Team:WHU-China/Poster

Poster: WHU-China

The Negotiator—A Synthetic Biology Strategy for Prophylactic Treatment to Ventilator-Associated Pneumonia

Presented by Team WHU-China 2020

Authors: Dingchen Yu, Yipeng Xie, Jiayang Li, Yuqing Liu, Boxuan Xia, Yuxin Qi, Xiaowen Shang, Liran Mao

Abstract

We aimed at rationally engineering probiotics to address the problem of nosocomial infections in respiratory tract, especially ventilator-associated pneumonia (VAP) during COVID-19 pandemic. The chassis of our project was Escherichia coli Nissle 1917, recognized as the most amenable probiotic, and the target pathogen was Pseudomonas aeruginosa, a representative nosocomial pathogen notorious for its quorum sensing-based virulence behaviours. We constructed two modules to endow our chassis with the capability as "the negotiator": (i) Quenching Module: heterologously overexpressing quorum quenching enzymes to parley with "criminals" (P. aeruginosa) by degrading AHLs; and (ii) Sensing Module: sensing PQS and excreting appropriate amounts of chemokines to recruit "police squads" (immune cells) for eradicating pathogens. Notably, we leveraged E. coli lysate-based cell-free system for rapidly prototyping genetic parts of interest, in order to accelerate the design-build-test-learn cycle of our project as well as to give insights into the elegance of cell-free expression renaissance.

Inspiration

This year, we have witnessed SARS-CoV-2 overwhelming the whole world and ensuing destructive influences brought by the severe pandemic. Meanwhile, deeply impressed by the heroic stories of medical staff and researchers battling the dangerous virus, we WHU-China iGEMers felt responsible for utilizing synthetic biology approaches to make our contributions. After enough investigations, we made nosocomial infections, serious problems usually neglected by the public in the epidemic, as our target and specifically focused on the Ventilator-Associated Pneumonia (VAP) caused by inhaling pathogen bacteria(Pseudomonas aeruginosa in our case) during ventilation which made up a large proportion of nosocomial infections in the COVID-19 pandemic. To solve this issue, we chose probiotics Escherichia coli Nissle 1917 as our chassis, considering their ability to stimulate and activate the immune systems and the possibility of harnessing engineered probiotics to directly solve pathogen infections.
This situation is much like an “arms race” with “criminals”(pathogen bacteria) using their quorum sensing system (regulatory expression of virulence genes controlled by the concentration of autoinducers) to “communicate”, increase the biofilm formation and virulence while “police squads”(immune cells) are recruited to directly kill the pathogens. What we want our engineered probiotics do is playing the role of “Negotiator” to parley with criminals as well as give instructions secretly to police squads which helps the balance lean towards the latter ones.

Introduction

Following the idea of arms race we come up with two modules as the weapons of the Negotiator. One is the Quenching Module: the crucial quorum sensing systems of P. aeruginosa are las and rhl systems including two kinds of N-acyl homoserine lactones (AHLs), 3-oxo-C12-HSL and C4-HSL respectively. To parley with criminals, quorum quenching enzymes(AHL acylases and AHL lactonases) will be used to degrade AHLs so that the communication among the pathogens will be inhibited and the virulence will decrease. As for selecting the best enzyme(s), we have designed experiments to obtain the Michaelis constant and built a quorum dynamic model to help us visualize it. Besides we try to reconstruct the ancestral enzymes to get one brand new enzyme with broad spectrum and enhanced activity for future usage.

Figure1: the Negotiator will express AHLase to degrade the signal molecules AHLs of P.aeruginosa and thus inhibit its toxicity.
The other is Sensing Module: equipped with the signal receptors expression and an activator device, the Negotiator will be able to sense the third quorum sensing signal molecules PQS of P.aeruginosa which will induce the expression and secretion of chemokines to recruit several immune cells and kill the pathogens directly.

Figure2: with PQS receptor expressed and an activator system to amplify the signals, the Negotiator is capable to sense the signal molecule PQS of P.aeruginosa and secret chemokines then to recruit immune cells.
In terms of the safety, TEV protease and its corresponding tag are used to control the secretion of chemokines in case of cytokine storm. And another measure is taking advantage of toxin-antitoxin systems to prevent horizontal gene transfer.

Figure3: TEV protease and its corresponding tag are utilized to control the secretion of chemokines whilst toxin-antitoxin system is added to prevent the horizontal gene transfer. They work together as our safety measures.
Besides these basic designs, we also propose combining the concepts of cell-free metabolic engineering with cell-free biosensor to build an in vitro platform for screening novel quorum sensing inhibitors(QSIs) as the increasing antimicrobial resistance in pathogens bacteria and fungi has become more and more severe. This way, we hope to expand our project’s influence and contribute more to the medical field.

Figure 4: a cell-free quorum sensing system is established to simulate the quorum sensing in P.aeruginosa and promote novel QSIs research.

Methodology

Quenching Module: High Performance Liquid Chromatography

In our design, Quenching Module displays considerable modularity, owing to the distinct catalytic activities of candidate quenching enzymes towards OdDHL and BHL. To optimize the construction of Quenching Module, high-accuracy enzymatic assays and reliable data for each candidate are needed. Therefore, we decide to utilize high performance liquid chromatography (HPLC) to measure the concentration change of substrates (AHLs), and calculate the enzymatic parameters accordingly. The obtained enzymatic parameters will later be used in our mathematical model, which describes the processes of quorum sensing and quorum quenching, for instructing us to select optimal enzymes or enzyme pairs.

Sensing Module: Transwell Test & Flow Cytometry Analysis

To select optimal chemokines for constructing Sensing Module, we plan to test the chemotactic activities of seven candidate chemokines to pathogen-killing immune cells, mainly monocytes and neutrophils. We choose THP-1 cell line as a model of monocytes for Transwell migration assay, and then perform flow cytometry analysis to quantify the results of chemotaxis. For each chemokine, 3 parallel experiments are performed, in which 0.2 mL THP-1 cell culture medium of certain density is added to the upper chamber and 0.6 mL chemokine solution of 10 nmol/L (dissolved in pure culture medium) is added to the lower chamber. We set 3 parallel control groups as well, in which the pure culture medium is added to the lower chamber. After about 15 hours of incubation, the number of cells in the lower chambers are counted.
Figure 5: The methods used to quantify chemotaxis. A. Transwell tests to measure the chemotactic functions of different chemokines for THP-1; B. Results generated by flow cytometry to quantify the chemotactic function of CCL2 for THP-1.
To better compare the results of each chemokine group, chemotactic index (CI) is utilized in subsequent analysis, which is defined as:

'Negotiators' and 'Criminals': Co-culture

To simulate the autoinducer-based interactions between 'Negotiators' and 'Criminals', we design and manufacture a co-culture device, made of low-cost materials. Later experiments illustrated its feasibility for adequate simulation.
Figure 6: A DIY co-culture device to perform relevant experiments.

Dry lab tools used:

SimBiology for mathematical simulation of quorum quenching;
gro for visualization of quorum quenching;
NCBI BLAST for sequences collection;
MEGA-X MUSCLE for multiple sequences alignment;
Mrbayes and GRASP server for ancestral sequences reconstruction;
Figtree and iTOL for tree drawing;
EXPASY SWISS-MODEL and Phyre2 for protein structure prediction;
Discovery Studio and PyMOL for structural alignment, virtual amino acid mutation, molecular docking and molecular dynamics.

Results

High Performance Liquid Chromatography:

Unfortunately, we only had time for preliminary experiments. As a proof-of-concept, 3-oxo-C12-HSL and C4-HSL bought from Sigma-Aldrich were used. We dissolved 2 mg of each in 2 mL chromatographic grade methanol to reach the final concentration of 1 mg/mL and quantified them by HPLC. The results showed that there’re two peaks for C4-HSL and one peak for 3-oxo-C12-HSL. This makes sense as the AHLs we bought both “D” and “L” configurations mixed together and the chemical property may influence the time that peaks appear. But for 3-oxo-C12-HSL, which has a longer acyl-chain, different configurations may not influence its property intensely, so only one peak appeared. The preliminary experiment results prove the feasibility of HPLC method to quantify AHL concentrations, and provide us the conditions needed. In the next phase of our project, HPLC will play a decisive role in the construction of Quenching Module.
Figure 7: HPLC analysis of the standard solutions of C4-HSL (A) and 3-oxo-C12-HSL (B) (1 mg/mL). A. the two peaks for C4-HSL occur at about 6 min and 8.4 min; B. the peak for 3-oxo-C12-HSL occurs at about 4.3 min.

Transwell Test & Flow Cytometry Analysis:

The number of cells in each lower chamber was collected according to the scatter diagrams and analyzed. The results suggested that at the concentration of 10 nmol/L, 3 chemokines from CCL family have the potential to attract THP1 cells, especially CCL2, which shows statistical significance (p < 0.05). However, 4 chemokines from CXCL family didn’t show a potential to attract THP-1 cells and even had the tendency to inhibit cell migration at the concentration of 10 nmol/L. Moreover, the data of several chemokines like CCL3, CXCL8 and CXCL3 showed great standard error, which indicates their unstable performance of chemotaxis at the concentration of 10 nmol/L. According to the results generated from Transwell tests and flow cytometry analysis, CCL2 was the optimal chemokine to attract THP-1 cells with the highest CI and showed the statistic significance. Although the CI of CCL3 was just a little lower than that of CCL2, its performance was considered unstable and showed no statistic significance compared with the control. Therefore, we decided to try CCL2 in ensuing construction of Sensing Module.
Figure 8: Quantification of chemotaxis. A. Chemotaxis of CCL2 to THP-1 (shown by cell number), chemokine concentration is 10 nmol/L (n=3); B. Chemotaxis of 7 chemokines to THP-1 (shown by chemotactic index), chemokine concentration is 10 nmol/L (n=3).

Co-culture of E. coli Nissle 1917 and P. aeruginosa PAO1:

To test the basic function of our hardware, we performed a simple co-culture experiment. First, we added 2 mL bacteria suspension (which was incubated overnight) to 120 mL fresh LB medium (two flasks for E. coli Nissle 1917 and one for P.aeruginosa PAO1) and incubated them in a shaker (37℃, 200 rpm) for 2 hours to obtain an initial bacteria density. Second, we poured one flask of E. coli Nissle 1917 and P. aeruginosa PAO1 in the left and right section of our hardware respectively. Third, we used the hardware to mix the medium and took 2 mL samples per 45 minutes to measure the OD600 value in a spectrophotometer. The same procedure was also used on the other flask of our probiotics (without being mixed with the medium of P. aeruginosa) as the control group. The growth curves showed no significant inhibition of E. coli Nissle 1917, and proved the basic function of our DIY hardware.
Figure 9: Growth curves for E. coli Nissle 1917 and P. aeruginosa PAO1 co-culture.

SimBiology: As can be seen in the comparison figures, after the addition of engineered bacteria, the equilibrium concentrations of LasR and RhlR as well as the time to reach the equilibrium do not change much. However, the equilibrium concentrations of OdDHL, BHL, both inside and outside pathogenic bacteria, decrease obviously, and the time to reach the equilibrium is shortened. This indicates that ‘negotiators’ can evidently change the equilibrium state of OdDHL and BHL, and have a significant effect on the reduced virulence expression, as the concentrations of LasR/OdDHL complex and RhlR/BHL complex also strongly decrease.
Figure 10: Simulation of concentrations of las-and-rhl-associated components before and after quorum quenching.

gro:

The simulation results inform us that, human cells nearly stop reproducing and growing in the beginning because of AHL and accompanying virulence, so the ratio of pathogens(Pseudomonas aeruginosa)/human cells keeps rising. However, ‘negotiators’ step in later and save human cells, after which the ratio keeps decreasing. Moreover, the visualized simulations vividly demonstrate these interactions.
Figure 11: gro simulation of quorum quenching. A: Simulation of the dynamic of different cells; B. Visualization of quorum quenching.

Ancestral protein reconstruction:

Using online database and bioinformatic tools, ancestral sequences of lactonase AiiA and acylase PvdQ were constructed. The ancestral sequences can either indicate the promiscuous mechanisms of quenching enzymes in subsequent structural analysis, or express ancestral enzymes in wet lab to provide extra choices for constructing Quenching Module.
Figure 12: Ancestral sequences generated and five candidates selected.

Structural analysis:

A series of structural analysis projects towards the broad-spectrum lactonase AiiA were performed. On the basis of molecular docking and molecular dynamics, virtual amino acid mutation was implemented in several important residues, implied by literature and ancestral sequences alignment. Notably, we discovered several residues worth further exploring in wet lab, including PHE68, GLU136, and TYR194.
Figure 13: Virtual amino acid mutation to improve AiiA catalysis. A. Virtual mutation of PHE68, and the ligand is C6-HSL; B. Virtual mutation of GLU136, and the ligand is C5-HSL; C. Virtual mutation of GLU136, and the ligand is C10-HSL.

Human Practices

In Human Practices, the principles of diversity and interactivity are highlighted:
(i) All student members in the team were asked to interview their family members with different occupations, to get a better understanding of nosocomial infections and ordinary people’s daily lives under COVID-19 pandemic.
(ii) To collect information about the public cognition of nosocomial infections and engineered probiotics, two integrated questionnaires were distributed online and offline. Subsequent analysis inspired us to create a popular science comic named ‘Ventilator-Associated Pneumonia’, in collaboration with a local artist.
(iii) We developed three social media accounts for our teams, including WeChat, Weibo and Bilibili, to provide different target audiences with correspondingly modified education materials. The related topics involved nosocomial infections, engineered probiotics, cell-free expression renaissance, etc.
(iv) We delivered a public lecture to students in Wuhan University, who showed strong interests in iGEM and synthetic biology.
(v) ‘A Handbook of Anti-Biofilm Community’ was designed this year by Anti-Biofilm Communtiy (a partnership run by us, composed of six iGEM teams), and we envisioned these brochures could have positive impacts on the public.

Meanwhile, our project has been advancing from theory to practice, motivated by experts from synthetic biology, immunology, critical care, biomedical engineering and business, in distinct stages. In the stage of Necessity, Prof. Liu Tiangang unraveled the severity of nosocomial infections and firmed our minds to develop ‘The Negotiator’. In the stage of Feasibility, Prof. Zhang Qiuping and Prof. Zhang Xiaolian optimized our design of Sensing Module and provided us with beneficial knowledges about chemotaxis; Prof. Peng Zhiyong confirmed our envisioned pattern of drug delivery; Prof. Chen Pu offered us suggestions on organ-simulating chips. In the stage of Applicability, Prof. Yang Daichang and Dr. Ma Zhaotang helped us in producing commercially feasible products.

Proposed Implementation

Evaluate the condition of patients to decide whether to use ventilators.

Ventilator is a kind of medical equipment that can prevent and treat respiratory failure, reduce complications and save and prolong the life of patients. According to the patients’ condition, it is used for respiratory failure of various causes, anesthesia during operation, respiratory management, respiratory support treatment and emergency resuscitation.

Choose invasive mechanical ventilation or noninvasive mechanical ventilation.

Non-invasive ventilation is mainly connected with the patient through face mask and nose mask, while invasive ventilation is mainly connected with the patient through endotracheal intubation or tracheotomy. Invasive mechanical ventilation is mainly used in hospitals, indications:
(1) Acute respiratory failure caused by various causes, including respiratory distress syndrome (ARDS).
(2) Acute exacerbation of chronic respiratory failure.
(3) Persistent state of severe acute pulmonary edema and asthma.
Non-invasive mechanical ventilation is mainly used in the home, indications:
(1) Sleep apnea syndrome (snoring);
(2) COPD;
(3) ALS;

Oral or nasal delivery of negotiator probiotics before ventilation, with the amount according to doctor’s prescriptions.

In this part, it is also needed to take the way of ventilation into consideration, for the fact that the two may differ in dose.
(1) D.P.I, dry powder inhaler
The airflow generated during inhalation is used to send drug particles into the airway and lung tissue, without the coordination of inhalation and manual control. The amount of drugs entering the airway and lung tissue is more than that of aerosols, while the drugs remaining in the oropharynx are less than that of aerosols, with better efficacy and reduced side effects such as oropharyngeal fungal infection.
(2) Nebulizer
Spray inhalation is powered by compressed air or hyperbaric oxygen and is based on jet interaction of gas to turn the liquid into mist particles, which are inhaled by patients through face mask. Nebulizer inhalation does not require active patient cooperation and can be used by patients with severe episodes or children who are unable to cooperate. But the atomization inhalation device is large and inconvenient to carry.
This product can effectively deal with the gradually rising drug resistance of pathogenic bacteria, and achieve better adjuvant therapeutic effect.

Implement ventilation.

In this part, our negotiator probiotics can enter the ‘battleground’-lower respiratory tract, and effectively deal with the tricky gram-negative bacteria and their increasing antimicrobial resistance. Furthermore, coupled with anti-inflammatory drugs (such as anti-IL-6 natural products), the strategy might avoid the potential side effects of over-activated chemotaxis. The feasibility of combinatorial usage will be discussed in the next section.

Future

With so many preliminary yet successful attempts this season, we are looking forward to turning them into practice next year. For Quenching Module, with special attention paid to the engineered ancestral enzymes, we envision that these novel sequences will be synthesized and tested in wet lab. And for Sensing Module, we hope that the microfluidic chips that simulate the lower respiratory tract can be made and help us verify the functions of this module in vitro. Moreover, through surveys we find Wuhan a potential city to develop a synthetic biology start-up for engineered probiotics products. Thus we really hope the future WHU-China iGEMers can give it a shot!
Besides, we also wish that the idea of using cell-free prototyping of signal transduction pathways to screen new drugs can be adopted, proved and expanded by more iGEM teams in the future. And for probiotics, the promising next generation chassis, we expect that more iGEMers will take part in characterizing and exploring their genetic parts and toolkits, which sounds really exciting because this may lead synthetic biology to a brighter future!

References, Acknowledgements and Sponsors

References

[1] Fetzner S, Quorum quenching enzymes. Journal of Biotechnology, 201 (2015): 2-14.
[2] Moura-Alves et al., Host monitoring of quorum sensing during Pseudomonas aeruginosa infection. Science, 366 (2019).
[3] Silverman AD et al., Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genetics, 21 (2020): 151-170.
[4] Karim AS, Jewett MC, A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metabolic Engineering, 36 (2016): 116-126.
[5] Lee J and Zhang L, The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell, 6 (2015): 26-41.
[6] Povoa H et al., COVID-19: An Alert to Ventilator-Associated Bacterial Pneumonia. Infectious Diseases and Therapy, 9 (2020): 417-420.
[7] Wypych TP, Wickramasinghe LC, Marsland BJ, The influence of the microbiome on respiratory health. Nature Reviews Immunology, 20 (2019): 1279-1290.
[8] Bober JR, Beisei CL, Nair NU, Synthetic biology approaches to engineer probiotics and members of the human microbiota for biomedical applications. Annual Review of Biomedical Engineering, 20 (2018): 277–300.
[9] https://2019.igem.org/Team:SUSTech_Shenzhen
[10] https://2007.igem.org/Cambridge

Acknowledgements

(in alphabetical order)
Dorothy Zhang
Dr. Zhaotang Ma
Graduate students Shiyu Teng, Ao Gong, Xingchen Dong, Qipeng Shu, Liu Ran, Mu Xin and Jie Chen
Prof. Daichang Yang
Prof. Pu Chen
Prof. Qiuping Zhang
Prof. Shuqiang Huang from SIAT, Chinese Academy of Sciences
Prof. Tiangang Liu’s Lab, College of Pharmacy, Wuhan University
Prof. Xiaodong Zhang’s lab, College of Life Sciences, Wuhan University
Prof. Xiaolian Zhang
Prof. Zhixiong Xie’s lab, College of Life Sciences, Wuhan University
Prof. Zhiyong Peng
Ran Zhang and teacher Jianshe He, Wuhan University Testing Center
Senior iGEMers Tianyi Chang, Renjie Zhou, Yiru Shen, Jiongyi He, Liangyue Song, Shunqing Tan
Teacher Yan Long, Researcher Mingyuan Yang, Teacher Jing Che, Teacher Xiaoyan Li

Sponsors