Overview

In order to address the challenges faced with our project idea - protection of the antigen through the mucosal route and ensuring our antigen is able to illicit the humoral immune response once it reaches its target, we developed the following solutions and got to work on designing the gene circuits.

Protection of antigen: Oral fed vaccines must overcome a variety of potential damaging factors along the digestive tract of fish. This was a concern for us as the antigen we want to express could all be damaged after eating. Our solution was to design an inducible promoter that would only express our antigen once it reached the intestines of fish. We explored the well documented Quorum Sensing (QS) system as it known in synthetic biology as a well-functioning population dependent gene circuit. We also then wanted to further control this system by adding an environment dependent parameter which would allow for the suppression of the QS system until it received an environmental stimulus similar to what would be experienced in vivo. For this we found that a lack of free iron is a signal we could use to incorporate into our design as many bacteria have evolved iron uptake mechanisms and promoters containing iron binding sequences to regulate genes and survive in such conditions. We called this system the Iron-QS system.

Display of antigen to illicit the immune response: It was discovered through literature exploration that gram positive bacterial strains such as Lactic Acid Bacteria are well suited for being recombinant bacterial vaccine vectors because they contain cell walls in which proteins can be anchored to. They are recognized as safe by the World Health Organization and some strains have probiotic properties meaning they have previously been studied for the delivery of antigens or other active molecules to mucosal tissue. We explored the options available to displaying a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall by a covalent link using sortase mediated anchoring via a LPXTG motif. The type of anchor used affects the stability of the protein while the type of signal peptide used can have great influence on the transport of the target protein to the cell surface. We therefore needed to consider these factors when choosing the optimal sequences to incorporate into our final designs. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli so we further explored what bacteria to use based on availability and the information available to us about anchors and signal peptides. We decided upon using Lactobacillus plantarum based primarily on its success at display surface antigens for inducing immune responses in previous studies on mice (see references), and also on the fact that many signal peptides have previously been characterized.

Other project desgin features: Later in our project design we explored the possibility of using bioencapsulation of our bacterial vaccine vectors to further protect them through the gastrointestinal tract of fish. We discussed this possibility with some companies as part of our integrated human practice work. However, in time for this year’s jamboree we needed to put this aspect of our project on hold until later in our academic year. We additionally explored the area of diagnosing when a fish has become infected with a pathogen. We researched the area of the immune response and biomarkers associated with this. We found that many species release Reactive Oxygen Species (ROS) in response to infection and also learned that ROS inducible genes could be used as reporters of infection in tissue. We further explored this idea by using modeling to attempt to improve upon biobrick parts which report oxidative stress in animal tissue. More information on this design process is found in modeling and our parts collection.

Click on the buttons below to learn more about each design.

Iron-QS System:

The aim here was for the expression and synthesis of our targeted antigen to be controlled by QS and iron starvation which mimics the in vivo conditions of the koi’s second gut. This system was designed with the intention to be used in gram negative bacteria such as E.coli or Edwardsiella tarda which has been used successfully as bacterial vaccine delivery strains previously.

To protect the antigen from the early break down in the stomach and for its higher vaccine efficacy, we want to design the antigen to be expressed in the highly tolerogenic gut environment of the Koi Fish. Quorum sensing allows bacteria to control gene expression according to their population density. In natural gram negative bacteria, their quorum sensing circuits contain homologues of two Vibrio fischeri, a bioluminescent marine bacterium, regulatory proteins called LuxI (BBa_C0061) and LuxR (BBa_C0062). LuxI is the autoinducer synthase that produce freely diffusible N-acylhomoserine lactone (AHL) molecules, which is consisting of a homoserine lactone (HSL) ring and an acyl chain that vary in length and degree of saturation. When the AHL reach a threshold concentration, which increases alongside the cell population density, two molecules of LuxR bind to two AHL autoinducers and form a complex, the transcription factor of Plux promoter (BBa_R0062). LuxR protein degrade rapidly if binding does not take place. AHL help the LuxR protein to stabilize and fold to activate the transcription of the target gene.

To ensure that the quorum sensing system would be only activated in vivo in iron starved conditions, we inserted a 19 bp sequence standard Ferric Uptake Regulator sequence (FUR box) into the -10 region. The position of the FUR box was inspired by our collaborators from last year ECUST who performed experiments on a range of newly designed promoters which incorporated the FUR box into them. In the classical FUR repression mechanism, iron-bound Fur binds to a Fur box sequence that overlaps with, or is proximal to, promoters of ironresponsive genes, thus preventing their transcription. When intracellular iron is depleted, Fe2+ is released from Fur, causing conformational changes in the protein resulting in dissociation from the Fur box.

In a low iron environment, through the regulation of our LuxI-Fur box promoter (BBa_K3031015), the LuxI protein would be produced, AHL. Then, AHL will bind and activate the LuxR protein, which is constantly produced by a constitutive promoter (BBa_J23101), and in this way turn on the last pLux promoter to express the antigen protein-ORF81. For our primary lab, we replace antigen with reporter gene GFP for certification of Iron QS system.Therefore, we design the piece of DNA contains GFP gene which is used as a reporter of expression with an appropriate promoter. Ideally, we may find active green fluorescent protein (GFP) under low iron condition and high cell density in the gene carrier E.coli.

Reference:

Chu, Teng, Ni, Chunshan, Zhang, Lingzhi, Wang, Qiyao, Xiao, Jingfan, Zhang, Yuanxing, & Liu, Qin. (2015). A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine. Microbial Cell Factories,14(1), 37.

Cui, L., Guan, X., Liu, Z., Tian, C., & Xu, Y. (2015). Recombinant lactobacillus expressing G protein of spring viremia of carp virus (SVCV) combined with ORF81 protein of koi herpesvirus (KHV): A promising way to induce protective immunity against SVCV and KHV infection in cyprinid fish via oral vaccination. Vaccine,33(27), 3092-3099.

Rai, N., Anand, R., Ramkumar, K., Sreenivasan, V., Dabholkar, S., Venkatesh, K., . . . Buchler, N. (2012). Prediction by Promoter Logic in Bacterial Quorum Sensing (Prediction by Promoter Logic in Quorum Sensing). PLoS Computational Biology,8(1), E1002361.

Cell wall anchor system:

We first identified a membrane protein of the Cyprinid herpes virus-3 that has shown to illicit an immune response in koi to the virus. This protein was chosen as our antigen because it was harmless to use by itself and has been reported to have a positive effect on immunity when administered through injection into fish in combination with another protein for spring viremia carp virus (SCCV) (Cui et al, 2015). This protein is called ORF81, is a major envelope protein of 256 amino acids. Its sequence was obtained from NCBI.

To tackle the problem associated with efficiency of cell wall anchoring and transport to the cell boundary associated with cell wall anchoring systems for surface displaying recombinant proteins, we designed several composite parts (BBa_K3031007, BBa_K3031008, BBa_K3031009, BBa_K30310010). These parts consist of a promoter, signal peptide, the ORF81 antigen, and a cell wall anchoring protein. These parts were designed to take advantage of the system whereby proteins are covalently attached to the cell wall by fusing the target antigen to an anchoring protein containing a LPXTG sequence motif and a hydrophobic amino acid tail. Once these fused proteins are shuttled to the cell periphery a transpeptidase called sortase cleaves between T and G of the motif and the protein is then bound to the peptidoglycan cell wall. Each of these composite parts contain the ORF81 antigen fused to a truncated version of a cell wall anchoring protein called Lp_2578 from L.plantarum. The difference between the four is that they contain different signaling peptides (BBa_K3031003, BBa_K3031004, BBa_K3031005, BBa_K3031006). These signal peptides are all native to the bacteria and were obtained from the genome of L. plantarum WCFS1. They have previously been shown function well at secreting heterologous proteins in L. plantarum by Fredriksen et al., (2010).

The composite parts differing in the signaling peptide were intended to be used to investigate signaling peptide efficiency in the future experiments. We also planned on comparing our new basic parts (the signal peptides) with the signal peptide Usp45 (BBa_K180002) which is a commonly used signal peptide for this purpose. In the design, we also made variations on the types of anchoring protein by replacing the truncated cell wall anchoring protein from Lactobacillus plantarum (BBa_K3031002) with cell wall anchoring protein from Streptococcus pyogenes (M6 Anchor) which is a well characterized biobrick part. We predicted that our new part would perform better for Lactobacillus plantarum as it is a native protein. We hoped to be able to provide in the registry another well characterized system for surface display of proteins or active molecules in addition to the M6 system. In the later experiment, the parts (BBa_K3031011, BBa_K3031012, BBa_K30310133, BBa_K3031014) will be compared to investigate the effect of our new signaling peptides with this common M6 anchoring protein adding new characterization to our new parts and these commonly used ones for surface display of antigens.

Our final design considerations for our composite part was to first include a 6 amino acid linker sequence consisting of a GGGGGS portion. This was to allow flexibility and reduce steric hinderance between the antigen and anchoring protein. We added a constitutive promoter to our original design as the iron QS system designed for gram negative strains may not work in lactic acid bacteria. Other inducible promoters would later be tested to ensure our design could be used in vivo.

References:

Jerry Eichler, and William Wickner. “Both an N-Terminal 65-KDa Domain and a C-Terminal 30-KDa Domain of SecA Cycle into the Membrane at SecYEG during Translocation.” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 11, 1997, pp. 5574–5581.

Fredriksen, Lasse, et al. “Cell Wall Anchoring of the 37-Kilodalton Oncofetal Antigen by Lactobacillus Plantarum for Mucosal Cancer Vaccine Delivery.” Applied and Environmental Microbiology, vol. 76, no. 21, 2010, pp. 7359–7362.

Marraffini, Luciano A, et al. “Anchoring of Surface Proteins to the Cell Wall of Staphylococcus Aureus. A Conserved Arginine Residue Is Required for Efficient Catalysis of Sortase A.” The Journal of Biological Chemistry, vol. 279, no. 36, 2004, pp. 37763–37770.

Michon, C., et al. “Display of Recombinant Proteins at the Surface of Lactic Acid Bacteria: Strategies and Applications.” Microbial Cell Factories, vol. 15, no. 73, 2016, p. 70.

Mobergslien, Anne, et al. “Recombinant Lactobacillus Plantarum Induces Immune Responses to Cancer Testis Antigen NY-ESO-1 and Maturation of Dendritic Cells.” Human Vaccines & Immunotherapeutics, vol. 11, no. 11, 2015, pp. 2664–2673.

Schneewind, Olaf, and Dominique Missiakas. “Sec-Secretion and Sortase-Mediated Anchoring of Proteins in Gram-Positive Bacteria.” BBA - Molecular Cell Research, vol. 1843, no. 8, 2014, pp. 1687–1697.

Schneewind, Olaf, et al. “Structure of the Cell Wall Anchor of Surface Proteins in Staphylococcus Aureus.” Science, vol. 268, no. 5207, 1995, pp. 103–106.