Team:WHU-China/Design
The design of "The Negotiator" encompasses four major aspects, i.e. Quenching Module, Sensing Module, Safety Measures and Cell-Free Quorum Sensing. With four aspects integrated together, we envision "The Negotiator" will become a novel and evolving prophylactic strategy to cope with ventilator-associated pneumonia, as well as nosocomial infections. The experimental workflows and achievements are mainly summarized in Engineering Success.
Notably, to demonstrate the biological designs mostly away from the laboratories, our team harnessed interdisciplinary methods and real-world perspectives for developing "The Negotiator", which are shown in relevant pages.
Pseudomonas aeruginosa is a slippery and dangerous criminal, whose quorum sensing networks comprise three interconnected systems, las, rhl and pqs [9]:
(i) las system is the dominant one in P. aeruginosa quorum sensing networks, regulating the activity of rhl and pqs systems, and its autoinducer OdDHL/3-oxo-12C-HSL belongs to N-acyl homoserine lactones (AHL);
(ii) rhl system is a major contributor to virulence, including biofilm formation and pyocyanine expression, and its autoinducer BHL/4C-HSL belongs to AHLs;
(iii) pqs system is also a major contributor to virulence, and its autoinducer Pseudomonas quinolone signal (PQS) is a characteristic molecule of Pseudomonas spp.
As a proof-of-concept of "The Negotiator", to cope with this pathogen with classic QS models, two modules are constructed within one of the most amenable probiotic chassis-Escherichia coli Nissle 1917, with their functions described whereafter.
Diffused in the environment, autoinducers could be taken into bacteria via different approaches [11], and guide cell density-dependent behaviours in its original host. However, several organisms develop natural mechanisms to interfere with quorum sensing of others, namely quorum sensing inhibitors (QSIs), in order to earn niches for themselves considering the limited resources. We plan to harness this natural mechanism to develop engineered modules.
To interfere with criminals’ communications for virulence inhibition, Quenching Module is constructed based on quorum quenching enzymes. These enzymes, which involve AHL acylases and AHL lactonases [1,2], can degrade gram-negative bacteria’s dominant communication tools-N-acyl homoserine lactones (AHLs), whereby those gram-negative bacteria cannot express virulence as the concentration of their AHLs lower. Aimed at degrading AHLs with diverse acyl-chain lengths, we select enzyme combinations with the help of our quorum sensing kinetic models. Furthermore, the promiscuity of enzymes captures our attentions, and we’re reconstructing ancestral proteins and designing engineered enzymes with enhanced performances from a perspective of molecular evolution.
Chemotaxis is a fascinating immune phenomenon. When outside objects (especially pathogen bacteria) invading human body, several immune cells (such as neutrophils) are recruited by chemokines excreted by cells in the invaded spot to clear the intruders. Notably, the strategies of trade-off exist in the immune response of healthy individuals-in the self-evolving arms race, chemokines will be secreted not immediately the invasion happens but just before the situation gets worse, to prevent unnecessary energy loss [3]. However, as for immunocompromised patients suffered from coronavirus, who lacked appropriate immune responses, chemotaxis will act in a strange way, resulting in surging probability of get infected by pathogen bacteria. Hence, a rational method to sustain their well-balanced chemotaxis activities to prevent nosocomial infections is needed.
Sensing Module is constructed based on chemotaxis. The logic of Sensing Module starts from Pseudomonas quinolone signal, PQS for abbreviation, an indicator of exacerbated infection (when natural chemotaxis should start). We design two approaches to make our negotiators react more sensitive than criminals in the same PQS concentration. First, we will overexpress the cognate transcription factor PqsR in our host by a strong constitutive promoter to capture PQS. Then, a phage-derived system will be harnessed to amplify the signals for robust expression of downstream recombinant chemokines, which comprise sec-tag DsbA for excretion. The chemotaxis of several specific pathogen-killing immune cells is utilized for eradicating P. aeruginosa. In our project, flow-cytometry analysis is utilized to evaluate the effects of different chemokines, and a part collection of chemokines is presented.
Two measures are designed to guarantee the safety of using negotiator probiotics inside human body:
The first safety measure is developing a flexible machinery to control the secretion of chemokines, which involves the expression of TEV protease to address the possible leakage of upstream pqs promoter and modulate chemokines secretion by cutting off the sec-tag, in case of cytokine storm. A model was designed to describe this process in a mathematic language.
The second safety measure is adding toxin-antitoxin systems to important modules, to prevent horizontal gene transfer. The loss of transformed plasmids will result in the suicide of engineered probiotics. We collected a large amount of information about toxin-antitoxin systems, and a toxin-antitoxin system, named MazE and MazF, was cloned from E. coli MG1655 to test its efficiency [5].
It is predicted that the abuse of conventional antibiotics renders increasing antimicrobial resistance in pathogen bacteria and fungi, which will make pathogen infection the rank first death cause in 2050 [10]. Thus developing novel drugs with targets less likely to cause resistant phenotypes in pathogens is emergent, and also in accordance with UN’s sustainable development goals. Quorum sensing inhibitors (QSIs) are one of the promising drugs, for their diverse anti-virulence functions and low selective pressure [3]. Besides quorum quenching enzymes in our Quenching Module, numerous small-molecule natural products are also potential quorum sensing inhibitors. However, to screen potent quorum sensing inhibitors, several obstacles exist in current in silico-in vivo methods, such as low throughput and tedious experimental cycle. Thus, we want to develop an in vitro platform to bridge traditional methods of in silico prediction and in vivo evaluation, to accelerate the design-build-test-learn cycle of QSI discovery.
Over the past decade, cell-free expression system has been going through its renaissance [6]. Cell-free expression system circumvents the barriers of cell wall, and bestows researchers with extraordinary conveniences, including high throughput, rapid workflow and straightforward quantification method. Tremendous researches have been performed on cell-free platforms to investigate metabolic pathways [7]. In addition, cell-free biosensors with fluorescent or colorimetric outputs were developed to promote small-molecule diagnosis [8]. What about combining the concepts of cell-free metabolic engineering with cell-free biosensor to establish a quantifiable signal transduction pathway?
To address antimicrobial resistance crisis and to provide our negotiators with an evolving arsenal, we’re reconstituting las and rhl systems of P. aeruginosa in E. coli lysate-based cell free expression systems for rapid and high-throughput screening of novel quorum sensing inhibitors. Additionally, an improved workflow integrating in silico, in vitro, and in vivo methods is proposed.
To reconstitute the basic logic of las and rhl systems of P. aeruginosa in cell-free systems, four plasmids will be constructed, with pSB1A3 as the backbone, which include: (i) Plasmid-T (K2965011+LasR; K2965011+RhlR) will overexpress LasR and RhlR in E. coli BL21(DE3) after IPTG induction, and the bacteria will then be lysed to prepare cell-free systems; (ii) Plasmid-L (J23100+LasI; K649000+LasI) will express LasI to synthesize OdDHL in cell-free systems, and achieve a positive feedback by its second device; (iii) Plasmid-R (K649000+RhlI; R0071+RhlI) will express RhlI to synthesize BHL in cell-free systems under the control of Plasmid-L, and achieve a positive feedback by its second device; (iv) Plasmid-F (K649000+GFPmut3b; R0071+mCherry) will express GFPmut3b and mCherry under the control of in vitro established las and rhl systems, and the green or red fluorescence will be measured by a microplate reader to quantify the activated level of these two systems.
1. Fetzner S, Quorum quenching enzymes. Journal of Biotechnology, 201 (2015): 2-14.
2. Grandclement C et al., Quorum quenching: role in nature and applied developments. FEMS Microbiology Reviews, 40 (2016): 86-116.
3. Remy B et al., Interference in Bacterial Quorum Sensing: A Biopharmaceutical Perspective. Frontier in Pharmacology, 9 (2018).
4. Moura-Alves et al., Host monitoring of quorum sensing during Pseudomonas aeruginosa infection. Science, 366 (2019).
5. Moll I, Engelberg-Kulka H, Selective translation during stress in Escherichia coli. Trends Biochem Sci, 37 (2012): 493-498.
6. Silverman AD et al., Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genetics, 21 (2020): 151-170.
7. Karim AS, Jewett MC, A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metabolic Engineering, 36 (2016): 116-126.
8. Wen KY et al., A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples. ACS Synth. Biol., 6 (2017): 2293-2301.
9. Lee J and Zhang L, The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell, 6 (2015): 26-41.
10. The Review on Antimicrobial Resistance Chaired by Jim O’Neil, 2014.
11. Pearson JP, van Delden C, Iglewski BH, Active Efflux and Diffusion Are Involved in Transport of Pseudomonas aeruginosa Cell-to-Cell Signals. Journal of Bacteriology, 181 (1999): 1203-1210.
