Team:INSA-UPS France/Description


Croc'n Cholera
Description

Cholera: still a widespread disease

Cholera is a worldwide diarrheal disease, caused by the ingestion of Vibrio cholerae in contaminated water. Nowadays cholera is still occurring in developing countries, war zones and natural disasters zones. The WHO reported over 1 million cases over the year 2015 and the mortality was around 1%1. In April 2017, a cholera epidemic burst in Yemen. In August, more than 500,000 cases have already been identified. These epidemics and crisis are sometimes still surpassing the abilities of the non-governmental organizations to help populations2 in the long-term. Drinking water shortages and the lack of hygienic facilities in developing countries are the main reasons explaining current outbreaks.

Current solutions

Therapeutics

While direct preventive methods such as vaccination are currently used, they have been shown to have low efficiency3. The most used treatment is the Oral Rehydratation Solution (ORS), composed of salts and glucose in order to fight the extreme loss of water due to cholera. It can be drunk or injected intravenously depending of the patient and his symptoms4.

Water treatment

Moreover, most of the detection or purification methods need professionals to be performed3. Currently the most efficient ways to eradicate cholera from water include sodium hypochlorite treatment or filters use. These existing prevention methods are expensive and difficult to set up. Finally, the main curative method, rehydrating patient with intravenous salted water, does not wipe out the disease vector. Even if the ORS treatment is really efficient, it would be more convenient for people to use prevention methods. However people living in remote villages don’t have easily access to these systems and it can take them days to reach a camp to be cured. Thus, new methods of prevention and treatment have to be developed. That’s why we decided to design a system that treats water and is suitable for these situations.

Research effort in synthetic biology

Recently, research teams started to focus on synthetic biology in order to find a way to deal with Vibrio cholerae5,6. Additionally, some iGEM teams also took the challenge of detecting V. cholerae, using E. coli as a host for the quorum sensing system, but it seems that this strategy did not succeed7 or showed mixed results8. Most of the iGEM teams intended to prevent the cholera infection thanks to gene targeting such as cleaving toxicity genes9 or inhibiting specific genes on the pathogen10.

The crocodile, an unexpected helper

Non-salty water is the natural habitat of V. cholerae21. Fortunatelty, Sobek, the Crocodile God of water and fertility which inspired us for our mascot Sobki, also likes those kind of environment. Indeed, crocodiles have an impressive immune system, that allows them to resist against lots of diseases vectors infections22,23,24. We took from their amazing immune system promising molecules: antimicrobial peptides (AMP) that have proven to have a good killing efficiency against V. cholerae13,14.

Our system : Croc'n Cholera

In contrast to current prevention systems, the iGEM INSA-UPS team of Toulouse would like to create a low-cost and easy-to-use device that could be able to both detect and destroy cholera to treat water. Our system implies interactions between three microorganisms:

1. Sense

Our first goal was to detect V. cholerae in water. To do so, we used its quorum sensing property: at low concentration, V. cholerae CqsS/CAI-1 pathway isn’t activated. AlphA is thus activated and HapR is inactivated (responsible for the bacterium virulence) thanks to a phosphorylation cascade. At high concentration, HapR makes V. cholerae virulent15.

Low CAI-1 concentration High CAI-1 concentration

For safety reasons, we were not able to manipulate V. cholerae in our lab. This is why we had to engineer E. coli to mimick V. cholerae in order to further test our system. The challenge was to make E. coli produce the specific quorum sensing molecules of V. cholerae: CAI-1.

We then used this quorum sensing mechanism in Vibrio harveyi, a non-pathogenic strain of Vibrio that is close enough from V. cholerae, to detect cholera in water. We chose this particular strain because it is easy to engineer its receptor to detect and respond to cholera, already having the capacity to detect such quorum sensing molecules.

2. Communicate

As we wanted to both detect and destroy cholera, one of our challenges was to create an eukaryote-prokaryote communication system, so that V. harveyi could trigger the production of antimicrobial peptides (AMPs) by P. pastoris upon cholera detection.

Team SCUT already described a binding-receptor system built on Saccharomyces cerevisiae18, based on the ODR10 receptor, a G Protein Coupled Receptor isolated from Caenorhabditis elegans19. The pathway engaged by the activation of ODR10 has been engineered in order to start the mating cascade which ends with the activation of the pFUS1 promoter by Ste1220.

As this mechanism relies on diacetyl detection for pFUS1 activation, we needed our sensing module to produce this molecule. Diacetyl is not produced by wild type Vibrio harveyi (Kegg pathway), but online tools predict that the missing enzyme to catalyse the production of this molecule is the acetolactate synthase (ALS). Indeed, this enzyme catalyses acetolactate production, which is then oxidized into diacetyl. Thus we decided to add the gene responsible for ALS production in the genetic construction we put in V. harveyi.

3. Treat

Instead of using gene targeting, we decided to eradicate the pathogen bacteria thanks to crocodile antimicrobial peptides (AMPs)13,14. Those peptides are cationic molecules and can target bacterium membranes, to create pores in it, leading to the lysis of the cells24.

Since these peptides have a high killing efficiency on V. cholerae it was obvious that our sensor, which is another species of Vibrio, could not stand any close contact with AMPs and would be killed by them. That’s why we needed to find another organism to produce it. We thus used a robust yeast as our effector, Pichia pastoris16,17 which is also famous for being a high protein producer.

Minimal inhibitory concentration 50 of selected antimicrobial peptides against Vibrio cholerae13,14,22.

References

  1. Ali M, Nelson AR, Lopez AL & Sack DA (2015) Updated Global Burden of Cholera in Endemic Countries. PLOS Neglected Tropical Diseases 9 e0003832
  2. WHO 23 SEPTEMBER 2016, 91thYEAR / No 38, 2016, 91, 433–440
  3. Clemens JD, Nair GB, Ahmed T, Qadri F & Holmgren J (2017) Cholera. The Lancet
  4. Davies HG, Bowman C & Luby SP (2017) Cholera - management and prevention. Journal of Infection
  5. Focareta A, Paton JC, Morona R, Cook J & Paton AW (2006) A Recombinant Probiotic for Treatment and Prevention of Cholera. Gastroenterology 130 1688–1695
  6. Holowko MB, Wang H, Jayaraman P & Poh CL (2016) Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli. ACS Synthetic Biology
  7. iGEM UI-Indonesia 2014
  8. iGEM Sheffield 2010
  9. iGEM UT-Dallas 2014
  10. iGEM Dundee 2016
  11. 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
  12. 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 6906–6918
  13. 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
  14. 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
  15. Ng W-L, Perez L, Cong J, Semmelhack MF & Bassler BL (2012) Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in Vibrios. PLoS Pathogens 8 e1002767
  16. Huang Y (2012) Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris. Experimental and Therapeutic Medicine
  17. Zhang Y, Teng D, Mao R, Wang X, Xi D, Hu X & Wang J (2014) High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus. Applied Microbiology and Biotechnology 98 681–694
  18. iGEM SCUT 2013
  19. 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
  20. Audet M & Bouvier M (2012) Restructuring G-Protein- Coupled Receptor Activation. Cell 151 14–23
  21. Robert Koch and the cholera vibrio: a centenary. N Howard-Jones, British Medical Journal (Clinical Research Edition)
  22. 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
  23. 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
  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