Team:INSA-UPS France/Description

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017


Description

Synthetic Consortia
A microbial consortium chassis against cholera
Mimicking Vibrio cholerae
The sensing organism: Vibrio harveyi
The effecting organism: Pichia pastoris
Our system
References

Synthetic biology: from unique chassis to synthetic consortia

Synthetic biology is based on Nature everlasting possibilities, usually by inserting genetic information from microorganisms into a single and unique chassis1,2,3. However, a single chassis could present inevitable limits (high genetic burden, incompatibility of some elements with the chassis, too complex design…). These start to be a limit in synthetic biology, its perspectives and applications 4. An emerging solution is the use of synthetic consortium (figure 1). Synthetic consortium have the advantage to require less amount of genetic information into a single chassis to achieve the required process since different microorganisms can share the genetic burden. Moreover, the components of the consortium could be selected to reduce the genetic modifications and increase the chance of success, for example, by taking advantage of already existing signaling or metabolic pathways. Several successes of those synthetic consortia, such as production of bio-electricity5 or shortening bio-manufacturing process like C-vitamin synthesis6 have provided insight into the strength of this approach.

Figure 1: General concept of the cell to cell communication. This cascade of events will be developed in our project.

Such approaches are still rare in the iGEM competition, maybe because they require to combine classic strain engineering with information processing strategies. So, our challenge was to demonstrate the power and feasibility of synthetic consortium approach to open new perspectives and applications to iGEMers.

As a proof of concept, we developed a strategy against cholera. It is based on a cascade of events starting from an engineered Escherichia coli strain mimicking Vibrio cholerae. It triggers a sensor bacterium, Vibrio harveyi, which in turn activates the effector cell (the yeast Pichia pastoris). The later eradicates Vibrio species by producing innovative antimicrobial peptides from crocodile.

Genesis of our molecular strategy

While we were defining our strategy, a cholera epidemia started unfortunately to expand in Yemen7. This terrible situation led us to focus on this problematic as it appears that current solutions are not efficient enough to deal with this situation. The bacteria V. cholerae, agent of the cholera disease, is usually found in water and infects more than a million people each year.

Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with V. cholerae8,9. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae10,11,12, using E. coli. They based their strategy on implementing the quorum sensing detection pathway of V. cholerae into E. coli to activate reporter gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough, likely due the complexity of introducing a large amount of DNA information in a single microorganism. This is the reason why we built a synthetic consortium of microorganisms. Using multiple microorganisms instead of one will allow us to choose existing species that are already specialized for their tasks, as well as reducing the amount of necessary modification to set up the required functions. The information processing steps have been split in two different microorganisms: V. harveyi as the detector and P. pastoris as the effector, with the system triggered by V. cholerae presence.

A microbial consortium chassis against cholera

The whole synthetic consortium is composed of three microorganisms. The first one should be V. cholerae but for safety reason, we engineered an E. coli strain to produce a V. cholerae molecular signal. The second bacteria required a quorum sensing pathway to detect V. cholerae signal. V. harveyi naturally possesses such a pathway and we decided to modify it toward V. cholerae detection, as well as to engineered V. harveyi to excrete a second molecule messenger. The third microorganism, P. pastoris, detects this messenger and induces the expression an secretion of a high amount of antimicrobial peptides to lyse V. cholerae.

We finally created an artificial consortium chassis to deal with cholera disease. The different partners are deeper described below.

Mimicking Vibrio cholerae using Escherichia coli

An interesting property of V. cholerae is its quorum sensing autoinducer system based on the production of CAI-1 molecule13. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is an efficient reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain E. coli in order to mimic V. cholerae. E. coli was transformed with the cqsA gene from V. cholerae. Since our sensor was V. harveyi, as a proof of concept, we also transformed an E. coli strain with the CqsA encoding gene of V. harveyi. This non-pathogen Vibrio synthetizes C8-CAI-1, an analog of V. cholerae CAI-114 thanks to CqsA enzyme (i.e. Vh_CqsA).

We therefore developed E. coli strains which produce markers simulating the presence of Vibrio species in the medium.

The sensing organism: Vibrio harveyi

The easiest way to detect CAI-1 or C8-CAI-1 is to use the quorum sensing pathway of the non-pathogen V. harveyi. As our project will deal directly with V. cholerae in real situation, the CqsS receptor of V. harveyi will have to also recognize the CAI-1 molecule13. To do so, a single mutation was introduced in the gene cqsS changing the phenylalanine 175 into a cysteine. We identified V. harveyi qrr4 as a gene whose expression depends on the binding of C8-CAI-1 on its receptor, CqsS14.

Figure 2: Closer view of the CAI-1/CqsS sensing of V. Cholerae15.
When C8-CAI1 binds to CqsS this activate a dephosphorylation cascade leading to the inhibition of the pQRR4 promoter and blocking the transcription of siRNA. In the absence of this siRNA, translation of the targets genes (i.e. virulence genes) is activated (See Figure 2). We used this system to produce the molecule (i.e. diactetyl) used to activate P. pastoris . The als coding sequence, encoding for the acetolactate synthase Als, is involved in the conversion of endogenous pyruvate into diacetyl (Figure 3). als was placed under the control of pQRR4 promoter. In this engineered V. Harveyi strain , diacetyl production will be produced in response to both CAI-1 or C8-CAI-1.

Figure 3: Production of diacetyl from pyruvate.

The effector organism: Pichia pastoris

The function of the third partner is to produce a killing molecule (i.e. anti-microbial peptides, AMPs) to lyse both V. cholerae and V. harveyi. Furthermore, this microorganism has to be resistant to the AMPs that are specific to prokaryotic cells. Therefore we chose an eukaryotic cell. Team SCUT 15 previously described a binding-receptor system involving diacetyl and a eukaryotic receptor, Odr-10 16,17. It is a G Protein Coupled Receptor isolated from Caenorhabditis elegans that once activated by diacetyl, lead to the activation of the pFUS1 promoter through the endogeneous Ste12 pathway (Figure 4). Finally we choose P. pastoris as the effector organism since it already possess the ste12 pathway and is also good protein producer 18,19.

Figure 4: The diacetyl/Odr-10 activation cascade.15.
We engineered the yeast to secret the AMPs under the pFUS1. We choose the AMPs from crocrodiles 20,21,22,23. Indeed crocodiles display a remarkable and efficient defense system, allowing the reptiles to resist to a large spectrum of bacterial infection. Thus, they produced antimicrobial peptides (AMPs) which are able to lyse bacteria such as V. cholerae. AMPs are cationic pore-forming molecules targeting bacterium membranes, causing bacterial lysis and death 24.

Our system

See our Design page for more informations about the genetic elements we used!

References

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    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/
  2. Deisseroth K (2015) Optogenetics: 10 years of microbial opsins in neuroscience. Nature Neuroscience.
    https://www.ncbi.nlm.nih.gov/pubmed/26308982
  3. Cameron E, Bashor C & Collins J (2014) A brief history of synthetic biology. Nature Reviews Microbiology https://www.ncbi.nlm.nih.gov/pubmed/24686414
  4. Hennig S, Rödel G & Ostermann K (2015) Artificial cell-cell communication as an emerging tool in synthetic biology applications. Journal of Biological Engineering
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/
  5. Liu T, Yu Y, Chen T and Chen W. (2016). A synthetic microbial consortium of Shewanella and Bacillusfor enhanced generation of bioelectricity. Biotechnology and Bioengineering, 114(3), pp.526-532. https://www.ncbi.nlm.nih.gov/pubmed/27596754
  6. Wang, E., Ding, M., Ma, Q., Dong, X. and Yuan, Y. (2016). Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation. Microbial Cell Factories, 15(1). https://www.ncbi.nlm.nih.gov/pubmed/26809519
  7. http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
  8. Focareta A, Paton JC, Morona R, Cook J & Paton AW (2006) A Recombinant Probiotic for Treatment and Prevention of Cholera. Gastroenterology 130 1688–1695
    https://www.ncbi.nlm.nih.gov/pubmed/16697733
  9. Holowko MB, Wang H, Jayaraman P & Poh CL (2016) Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli. ACS Synthetic Biology http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00079
  10. https://2014.igem.org/Team:UI-Indonesia
  11. https://2010.igem.org/Team:Sheffield
  12. https://2014.igem.org/Team:UT-Dallas
  13. Bolitho ME, Perez LJ, Koch MJ, Ng W-L, Bassler BL & Semmelhack MF (2011) Small molecule probes of the receptor binding site in the Vibrio cholerae CAI-1 quorum sensing circuit. Bioorganic & Medicinal Chemistry 19 6906–691
    https://www.ncbi.nlm.nih.gov/pubmed/22001326
  14. Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417
    https://www.ncbi.nlm.nih.gov/pubmed/21219472
  15. https://2013.igem.org/Team:SCUT
  16. Zhang Y, Chou JH, Bradley J, Bargmann CI & Zinn K (1997) The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells. Proceedings of the National Academy of Sciences 94 12162–12167
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/
  17. Audet M & Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. Cell 151 14–2
    https://www.ncbi.nlm.nih.gov/pubmed/2302121
  18. Kang Z, Huang H, Zhang Y, Du G & Chen J (2017) Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications. World Journal of Microbiology and Biotechnology
    https://www.ncbi.nlm.nih.gov/pubmed/27905091
  19. Huang Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris. Experimental and Therapeutic Medicine
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/
  20. Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T & Thammasirirak S (2011) Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Developmental & Comparative Immunology 35 545–553
    https://www.ncbi.nlm.nih.gov/pubmed/21184776
  21. Preecharram S, Jearranaiprepame P, Daduang S, Temsiripong Y, Somdee T, Fukamizo T, Svasti J, Araki T & Thammasirirak S (2010) Isolation and characterisation of crocosin, an antibacterial compound from crocodile (Crocodylus siamensis) plasma: CROCODILE PLASMA ANTIBACTERIAL COMPOUND. Animal Science Journal 81 393–401
    https://www.ncbi.nlm.nih.gov/pubmed/2059789
  22. Prajanban B, Jangpromma N, Araki T & Klaynongsruang S (2017) Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins. Biochimica et Biophysica Acta (BBA) - Biomembranes 1859 860–869
    https://www.ncbi.nlm.nih.gov/pubmed/28159460
  23. Yaraksa N, Anunthawan T, Theansungnoen T, Daduang S, Araki T, Dhiravisit A & Thammasirirak S (2014) Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts. Journal of Antibiotics 67 205
    https://www.ncbi.nlm.nih.gov/pubmed/24192554
  24. Marín-Medina N, Ramírez DA, Trier S & Leidy C (2016) Mechanical properties that influence antimicrobial peptide activity in lipid membranes. Applied Microbiology and Biotechnology 100 10251–10263
    https://www.ncbi.nlm.nih.gov/pubmed/27837316