NAU-iGEM

DESIGN Overview

MARS, the Malaria Averting with Re-engineered Serratia, is a strategy introduced to cut off the transmission route of malaria.
Considering the disadvantages of traditional anti-malarial methods, we take advantage of synthetic biology by highlighting three aspects in our gene circuit: TEV protease cleavage system, the combination of anti-plasmodium effector proteins and HasA secretion system.
In terms of chassis organism, we select Serratia marcescens, one of the symbiotic bacteria isolated from anopheline mosquitoes which has the ability to colonize in the midgut of mosquitoes^[1-2].

TEV Protease Cleavage System

Tobacco Etch Virus protease, also known as TEV protease or TEVp, is a site-specific protease found in Tobacco Etch Virus which can cleave fusion proteins to form functional viral components with consistent stoichiometry.[3] TEVp is able to cleave its substrate ENLYFQS between the Q and S residues with high specificity and efficiency, leaving an ENLYFQ-tail on the C-terminus of the protein encoded upstream and a serine residue on the N-terminus of the downstream-encoded protein.[4]

So far, no cases of cleavage at other sites have been reported, indicating that TEVp is an extremely specific enzyme, thus guaranteeing the orthogonality of our designed circuit.

Combination of Anti-Plasmodium Effector Proteins

Studies have shown that Plasmodium is likely to develop drug resistance due to the huge amount of mutations. In response to that, the combination of more than one effector protein is applied by us since it’s is quite difficult for one Plasmodium cell to obtain all these resistance features at the same time. Nine efficient anti-Plasmodium effector proteins[5-12] are involved and assembled as a fusion protein with a TEVp cleavage site between every two adjacent effector genes.

HasA Secretion System

As our chassis organism, S. marcescens has its secretion systems which can be potentially used by us. Among them, the Type 1 HasA secretion system is finally selected for its unique characteristics. The HasA secretion system consists of three transmembrane components, HasD, HasE and HasF that form a membrane pore so that the HasA C-terminal secretion signal domain can guide the fusion protein through the pore and out of the bacterial cell.[1,13] After being transported to the extracellular environment, the HasA won’t be cleaved, but is kept to the C-terminal of the protein.

MARs’ Gene Circuit

1.Fusion effector genes. Nine anti-Plasmodium effectors with various mechanisms of action are connected in series, and each two adjacent genes are linked with TEVp recognition sites.
2.TEV protease. TEVp can cleave the TEVp recognition sites inside the fusion effector proteins to form effector monomers, thus each monomer will come into effect separately.
3.HasA secretion signal. HasA is linked at the C-terminal of both fusion effector protein and TEVp. In this way, these two parts can be secreted to the extracellular environment and function .
4.Promoter combinations. Different expression ratios of TEVp and fusion protein will lead to different allocation situations of cell resources, thus affecting the effect of our MARS. If TEVp is excessive, the fusion protein can be completely cleaved but the number of effector monomers cannot reach its peak value. On the contrary, if the fusion protein is excessive, the cleavage can be incomplete, and the number of effector monomers cannot reach its peak value either. By testing different promoter combinations, we can find out the most efficient one, where the extracellular numbers of fusion effector protein and TEVp achieve a balance, so that no extra TEVp or fusion protein will remain as a waste.

Chassis with Ecological Advantages

S. marcescens is chosen as our chassis with its unique ecological advantages. Of those, the most important ones are shown below:
1.As a kind of symbiotic bacteria isolated from mosquitoes, the bacteria have an ability to colonize in the midgut of mosquitoes. It can also survive in the male accessory glands, female ovaries, hemolymph, and salivary glands.
2.With its enough viability within the sexual glands and their secrets, the strain can be transmitted both horizontally (sexually) and vertically (mother-to-child).
3.It does no harm to the host mosquitoes, to both aspects of viability and fertility.

Application (Simulation)

In consideration of biosafety and rules of iGEM, we are not planning to infect mosquitoes with our engineered S. marcescens and collect experimental data in the real world temporarily, but to simulate the effect of MARS based on the mathematic model. We established an improved cellular automaton model to explore the final implementation effect and suggested the optimal strategy for mosquito release in the maturity of the project. Based on the sensitivity analysis, a direction was given to further optimize the project design and the experimental focus.

References

[1] Sibao Wang,André L. A. Dos-Santos,Wei Huang,Kun Connie Liu,Mohammad Ali Oshaghi,Ge Wei,Peter Agre,Marcelo Jacobs-Lorena. Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria[J]. Science,2017,357(6358).

[2] Ganley Jack G,Carr Gavin,Ioerger Thomas R,Sacchettini James C,Clardy Jon,Derbyshire Emily R. Discovery of Antimicrobial Lipodepsipeptides Produced by a Serratia sp. within Mosquito Microbiomes.[J]. Chembiochem : a European journal of chemical biology,2018,19(15).

[3] Taxis C , Knop M . TIPI: TEV protease-mediated induction of protein instability[J]. Methods in molecular biology (Clifton, N.J.), 2012, 832(832):611-626.

[4] Yang Jianguo,Xie Xiaqing,Xiang Nan,Tian Zhe-Xian,Dixon Ray,Wang Yi-Ping. Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.[J]. Proceedings of the National Academy of Sciences of the United States of America,2018.

[5] Gao B, Rodriguez M D, Lanzmendoza H, et al. AdDLP, a bacterial defensin-like peptide, exhibits anti-Plasmodium activity[J]. Biochem Biophys Res Commun, 2009, 387(2):393-398.

[6] Possani L D, Zurita M, Delepierre M, et al. From noxiustoxin to Shiva-3, a peptide toxic to the sporogonic development of Plasmodium berghei.[J]. Toxicon, 1998, 36(11):1683-1692.

[7] Ghosh A K, Ribolla P E M, Jacobs-Lorena M. Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(23):13278-13281.

[8] Moreira C K, Rodrigues F G, Ghosh A, et al. Effect of the antimicrobial peptide gomesin against different life stages of Plasmodium spp[J]. Experimental Parasitology, 2007, 116(4):346-353.

[9] Tian C, Gao B, Rodriguez M C, et al. Gene expression, antiparasitic activity, and functional evolution of the drosomycin family.[J]. Molecular Immunology, 2008, 45

[10] Design and Activity of Antimicrobial Peptides against Sporogonic-Stage Parasites Causing Murine Malarias. Romanico B. G. Arrighi, Chikashi Nakamura, Jun Miyake, Hilary Hurd, J. Grant Burgess. Antimicrobial Agents and Chemotherapy Jul 2002, 46 (7) 2104-2110; DOI: 10.1128/AAC.46.7.2104-2110.2002 Vida3

[11] Thiostrepton and Derivatives Exhibit Antimalarial and Gametocytocidal Activity by Dually Targeting Parasite Proteasome and Apicoplast. Makoah N. Aminake, Sebastian Schoof, Ludmilla Sologub, Monika Leubner, Marc Kirschner, Hans-Dieter Arndt, Gabriele Pradel. DOI: 10.1128/AAC.01096-10

[12] Shunyi Zhu, Bin Gao, André Aumelas, Maria del Carmen Rodríguez, Humberto Lanz-Mendoza, Steve Peigneur, Elia Diego-Garcia, Marie-France Martin-Eauclaire, Jan Tytgat, Lourival D. Possani, MeuTXKβ1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1804, Issue 4, 2010, Pages 872-883, ISSN 1570-9639, https://doi.org/10.1016/j.bbapap.2009.12.017.

[13] Létoffé S, Ghigo J M, Wandersman C. Secretion of the Serratia marcescens HasA protein by an ABC transporter.[J]. Journal of Bacteriology, 1994, 176(17):5372-7.

Team:NAU-CHINA/Design

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