Leishmaniasis and Synthetic Biology
Before we started our circuit's design, we searched for efficient strategies to treat parasitic diseases. These strategies are a big challenge to leishmaniasis, considering that the generalist and diversified way of the parasite's infection demands robust treatment options. We were introduced to paratransgenesis in a meeting with specialists and advisors, learning it was a way to control the parasite's transmissibility with engineered bacteria into the vector population. This technique was explored by iGEM USP 2017 and motivated us to design our paratransgenic platform to fight leishmaniasis.
BioPank's engineering cycle expanded to different modules, each one of them designed to make the traditional paratransgenesis technique more robust, being ready to face the adversities of the real world. Considering all the problems that could arise in a real-world application, we traced strategies that led us to design four different genetic circuit modules and utilized different in silico and in vitro approaches to assess each one of them.
Modules Design
The circuits modularization favored a robust technique that would demand planning of "design, Model, Build and Test" for each module. This design framework allowed us to initiate four different engineering cycles to Protection, Detection, Overview, and Modeling. However, we completed the engineering cycle only for the Elimination Module because of our difficulty going to the lab due to the COVID-19 pandemic.
Engineering Success and Elimination Module
The Elimination Module is responsible for acting against Leishmania inside the vector's midgut. Its primary function is to produce an effector molecule, the antimicrobial peptide (AMP) DRS-N1, in its deactivated form (proAMP), linked to an acidic pro-peptide, which will be separated at the moment of enzymatic digestion in the midgut. This mechanism was assessed in the lab by the expression of proAMP, trypsinization assay, and bioassay against Leishmania.
DESIGN
The in vitro production of recombinant antimicrobial peptides in bacteria is a challenge. In a specific concentration, these peptides can considerably harm the chassis growth and derail its production [1]. Now imagine an engineered bacteria in the sandfly's midgut to produce enough AMPs to kill any parasitic cell. Probably, high quantities would diminish the vector's persistency and affect the bacteria's efficiency action. This context could put at risk the paratransgenesis technique, and that's why we have a strategy to avoid the situation.
In a talk with specialists, we realized that it was possible to produce these AMPs in a deactivated form, connected to pro-peptides that neutralize the cationic charge [2], and to induce their activation through enzymatic digestion that will split them up. We chose trypsin, an already highly present enzyme in the Luztomyia longipalpis midgut that rises its production after the blood meal.
The final Elimination Module design has the leishmanicidal activity AMPs and its related pro-peptides, according to the table:
The circuit control is done by the Detection Module and for the implementation in Bacillus subtilis.
Nonetheless, the leishmanicidal AMPs have trypsin cleavage sites, harming any AMP strategy. Our team discussed this aspect with specialists who pointed out that the pro-peptides provide protection against digestion and raise the stability of the AMP. This corroborates with the idea of activating our system through limited ingestion, a natural event in the production of AMPs in different organisms [3].
Therefore, we decided to assess the proAMP activation through trypsinization and its return to a leishmanicidal activity through a cytotoxic assay.
MODEL
From our circuit's design, we assessed the paratransgenesis system operation through a kinetic model. We saw that one of the main factors that could influence our system efficiency is the AMP cooperativity. This factor showed us the importance of testing the elimination module to assess the kinetic action characteristics of AMPs.
TEST
Despite our original chassis being Bacillus subtilis, we decided to express the proAMPs in E. coli BL21(DE3) due to available time and lab resources. This fact did not disturb our goals since the strategy was to assess the AMP activation and deactivation. The selected AMPs had pro-peptides inducing their neutralization and the OmpA [4] signal peptide, with the function of secreting to the extracellular region or allocating the peptide in the periplasm. We used natural pro-peptides for the dermaseptins (DRSs) and a synthetic pro-peptide model for the CAM-W. As the promoter, we chose PT7_LacO (BBa K2406020) that activates with IPTG presence.
The cloning of our circuit containing the proDRS-N1 (BBa_K4075008) and the circuit with the reporter gene was made in NEB® 5-alpha Competent E. coli. In E. coli BL21(DE3), we succeeded only in the cloning of the proDRS-N1 construct.
With the assembly and cloning done, we obtained the proAMP, leading us to the final steps of our experiments. Those consisted of the enzymatic treatment with trypsin and leishmanicidal assay with the previous treatment product. We had three goals to demonstrate: 1) our effector molecule activation capacity in the presence of trypsin; 2) the relation between trypsin concentration and activation through partial digestion; and 3) the activated AMP leishmanicidal activity.
References
JI, S. et al. Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700. Nature Scientific Reports, 7(40587), 2017. https://www.nature.com/articles/srep40587
JUNIOR, N. G. O. et al. An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of crotalicidin fragment. Nature Scientific Reports, 8(11127), 2018. https://www.nature.com/articles/s41598-018-29444-0
Mahlapuu, M. et al. Antimicrobial Peptides: An Emerging Category of Therapeutic Agents. Frontiers in Cellular and Infection Microbiology. 6:194, 2016. https://www.frontiersin.org/articles/10.3389/fcimb.2016.00194/full
Pechsrichuang, P. et al. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus, 5:1200, 2016. https://springerplus.springeropen.com/articles/10.1186/s40064-016-2893-y