Elimination

For our project, we decided to focus our efforts on expressing and characterizing four effector proteins. Two against malaria, and two others against Chagas' disease and Dengue, as these diseases of global importance are specially significant to Brazil. The effectors we chose to work with were based on previous literature characterizon, with promising results for disease control:

Scorpine (Malaria)(4)
EPIP₄ (Malaria)(4)
Cecropin A (Chagas)(1, 2)
DN59 (Dengue)(3)

Along with the effectors, we decided to include in our synthesized sequences a 6xHis tag and a HlyA signal peptide, so that we could make our bacteria secrete them, and that we could easily purify it

The four effector parts were cloned and sent to the registry, but to try to express and secrete one of our effectors, we needed to clone their sequence together with a RBS, a promoter and genes for assembling the rest needed for the T1SS (HlyB and HlyD). We focused on cloning this elements with Scorpine, as it showed to be the most efficient effector of the two against Plasmodium. We checked the length of pSB1C3 with our parts and sequenced them, which showed that they were as we expected:

Agarose gel results: all at the right size

A little bit more into the choice to work with each peptide:

Scorpine, our pride and joy

Our candidate for an antimicrobial peptide was scorpine, derived from Scorpion venom. Why? Well, first because it’s a really strong antimicrobial. And we mean REALLY strong. Secondly, this type of protein can be used against a wide variety of pathogenic agents, since its natural function is exactly to protect the scorpion from a wide variety of pathogens. And in fact, it has been reported to work against Plasmodium falciparum, the malaria parasite, which makes it perfect for our project!

So we decided to try and express it, and we got it!

So we now knew that we could express Scorpine in E. coli, but what about secreting it? For the paratransgenesis system to work and effectively block malaria transmission, we needed to get the produced Scorpine out of our chassis and onto the parasite:

Cecropin A, from insects to (the inside of) insects

This antimicrobial peptide,initially isolated Hyalophora cecropia interects with cell membranes, depolarizing and permeabilizating them. It showed promising activity when used against Trypanosoma cruzi, in a paratransgenic system where the bacterium Rhodococcus rodnii was used. It also shows activity against a range of bacteria, so if you want to use this you will need to choose well your chassis, but then you will be able to target many diseases!

EPIP4, a synthetic solution for malaria

Another way of targeting malaria was with the "Enolase-plasminogen interaction peptide" (EPIP), as it binds surface proteins of Plasmodium and inhibits its penetration in the mosquito midgut's cell walls. The peptide we tried to use was a fusion of 4 variations of EPIP in the same peptide, which ends up exhibiting great effectiveness stopping malaria's life cicle in the mosquitoes.

DN59, an effector against an endemic problem

This is a peptide that mimics a membrane-interacting region of a dengue virus envelope protein. It inhibits all of the Dengue virus serotypes and other flaviviruses. Although strongly interacting with viral and synthetic membranes, disrupting them and releasing its contents (in the case of viruses, its genome), it shows no such effect in mammalian or insect membranes, so its a powerful and specific way of fighting these viruses.

References

Bulet P, Hetru C, Dimarcq JL, Hoffmann D. "Antimicrobial peptides in insects; structure and function." Developmental and Comparative Immunology. 1999; 23:329–344.
Fieck, Annabeth et al. “Trypanosoma Cruzi: Synergistic Cytotoxicity of Multiple Amphipathic Anti-Microbial Peptides to T. Cruzi and Potential Bacterial Hosts.” Experimental parasitology 125.4 (2010): 342–347. PMC. Web. 30 Oct. 2017.
Lok, Shee-Mei et al. “Release of Dengue Virus Genome Induced by a Peptide Inhibitor.” Ed. Young-Min Lee. PLoS ONE 7.11 (2012): e50995. PMC. Web. 30 Oct. 2017.
Wang, Sibao, et al. "Fighting malaria with engineered symbiotic bacteria from vector mosquitoes." Proceedings of the National Academy of Sciences 109.31 (2012): 12734-12739

[1] Bulet P, Hetru C, Dimarcq JL, Hoffmann D. "Antimicrobial peptides in insects; structure and function." Developmental and Comparative Immunology. 1999; 23:329–344.

[2] Fieck, Annabeth et al. “Trypanosoma Cruzi: Synergistic Cytotoxicity of Multiple Amphipathic Anti-Microbial Peptides to T. Cruzi and Potential Bacterial Hosts.” Experimental parasitology 125.4 (2010): 342–347. PMC. Web. 30 Oct. 2017.

[3] Lok, Shee-Mei et al. “Release of Dengue Virus Genome Induced by a Peptide Inhibitor.” Ed. Young-Min Lee. PLoS ONE 7.11 (2012): e50995. PMC. Web. 30 Oct. 2017.

[4] Wang, Sibao, et al. "Fighting malaria with engineered symbiotic bacteria from vector mosquitoes." Proceedings of the National Academy of Sciences 109.31 (2012): 12734-12739