Design

Our project created from ground a synthetic consortium to demonstrate the power of this approach through its application to the cholera thematic. Our proof of concept consortium involves three microorganism: i) an engineered E. coli to mimic V. cholerae ii) an engineered V. harveyi to sense the presence of the engineered E. coli and produce diacetyl in response iii) an engineered yeast P. pastoris modified to detect diacetyl and to induce the production of antibacterial peptides (AMPs) in order to trigger lysis of Vibrio species. Here is presented a closer view of the molecular details of our project for each micro-organism, and our experimental plan.

Overview

Organisms

Escherichia coli

For safety reasons, the bacteria gram negative E. coli was chosen to mimic V. cholerae. Moreover E. coli is an easy organism to deal with, especially as it is well documented, easy to transform with exogenous DNA and easy to culture. The strain K-12 MG1655 was transformed with a plasmid allowing expression of the protein CqsA from V. cholerae, the enzyme responsible for the synthesis of CAI-1. However, as a proof of concept in our project, we also transformed our E. coli strain with the gene coding for the CqsA of V. harveyi, a non-pathogen strain, producing the molecule C8-CAI-1 (an analogue of the V. cholerae CAI-1)^1,2. This molecule is a carbohydrate chain based displaying an hydroxyl group on carbon 3 and ketone function on carbon 4. The CqsA synthetase from V. harveyi is able to produce C8-CAI-1 from endogenous E. coli (S)-adenosylmethionine (SAM) and octanoyl-coenzyme. Quorum sensing producing systems were placed under the pLac promoter and we used plasmid pSB1C3 to maintain compatibility with the iGEM registry.

V. harveyi

V. harveyi is a gram negative bacteria, well studied for its quorum sensing system. This bacteria displays its own pathway for the detection of C8-CAI-1. The gene cqsS encodes for the sensor C8-CAI-1 and a single point mutation in it sequence allows V. harveyi to detect both C8-CAI-1 and CAI-1 from V. cholerae. To avoid auto-activation of V. harveyi, we used the JMH626 strain, in which the cqsA gene, encoding the enzyme responsible for the production C8-CAI-1, has been deleted. Furthermore, additional genes luxS and luxS coding for key enzymes involved in the expression of other quorum sensing molecules have been deleted, making the strain JMH626 specific for detecting non-endogenous C8-CAI-1. V. harveyi is also able to regulate the activation of genes under the control of the promoter pQRR4, in a C8-CAI-1 concentration dependent manner ¹. At high C8-CAI-1 concentration, the promoter is inactivated. Thus we added the inverter tetR/pTet to activate a gene of interest in presence of C8-CAI-1. The gene of interest is als. This gene encodes for the acetolactate synthase (Als) involved in the synthesis of diacetyl from pyruvate³, our transmitter molecule (Figure 1).

The pBBR1MCS-44⁴ broad host range plasmid was chosen to allow the transfer of the system into V. harveyi by conjugation (i.e. this is the only way to modify the V. harveyi chassis).

In conclusion, we designed a V. harveyi strain enable to detect both exogenous CAI-1 or C8-CAI-1, and to produce diacetyl as a molecular response.

P. pastoris

V. harveyi cannot not be used as the effector since production of antimicrobial peptides (AMP) aimed at V. cholerae would be lethal also to its producer. P. pastoris is a yeast commonly used in academic laboratories and industry for high protein expression. The yeast was previously described to produce a wide range of antimicrobial peptides^5,6. Furthermore, the diacetyl-dependant Odr-10 receptor system has been described as a useful tool for prokaryotic/eukaryotic communication system⁷. This receptor has been reported as a gene expression activator in yeasts through the Ste12 pathway, leading to the activation of the promotor pFUS1 (Figure 2).

The constitutive pGAP promoter allows a continuous expression of the receptor Odr-10, making P. pastoris in theorie highly sensitive to diacetyl.

To kill V. cholerae, we searched for new and innovative antibiotic solution to limit the risk of acquired-resistance. We went for antimicrobial peptides (AMPs), that are small membrane disrupting molecules toxic for a large panel of microorganisms⁸. We selected for this project AMPs from crocodiles. Crocodiles live in harsh environment and are known to possess an impressive immune system, that allows them to catch very few disease and antimicrobial peptides are related to this immunity⁹. We chose to focus on 3 different AMPs described to have the best efficiency against V. cholerae. Those AMPs are Leucrocin I¹⁰, D-NY15¹¹ and cOT2¹². Leucrocine I possess one cationic charge for 7 amino acids. D-NY15 is its optimized counterpart with 4 cationic charges and a sequence of 15 amino-acid long. Finally, cOT2 is 29 amino acid long and possesses 6 cationic charges helping to diversify our pool of AMP. These AMPs will be placed under control of the pGAP constitutive promoter for preliminary tests, then under pFUS1 promoter to initiate the expression in response to diacetyl. The genetic constructions were built on the integrative pPICZα plasmid as a good plasmid for protein production. The signal peptide α-factor was fused to the AMPs to allow for secretion of the peptides.

The action of these AMPs is the last event of our synthetic consortium.

Experimental plan

E. coli

Quorum sensing molecule production

• C8-CAI-1 & CAI-1 NMR
• Bioluminescence

V. harveyi

Conjugation

• Conjugation test with RFP fluorescence
• CCqsS* pathway test with RFP fluorescence

diacetyl production

• diacetyl NMR (E. coli and V. harveyi)
• pTet characterization in V. harveyi by reporter gene

P. pastoris

diacetyl detection

• pGAP validation
• ODR10/pFus test with fluorescence

Antimicrobial peptides (AMP)

• AMP gene expression
• AMP activity: halo assay