Poster Presented by: SUSTech_Shenzhen 2020
Zherui Li1, Jiaxi Zhang1, Shiqi Liang2, Linsheng Zhang2, You He3, Yunxuan Wang2, Linzhou Wang4, Ziyi Yin3, Yalikunjiang Aizezi2, Xvkun Yuan2, Yuao Zhu3
College of engineering, Southern University of Science and Technology, Shenzhen 518055,
Guangdong, China
Department of Biology, Southern University of Science and Technology, Shenzhen 518055,
Guangdong, China
School of medicine, Southern University of Science and Technology, Shenzhen 518055,
Guangdong, China
College of science, Southern University of Science and Technology, Shenzhen 518055,
Guangdong, China
Abstract: Sewage treatment is one of the most essential parts of water governing strategy. And among all microbes existing in the biofilm that used in wastewater treatment, Pseudomonas putida contributes vastly to waste processing due to its complex metabolism. The overgrowth of biofilms may block sewage treatment pipelines. An interesting idea arises that we can utilize phages to control the growth of biofilms. Meanwhile, stopping the excessive damage trend is also important. In our project, we aim to find a compound to protect Pseudomonas from phage infection through disrupting the expression of a critical transcription factor in phages.
Team:SUSTech Shenzhen/Poster
Poster: SUSTech_Shenzhen
Developing a Small Molecule to Inhibit Phage
Infection of Bacterium Pseudomonas
Poster Presented by: SUSTech_Shenzhen 2020
Developing a Small Molecule to Inhibit Phage
Infection of Bacterium Pseudomonas
Infection of Bacterium Pseudomonas
Introduction
To promote the usage of biofilm in sewage treatment
To shutoff the decline of Pseudomonas population caused by phages
To design a composite part for the screening of transcription factors inhibitors
Introduction
1. Sewage treatment has been regarded as one of the most essential parts water governing strategy, while the use of biofilm is an environmentally friendly way to treat the sewage.
2. The overgrowth of biofilms will block pipelines, while phages added artificially cannot be controlled and the biofilm used for sewage treatment will be completely destroyed. Therefore, we need to shutoff the damage process whenever we need.
3. We designed a composite part (BBa_K3423005), it was used for the screening of inhibitors against Aca1. One potential Aca1 inhibitor (Navitoclax) was found using this composite part, which proved the feasibility of this in vivo screening system, similar system can be derived form BBa_K3423005 for the screening of other transcription factors inhibitors.
2. The overgrowth of biofilms will block pipelines, while phages added artificially cannot be controlled and the biofilm used for sewage treatment will be completely destroyed. Therefore, we need to shutoff the damage process whenever we need.
3. We designed a composite part (BBa_K3423005), it was used for the screening of inhibitors against Aca1. One potential Aca1 inhibitor (Navitoclax) was found using this composite part, which proved the feasibility of this in vivo screening system, similar system can be derived form BBa_K3423005 for the screening of other transcription factors inhibitors.
Inspiration
Inspiration
The conserved Aca protein in Pseudomonas phage acts on the activity of acr (anti-CRISPR)-promoter in the anti-CRISPR-Cas system in Pseudomonas phage. The CRISPR-Cas system is an acquired immune system in bacteria and archaea that helps them resist the invasion of foreign genetic material, such as bacteriophage, by integrating the foreign DNA sequence into its own genome. Phages express anti-CRISPR (Acr) proteins to inhibit the CRISPR-Cas system, preventing their own genomes from being destroyed. acr genes encode proteins that bind to acr-promoter and regulate adjacent anti-CRISPR-associated (aca) genes. Through binding to acr-promoter, Aca proteins could act as crucial inhibitors of the transcription of anti-CRISPR genes. After the DNA of phage is injected into host cells, immediate high-level transcription of acr gene helps phage to survive under the pressure of the CRISPR-Cas system in host cells, but such high transcription level of acr gene induces dysregulation of other critical genes, leading to the death of phage. With the presence of Aca proteins, transcription of acr gene is downregulated and phage can express essential genes for survival.
In all, if the function of Aca proteins can be artificially inhibited via small molecule compounds, we will be able to increase the defense system of the bacterium against phages.
In all, if the function of Aca proteins can be artificially inhibited via small molecule compounds, we will be able to increase the defense system of the bacterium against phages.
Problem
Problem
Sewage treatment has been regarded as one of the most essential parts of Shenzhen water governing strategy, and biofilm, which is composed of numerous microbes and multiple types of organic matters, plays an important role in the bioremediation of sewage. The use of biofilm is an environmentally friendly way to treat the sewage, while the overgrowth of biofilms will block sewage treatment pipelines. One proposed solution is to constantly replace the blocked pipeline. However, this method is not only too costly for large-scale applications but will also completely destroy the structure of biofilm, which can hardly be regenerated in a short period of time.
We proposed to utilize phages to limit the growth of biofilms, but phages added artificially cannot be controlled and the biofilm used for sewage treatment will be completely destroyed. Therefore, we need to shutoff the damage process whenever we need.
Idea
Idea
The conserved Aca protein in Pseudomonas phage is related with the CRISPR-Cas system in Pseudomonas. If the function of Aca proteins can be artificially inhibited via small molecule compounds, we will be able to increase the defense system of the bacterium against phages. With the conserved aca genes in Pseudomonas phage, we are inspired to find an effective drug to protect Pseudomonas, an important bacterium in the sewage treatment system, from excessive phage infection in our expected system.
Methodology
Methodology
Engineering & Parts
Engineering & Parts
Aca1 is constitutively expressed by T7 promoter, it will subsequently inhibit the anti-CRISPR (acr) promoter, so the gene expression downstream anti-CRISPR promoter will be inhibited until Aca1 inhibitor is added artificially. Without the inhibition from Aca1, the expression of genes downstream anti-CRISPR promoter will increase significantly. Therefore, protein expression can be regulated artificially. In this case, the activity of the acr promoter is estimated through measuring the intensity of green fluorescence.
Results & Conclusions
Results & Conclusions
1. In silico screening was performed based on the Aca1 structure solved by our primary PI using the software Molecular Operating Environment (MOE) and several promising candidates were identified.
2. Then, 30 candidates we got form the virtual screening is tested using composite part BBa_K3423005, we applied our model to evaluate the efficiency of 10 different PDI inhibitor candidates and found that 1400-C4 has the smallest in vivo KI among those candidates. Therefore, we concluded that 1400-C4 could have the best inhibition efficiency and focused on 1400-C4 in the further experiment process.
3. 1400-C4 (Navitoclax) is tested for three times independently, R44A is the positive control, in which Aca1 is mutated to Aca1R44A, which lose its binding affinity to the acr promoter region. Data are represented as mean ± SEM from three independent experiments.
4. Navitoclax’s binding to Aca1 is verified using Differential Fluorescence Scanning Assay.
5. Based on the results from computer virtual molecular docking model of Navitoclax in complex with Aca1, we designed 60 inhibitors derived from Navitoclax, which is going to be tested latter.
Conclusion: A promising hit of Aca1 inhibitor was found by SUSTech_Shenzhen from a very early step (target structure obtaining), a drug used to inhibit Pseudomonas phages will be found in the near future.
2. Then, 30 candidates we got form the virtual screening is tested using composite part BBa_K3423005, we applied our model to evaluate the efficiency of 10 different PDI inhibitor candidates and found that 1400-C4 has the smallest in vivo KI among those candidates. Therefore, we concluded that 1400-C4 could have the best inhibition efficiency and focused on 1400-C4 in the further experiment process.
3. 1400-C4 (Navitoclax) is tested for three times independently, R44A is the positive control, in which Aca1 is mutated to Aca1R44A, which lose its binding affinity to the acr promoter region. Data are represented as mean ± SEM from three independent experiments.
4. Navitoclax’s binding to Aca1 is verified using Differential Fluorescence Scanning Assay.
5. Based on the results from computer virtual molecular docking model of Navitoclax in complex with Aca1, we designed 60 inhibitors derived from Navitoclax, which is going to be tested latter.
Conclusion: A promising hit of Aca1 inhibitor was found by SUSTech_Shenzhen from a very early step (target structure obtaining), a drug used to inhibit Pseudomonas phages will be found in the near future.
Application and Extension
Application and Extension
Application: First of all, we expect to implement our project in the sewage treatment plants that face the problems of the requirement of regularly replacing old, overgrown biofilm with a new one. With our inhibitors that can effectively prevent phage infection of Pseudomonas, we can reduce overgrown Pseudomonas used in the biofilm for waste-processing by adding bacteriophage specific to Pseudomonas into the overgrown biofilm and then adding our inhibitor to prevent too much damage from phage infection.
Also, as our inhibitor can effectively protect Pseudomonas from phage infection, we propose that we can implement a strategy for Pseudomonas protection in the industry with massive cultivation of Pseudomonas, which is to add effective phage inhibitors during the cultivation.
Extension: With such a system, we can focus on another type of protein, the transcription factors that often act as the final executors of certain signaling pathways by either promoting or inhibiting the expression of certain sets of genes. Using a two-plasmid fluorescence-based reporter system, we are able to perform easy and convenient high-throughput chemical screening for inhibitors of transcription factors. And the corresponding small effector molecules could be utilized to exert precise control over the expression of certain sets of genes.
Also, as our inhibitor can effectively protect Pseudomonas from phage infection, we propose that we can implement a strategy for Pseudomonas protection in the industry with massive cultivation of Pseudomonas, which is to add effective phage inhibitors during the cultivation.
Extension: With such a system, we can focus on another type of protein, the transcription factors that often act as the final executors of certain signaling pathways by either promoting or inhibiting the expression of certain sets of genes. Using a two-plasmid fluorescence-based reporter system, we are able to perform easy and convenient high-throughput chemical screening for inhibitors of transcription factors. And the corresponding small effector molecules could be utilized to exert precise control over the expression of certain sets of genes.
Human Practices
Human Practices
Integrated Human Practices helps us avoid isolated works. We continuously consult different people from academia and industry in every important step of our project.
We focus on three main questions:
Is our idea of this project reasonable and does it have practical significance?
What can we do for improvement?
How can we develop a plan for an extension?
In the part of science communication, we mainly carried out three meaningful interaction activities. We designed an educational event for undergraduate students who are interested in biology, aiming to spread the concept of synthetic biology and make more students interested in synthetic biology and our project. Also, we produced and distributed science brochures on campus. Furthermore, we uploaded 2 small videos on the Bilibili website to introduce our project to the public.
Reference
References
1. Stanley, S. Y., et al. Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription. Cell 178, 1452-1464.e1413, doi:https://doi.org/10.1016/j.cell.2019.07.046 (2019).
2. Zhang, G., Gurtu, V. & Kain, S. R. An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical and Biophysical Research Communications 227, 707-711, doi:10.1006/bbrc.1996.1573 (1996).
3. Kämpfer, P. et al. Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes. Microbial Ecology 32, 101-121, doi:10.1007/BF00185883 (1996).
4. Sehar, S. & Naz, I. Role of the Biofilms in Wastewater Treatment. in Microbial Biofilms-Importance and Applications (eds. Dhanasekaran, D. & Thajuddin, N.) (IntechOpen, 2016). doi: 10.5772/63499
5. Martens, .J., Barg, .H., Warren, .M. et al. Microbial production of vitamin B12. Appl Microbiol Biotechnol 58, 275–285 (2002). doi:10.1007/s00253-001-0902-7
6. Savoldelli, J., Tomback, D. & Savoldelli, H. Breaking down polystyrene through the application of a two-step thermal degradation and bacterial method to produce usable byproducts. Waste Management 60, 123-126, doi:1016/j.wasman.2016.04.017 (2017).
7. Vaidya, Aditya S, Helander, Jonathan D. M, Peterson, Francis C, Elzinga, Dezi, Dejonghe, Wim, Kaundal, Amita, . . . Cutler, Sean R. (2019). Dynamic control of plant water use using designed ABA receptor agonists. Science (American Association for the Advancement of Science), 366(6464), Eaaw8848.
8. Sun, Xiangzhong, Li, Yaxin, He, Wenrong, Ji, Chenggong, Xia, Peixue, Wang, Yichuan, . . . Guo, Hongwei. (2017). Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase. Nature Communications, 8(1), 15758.
9. He, Wenrong, Brumos, Javier, Li, Hongjiang, Ji, Yusi, Ke, Meng, Gong, Xinqi, . . . Guo, Hongwei. (2011). A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis. The Plant Cell, 23(11), 3944-3960.
2. Zhang, G., Gurtu, V. & Kain, S. R. An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical and Biophysical Research Communications 227, 707-711, doi:10.1006/bbrc.1996.1573 (1996).
3. Kämpfer, P. et al. Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes. Microbial Ecology 32, 101-121, doi:10.1007/BF00185883 (1996).
4. Sehar, S. & Naz, I. Role of the Biofilms in Wastewater Treatment. in Microbial Biofilms-Importance and Applications (eds. Dhanasekaran, D. & Thajuddin, N.) (IntechOpen, 2016). doi: 10.5772/63499
5. Martens, .J., Barg, .H., Warren, .M. et al. Microbial production of vitamin B12. Appl Microbiol Biotechnol 58, 275–285 (2002). doi:10.1007/s00253-001-0902-7
6. Savoldelli, J., Tomback, D. & Savoldelli, H. Breaking down polystyrene through the application of a two-step thermal degradation and bacterial method to produce usable byproducts. Waste Management 60, 123-126, doi:1016/j.wasman.2016.04.017 (2017).
7. Vaidya, Aditya S, Helander, Jonathan D. M, Peterson, Francis C, Elzinga, Dezi, Dejonghe, Wim, Kaundal, Amita, . . . Cutler, Sean R. (2019). Dynamic control of plant water use using designed ABA receptor agonists. Science (American Association for the Advancement of Science), 366(6464), Eaaw8848.
8. Sun, Xiangzhong, Li, Yaxin, He, Wenrong, Ji, Chenggong, Xia, Peixue, Wang, Yichuan, . . . Guo, Hongwei. (2017). Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase. Nature Communications, 8(1), 15758.
9. He, Wenrong, Brumos, Javier, Li, Hongjiang, Ji, Yusi, Ke, Meng, Gong, Xinqi, . . . Guo, Hongwei. (2011). A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis. The Plant Cell, 23(11), 3944-3960.
Acknowledgements
Acknowledgements
Liang Yang, Ph.D., Professor
School of Medicine, Southern University of Science and Technology
Mr. Shengchun Wu, Vice manager
Shenzhen Techand Ecological and Environmental Co. Ltd.
Hongwei Guo, Ph.D, Chair Professor
Department of Biology, Southern University of Science and Technology
QuanLiu, M.D, Ph.D, Associate Professor
School of Medicine, Southern University of Science and Technology
Kai Jiang, Ph.D, Research Assistant Professor
Department of Biology, Southern University of Science and Technology
Shaohong Zhang
Undergraduate majoring in Biology Science, member of 2019 SUSTech_Shenzhen iGEM team
Yi Zhang
Undergraduate majoring in Biology Science, member of 2019 SUSTech_Shenzhen iGEM team
Huazhang Shu
Undergraduate majoring in Biology Science, member of Dr. Hongwei Guo’s Lab
Shangzhen Yu
Undergraduate majoring in Biology Science, member of Dr. Hongda Huang’s Lab
Hongyu Bao, Ph.D
Postdoc Fellow, Department of Biology
Xiaoqian Xue, Ph.D
Postdoc Fellow, Department of Chemistry
SUSTech Presidential Foundation
For generously providing funds for research.