Team:INSA-UPS France/test

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017


Description

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

Synthetic biology: to the multi-organisms communication and beyond

Nature is still developing a wide large diversity of remarkably efficient pathways in order to sense presence of specific chemical, or even physical parameters such as temperature, pressure and light1,2. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature’s tricks, basically by inserting genetic information from microorganisms into a single and unique one, most of the time Escherichia coli3. However, focusing only on this type of bacteria is not appropriate to reflect the large complexity of living organisms and more, their intimate relationship in Nature. This aspect starts to be a limiting border in the way of the development of the synthetic biology4.

Then, our iGEM project focused on a multi organisms communication pathway, especially between prokaryotic and eukaryotic cells. Thus, we developed a strategy using a cascade of events from a sensor cell (Vibrio harveyi) to an effector cell (Pichia pastoris) in order to detect and eradicate a Vibrio cholera mimicking cell (Escherichia coli) using an antimicrobial peptides from crocodile.

Genesis of our molecular strategy

During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen5. Actually, the bacteria Vibrio cholerae that causes cholera disease is usually found in water and infects more than a million of people each year. This terrible situation led us to focus on this problematic and it appeared that current solutions were not efficient enough to deal with this situation.

Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with Vibrio cholerae6,7. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae8,9,10, using E. coli. They based their strategy around the quorum sensing system of V. cholerae in order to detect it, implementing CqsS receptor and the LuxU/O pathway into E. coli in order to activate gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough maybe due to the process complexity of introducing a large amount of DNA information in a single microorganism. That is why we built a synthetic consortium of microorganism against Vibrio cholerae.

A microbial consortium chassis against cholera

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

  • To mimic V. cholerae by producing CAI-1 molecule. This will be done in E. coli
  • A bacteria with a quorum sensing pathway activated on the V. cholerae presence on which CAI-1 bind. Vibrio harveyi naturally possess that pathway. It will lead to the production of a messenger molecule: that we choose to be diacetyl.
  • The diacetyl binds to the Odr-10 receptor that can be expressed on yeast such as Pichia pastoris and start a molecular pathway.This pathway lead to the activation of pFUS1 and will produce our antimicrobial peptide with a secretion cassette. Those peptides will kill Vibrio cholerae.

Mimicking Vibrio cholerae using Escherichia coli

An interesting property of Vibrio cholerae is its quorum sensing autoinducer system based on the production of CAI-1 molecule11. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is a good reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain Escherichia coli in order to mimic V. cholerae. Thus, we transformed E. coli strain with the CqsA synthase coding gene of Vibrio harveyi, non-pathogen bacteria. CqsA from V. harveyi produces an analog of CAI-1, the molecule C8-CAI-1, from (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A12. Finally, we developed an E. coli strain which produces a marker simulating the presence of the pathogen V. cholerae in the medium. This is the first step of our molecular cascade.

The sensing organism: Vibrio harveyi

Once we developed E. coli to produce the V. harveyi C8-CAI-1, this molecule as to be detected in the medium. The easiest way to do it is to use directly the quorum sensing of the non-pathogen V. harveyi. This bacteria is an advantageous good engineerable chassis. We identified that V. harveyi already possesses gene expression depending on the binding of C8-CAI-1 on its receptor, CqsS12. For example, pQRR4 is a promoter which activation depends on the presence of C8-CAI-1. To fit with CAI-1 molecule, the CqsS receptor of V. harveyi only needed to be mutated on a single amino acid. We only had to mutate CqsS changing the phenylalanine 175 into a cystein and to integrate the ALS gene under the control of pQRR4 to trigger diacetyl production in presence of CAI-112.

Cascade of events depending on the CAI-1/CqsS binding in V. cholerae12. the CAI-1/CqsS binding will start a dephosphorylation cascade leading to the inhibition of pQRR4 and its depending siRNA. The lack of his siRNA will allow the translation of their targeted mRNA.

We checked the metabolism of diacetyl of V. harveyi on KEGG Pathway, and identified that the acetolactate synthase (ALS) alone allowed the production of diacetyl from pyruvate, a ubiquitous metabolite.This is the second step of our molecular cascade.

Production of diacetyl from pyruvate. The addition of the acetolactate synthase (ALS) can lead to the production of acetolactate which convert itself into diacetyl without enzymatic process.

The effecting organism: Pichia pastoris

The molecular response to the presence of the mimicking vibrio E. coli strain is the production by V. harveyi of diacetyl. We then need a third partner to produce toxic molecule to kill V. cholerae. This last partner needs to be resistant to the toxic molecule so we choose an eukaryotic cell. Team SCUT13 previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, the Odr-10 receptor14,15. It is a G Protein Coupled Receptor isolated from Caenorhabditis elegans that once activated by diacetyl, lead to the activation of the pFUS1 promoter by Ste12. Pichia pastoris has been chosen as it displays already the Odr-10/pFUS1 pathway.

Activation cascade on the dependence of Diacetyl/Odr-10 binding13. Once diacetyl bind to Odr-10 a cascade of activation of Ste proteins will lead to the binding of Ste12 on pFUS1 promoter, and so to the expression of gene of interest.

Moreover, P. pastoris is a good protein producing organism16,17. We engineered the yeast to secret the toxic molecule under the promoter of Ste12. The toxic molecule secreted by P. pastoris is originated from crocodiles18,19,20,21. Crocodiles display a remarkable and efficient immune system, allowing the reptiles to resist to a large spectrum of diseases. 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 death22. This is the third step of our cascade.

Mechanism of action of antimicrobial peptide and their effects on cells22. Antimicrobial peptides are making pore formation into the membrane leading to death of the cell. Transmission electron microscopy provide an insight of the effect of the peptide on the cell.
efficiency of the antimicrobial peptide from crocodile on V. cholerae18,20,21.The three peptides display and minimal inhibitory concentration 50 in the scale of mg/L.

Our system

See our Design page for more informations of the genetic engineering we used!

References

  1. Brogi S, Tafi A, Désaubry L & Nebigil CG (2014) Discovery of GPCR ligands for probing signal transduction pathways. Frontiers in Pharmacology
    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. http://www.emro.who.int/yem/yemeninfocus/situation-reports.html
  6. 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
  7. 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
  8. https://2014.igem.org/Team:UI-Indonesia
  9. https://2010.igem.org/Team:Sheffield
  10. https://2014.igem.org/Team:UT-Dallas
  11. 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
  12. 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
  13. https://2013.igem.org/Team:SCUT
  14. 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/
  15. Audet M & Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. Cell 151 14–2
    https://www.ncbi.nlm.nih.gov/pubmed/2302121
  16. 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
  17. 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/
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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