B effect on biofilm disassembly. The biofilm biomass was measured by CV assay.
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Figure 3. Time course of PslG，FL，AB，A，B effect on biofilm growth. The biofilm biomass was measured by CV assay.

Section 2: Intuitive Observation by Confocal Microscopy

In order to visualize the changes in the biofilm intuitively, confocal microscopy was employed in our experiments. With the assistance of this imaging technique, we successfully proved the elimination of mature biofilms by these proteins. The biofilm biomass was significantly reduced following treatment with these proteins compared to the control. What we can observe is that the network of DNA and polysaccharide has disappeared, while the fluorescent dots representing P. aeruginosa still exist, indicating that our system is indeed working to break down the biofilm.

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Figure 4. HMGB1 and PslG contribute to conspicuous clearance of biofilm. DNA is stained by SYTO9 (green), and polysaccharide is stained by HHL (red).

Section 3: Side Effects Examination and Sensor Validation

3.1 Cytotoxicity Test and Inflammation Verification

Given that we expect engineered bacteria to secrete these proteins in the human intestine, ensuring that these proteins do not have adverse effects on human cells is imperative. We experimentally demonstrated that these proteins exhibit little toxicity, as they cause cell death only at very high concentrations. In addition, these proteins do not cause a significant inflammatory response as well.

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Figure 5. Results of cytotoxicity test. Different curves indicate the toxicity of different substances for cells. Error bar represents s.d.
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Figure 6. a, Engineered HMGB1 (full length) exhibits an alleviated pro-inflammatory effect. Cells are treated with HMGB1 (fl) for 2 hours (n = 3). **P < 0.01. Error bar represents s.d.
b, PslG exhibits little pro-inflammatory effect on human intestinal cells. Cells are treated with PslG for 2 hours (n = 3). *P < 0.05. Error bar represents s.d.

3.2 Quorum Sensing System

We chose the quorum sensing system as the sensor and subsequently proved that our system is able to sense the presence of pathogenic bacteria. We first explored the optimal concentration range for the operation of the quorum-sensing system, and then successfully demonstrated that the presence of pathogenic bacteria (Pseudomonas aeruginosa) could be efficiently sensed by the system.

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Figure 7. Characterization results of quorum sensing device. a, GFP production rate per cell over time at different concentration of 3OC12HSL.
b, Time-average GFP production rate per cell over time at different concentration of 3OC12HSL. Error bar represents s.d.

Section 4: Protein Secretion via Lysis E7

Finally, our engineered bacteria can release these proteins after induction by arabinose. We first demonstrated that successful expression of Lysis E7 in response to arabinose resulted in the massive death of engineered bacteria. We then experimentally confirmed that this death was due to lysis of bacteria by Lysis E7.

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Figure 8. Upon arabinose induction, Lysis E7 is expressed followed by massive death of engineered bacteria. Error bar represents s.d.

For complete results, check out our "Results" page please.

Reference

Section 1: Quantitative Analysis by Crystal Violet Staining

  1. Blair, R. H., Horn, A. E., Pazhani, Y., Grado, L., Goodrich, J. A., & Kugel, J. F. (2016). The HMGB1 C-Terminal Tail Regulates DNA Bending. Journal of molecular biology, 428(20), 4060–4072. https://doi.org/10.1016/j.jmb.2016.08.018
  2. Buzzo, J. R., Devaraj, A., Gloag, E. S., Jurcisek, J. A., Robledo-Avila, F., Kesler, T., Wilbanks, K., Mashburn-Warren, L., Balu, S., Wickham, J., Novotny, L. A., Stoodley, P., Bakaletz, L. O., & Goodman, S. D. (2021). Z-form extracellular DNA is a structural component of the bacterial biofilm matrix. Cell, 184(23), 5740–5758.e17. https://doi.org/10.1016/j.cell.2021.10.010
  3. Devaraj, A., Novotny, L. A., Robledo-Avila, F. H., Buzzo, J. R., Mashburn-Warren, L., Jurcisek, J. A., Tjokro, N. O., Partida-Sanchez, S., Bakaletz, L. O., & Goodman, S. D. (2021). The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation. The Journal of Clinical Investigation, 131(16), e140527. https://doi.org/10.1172/JCI140527
  4. Thomas, J. O., & Travers, A. A. (2001). HMG1 and 2, and related 'architectural' DNA-binding proteins. Trends in biochemical sciences, 26(3), 167–174. https://doi.org/10.1016/s0968-0004(01)01801-1
  5. Yu, S., Su, T., Wu, H., Liu, S., Wang, D., Zhao, T., Jin, Z., Du, W., Zhu, M. J., Chua, S. L., Yang, L., Zhu, D., Gu, L., & Ma, L. Z. (2015). PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix. Cell Research, 25(12), 1352–1367. https://doi.org/10.1038/cr.2015.129

Section 2: Intuitive Observation by Confocal Microscopy

  1. Devaraj, A., Novotny, L. A., Robledo-Avila, F. H., Buzzo, J. R., Mashburn-Warren, L., Jurcisek, J. A., Tjokro, N. O., Partida-Sanchez, S., Bakaletz, L. O., & Goodman, S. D. (2021). The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation. The Journal of Clinical Investigation, 131(16), e140527. https://doi.org/10.1172/JCI140527
  2. Yu, S., Su, T., Wu, H., Liu, S., Wang, D., Zhao, T., Jin, Z., Du, W., Zhu, M.-J., Chua, S. L., Yang, L., Zhu, D., Gu, L., & Ma, L. Z. (2015). PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix. Cell Research, 25(12), Article 12. https://doi.org/10.1038/cr.2015.129

Section 3: Side Effects Examination and Sensor Validation

3.1 Cytotoxicity Test and Inflammation Verification
  1. Gdynia, G., Sauer, S. W., Kopitz, J., Fuchs, D., Duglova, K., Ruppert, T., Miller, M., Pahl, J., Cerwenka, A., Enders, M., Mairbäurl, H., Kamiński, M. M., Penzel, R., Zhang, C., Fuller, J. C., Wade, R. C., Benner, A., Chang-Claude, J., Brenner, H., … Roth, W. (2016). The HMGB1 protein induces a metabolic type of tumor cell death by blocking aerobic respiration. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10764
  2. Park, J. S., Arcaroli, J., Yum, H.-K., Yang, H., Wang, H., Yang, K.-Y., Choe, K.-H., Strassheim, D., Pitts, T. M., Tracey, K. J., & Abraham, E. (2003). Activation of gene expression in human neutrophils by high mobility group box 1 protein. American Journal of Physiology-Cell Physiology, 284(4), C870–C879. https://doi.org/10.1152/ajpcell.00322.2002
  3. Wang, H., Bloom, O., Zhang, M., Vishnubhakat, J. M., Ombrellino, M., Che, J., Frazier, A., Yang, H., Ivanova, S., Borovikova, L., Manogue, K. R., Faist, E., Abraham, E., Andersson, J., Andersson, U., Molina, P. E., Abumrad, N. N., Sama, A., & Tracey, K. J. (1999). HMG-1 as a Late Mediator of Endotoxin Lethality in Mice. Science, 285(5425), 248–251. https://doi.org/10.1126/science.285.5425.248
3.2 Quorum Sensing System
  1. Chang, W., Small, D. A., Toghrol, F., & Bentley, W. E. (2005). Microarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide. BMC Genomics, 6, 115. https://doi.org/10.1186/1471-2164-6-115
  2. Gray, K. M., Passador, L., Iglewski, B. H., & Greenberg, E. P. (1994). Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology, 176(10), 3076–3080. https://doi.org/10.1128/jb.176.10.3076-3080.1994
  3. Pearson, J. P., Passador, L., Iglewski, B. H., & Greenberg, E. P. (1995). A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences, 92(5), 1490–1494. https://doi.org/10.1073/pnas.92.5.1490
  4. Saeidi, N., Wong, C. K., Lo, T.-M., Nguyen, H. X., Ling, H., Leong, S. S. J., Poh, C. L., & Chang, M. W. (2011). Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Molecular Systems Biology, 7(1), 521. https://doi.org/10.1038/msb.2011.55
  5. Schuster, M., Urbanowski, M. L., & Greenberg, E. P. (2004). Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR. Proceedings of the National Academy of Sciences, 101(45), 15833–15839. https://doi.org/10.1073/pnas.0407229101