Chassis

In paratransgenesis, the chassis of choice needs to be very well thought, as the efficiency of the strategy is directly related to the survival rate of bacteria and the production rate of the effector molecule.

The ideal for paratransgenesis is to use symbiotic microorganisms of vectors as chassis, as these organisms are more likely to stay longer within the vector. However, Lutzomyia longipalpis (visceral leishmaniasis vector) does not have any symbionts, so this search is extended to commensal organisms.

With this in mind, we did extensive bibliographical research on papers that identified sandfly microbiota and papers that indicated possible model bacteria for paratransgenesis application.

As a result, we obtained three candidates: Enterobacter cloacae dissolvens, Pantoea agglomerans and Bacillus subtilis. The advantages and disadvantages of these chassis are as follows:

E. cloacae dissolvens

Advantages

• Most abundant bacteria in the intestine of some insects, including L. longipalpis

• Was used in other paratransgenesis researches

Disadvantages

• In a study, P. papatasi was not attracted to food containing E. cloacae. This may have been caused by the odor of the bacteria.

• Some other strains are pathogenic.

• hard to get a strain

• Poorly characterized

Pantoea agglomerans

Advantages

• Used by the USP-Brazil 2017 team (BioTROJAN) and other researches with paratransgenesis

• Similar to E. coli

• Naturally found in L. longipalpis

• We could get the strain easily

Disadvantages

• Gram-negative - Less resistant to our AMP

• Poorly characterized

• Does not sporulate

Bacillus subtilis

Advantages

• GRAS (Generally recognized as safe)

• We would get the strain easily

• Was already used paratransgenesis researches

• Gram-positive - More resistant to our AMP

• Well Characterized Genetic Parts

• Great protein producer

• Sporulation may be advantageous for field application.

Disadvantages

• It is not naturally found in L. longipalpis, but studies show that it can survive in the midgut.

• Not similar to E. coli

B. subtilis we choose you!

Comparing the advantages and disadvantages of our candidates, B. subtilis was an easy choice to make. It's amenable to genetic modifications, relatively easy to work, and its sporulation trait could protect our circuit in unstable environments.

Protection Module

In the implementation section, we discussed the contribution of sugar baits to the application of our project. These structures can be distributed in hotspots and carry out vector control normally through toxin ingestion[1]. Here, sugar baits are implemented differently, where we only use a sugar solution containing a concentration of our chassis without any toxin. Nonetheless, it's necessary to consider that these sugar baits are subjected to climatic variations and are not amenable to maintaining stable conditions for microorganisms' cultivation. This variation can affect the chassis viability and the entire system's efficiency, as it needs to be viable for leishmanicidal production when within the vector.

However, our chassis can differentiate between the vegetative to the sporulated form. This sporulation mechanism provides resistance to stresses generated in the environment (i.e., nutrient-limitation, extreme conditions, etc.), allowing them to withstand the different stresses events[2]. To optimize the chassis delivery to the vector population and to take advantage of the differentiation capacity of B. subtilis, we introduced the Protection Module.

Design

The Protection Module idealization came from a meeting with our advisor Tiago Lubiana. We discussed the instability of conditions in sugar baits and the costs to maintain the cultivation of B. subtilis. Then, we saw an advantage in using this chassis' differentiation mechanism, where we could produce the sporulated form to fill the sugar baits and engineer a module to induce germination upon entry into the vector.

The iGEM SZU 2017 Team described the PsspB-gerA part BBa_K2232020 to induce germination in B. subtilis, and we used it as the basis for this Module. The main component is the gerA receptor, an endogenous Bacillus subtilis receptor located on spores coat, which recognizes nutrients in the medium and triggers germination[3]. L-alanine is the main nutrient that triggers differentiation from binding to gerA and probably an abundant amino acid in the midgut of the vector (or any other organism). The PsspB promoter overexpresses this receptor during the forespore stage formation and with that, we can produce the chassis in its sporulated form and store it in sugar baits, waiting for the sand fly's digestion.

Indeed, L-alanine is the main nutrient that triggers differentiation from binding to gerA and abundant amino acid in the midgut of the vector (or any other organism). It is well known that the spores of B. subtilis can support enzymes and gastric fluids in humans, remaining viable and germinating in the intestine[4][5]. This suggests that an analogous process occurs in the sandflies, as this circuit will guarantee these sporulation-germination cycles.

Figure 1: Germination Measurement Device

We designed a Germination Measurement Device BBa_K4075555 to assess sporulation and the germination process, which has potential for Paratransgenesis assays in sandflies or mosquitos with Bacillus subtilis. The system is composed of the PsspB and gerA promoter with a GFP reporter expressed downstream and a mCherry reporter expressed by the constitutive promoter Pveg. This promoter has activity only in the vegetative phase, that is, when the spores underwent differentiation within the vector.

Detection

Overview

Synthetic biology as a platform for developing paratransgenesis technologies can promote an efficient application in the control of vector-borne diseases and, especially, guarantee a safe application. Safety is the main challenge of paratransgenesis, as releasing engineered organisms into the environment can be harmful. Our first biocontainment against this event is a killswitch mechanism (see Biocontainment Module), and the second one is this module that identifies the vector's midgut.

The Detection Module was designed to be specific to Lutzomyia longipalpis, the vector target. Although it is challenging to find biomarkers in this insect, through the literature, we have identified putative conserved miRNAs present in the midgut (referred here as miRNA-Lulo). Then, we built a library of de-novo-designed Toehold RNA switches specifically for miRNA-Lulo using our (slow but efficient) Toehold Switch Generator, the Carpincho Tool.

How we design it?

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The Toeholds design for the recognition of miRNA represents a good alternative for the detection of Leishmania infantum within the sandfly; however, several factors need to be evaluated. One of them is the ability of our Bacillus subtilis chassis to internalize the extracellular RNA to the intracellular medium to be recognized and activate the Riboswitch. This was mentioned by the Stanford team, in which a mechanism for the import of DNA is cited, it is suggested that B. subtilis has a natural competence to internalize extracellular DNA sequences from the environment.

However, in a meeting with Professor Danielle Pedrolli, we perceived that the importation of RNA can follow different mechanisms. There are studies on the interactions between the microbiome and the host through miRNA, acting as molecular regulators and gene expression, the host exerts signaling mechanisms through the release of miRNA in extracellular vesicles or those associated with high-density lipoproteins or argonaut protein, which are taken up by microorganisms in the environment[8][9]. These interactions require different mechanisms of internalization of extracellular RNA within Bacillus subtilis that involve transcytosis processes, such as clathrin-dependent endocytosis, caveolin or other independent mechanisms.[10][11]. More tests are needed to verify that the competition of Bacillus subtilis allows the internalization of sufficient quantities of miRNAs for the detection and activation of our system.

Design

Figure 2: Elimination Module constitutively expresses proAMPs upon activation of the Detection Module. The digestion of AMPs causes the separation of propeptide and AMP through cleavage by trypsin, enzymes that increase their activity after the vector's blood meal.

The Ellimination Module was the first part of the system designed this season. We had several meetings with our advisor, specialist in paratransgenesis, Ph.D Marcelo Ortigao. In these meetings, Prof. Marcelo suggested that AMP can be produced in its inactivated form (proAMP) and activated at a specific moment of interest. The chassis could have a killing response to the parasite without harming itself or even sandfly.

Following the suggestion of Prof. Marcelo, who described how the change of digestive enzymes occurs in the midgut post blood meal, we used the increase of trypsin to activate the proAMP [11]. In our system, the proAMP becomes activated (AMP) seconds after the blood meal, ensuring that if there is a parasite in the blood, it does not develop in the vector after the bite of an infected animal/human (Figure 1). Thus, the mechanism quickly reached toxic quantities, considering that proAMP, the inactivated form, will have a faster enzymatic activation than instantaneous transcription/translation activation; and modular, as we can use the regulation of digestive enzymes to define the moment of activation.

The Elimination Module is linked to the Detection Module constitutively expressed by the promoter Pveg. The chassis recognizes that it is inside the vector's midgut with the Detection Module with Toehold RNA switch and starts the proAMP translation (Click here for more information). This inactivated form of AMP enables its production without harming chassis growth and promotes sufficient levels to kill the parasite efficiently and quickly after trypsin cleavage.

The proAMP consists of three components: the acidic propeptide, a sequence of peptides used to neutralize the AMP charge and reduce toxicity; a trypsin cleavage site, a sequence located between acidic propeptide and AMP; and the AMP itself, a sequence of peptides release after trypsin digestion (Figure 2). All proAMPs contain an upstream signal peptide that guides the secretion pathway. These assembled parts form our main component in the elimination mechanism as a fast and modular response against the parasite.

Figure 3: proAMP and AMP representation.

In a meeting with AMP expert Dr. Guilherme Brand, we found that limited digestion is a common reaction for obtaining AMPs in nature and in fact, it would have a similar behavior in the midgut of the vector with trypsin digestion. This is because the propeptide aggregates the stability of AMP, making enzymatic digestion difficult. Therefore, we decided to evaluate this mechanism in our laboratory experiments, check our Results section.

Biocontainment Module

Overview

Programming a system to act only within the vector (i.e. mosquitos, sandflies, etc) is the main challenge to control undesirable GMOs spreading in paratransgenesis strategies even though it has few strategies to solve it. Kill switches (KS) are programmed death circuits, which prevent engineered bacteria from escaping into the environment. This Module can enhance the biocontainment of paratransgenesis strategies. Therefore, based on UNESP’s 2018 team idea, our team designed a photoactivatable CRISPR-Cpf1 to prevent our chassis from remaining functional when getting out of the midgut.

Design

CRISPR-based photo-switches have been used in optogenetic control, which offer photo-sensitive devices that are activated with post-translational modifications. CRISPR nuclease is produced into two fragments (C-terminal and N-terminal) connected to a paired photoswitchable dimerization domain. Upon exposure to light, the parts heterodimerize, connect the N-terminal and C-terminal fragments and make it functional. After that, a single-guide RNA (sgRNA), composed of crRNA and trancrRNA, guides the nuclease functional to bind and cleave a target DNA sequence. This system was already tested with split Cas9 and other variants with photoswitches such as Cryptochrome 2 (CRY2) and CIB1, a blue light-dependent protein and its binding partner from Arabidopsis thaliana, or Vivids (VVDs), the smallest photoreceptors already tested[1].

Based on the optogenetic tool from Nihongaki et al. (2019), we designed a photoactivatable KS Module containing split CRISPR-Cpf1 fragments ( BBa_K4075100 and BBa_K4075101). These fragments are connected to photoswitches Magnets (known as pMag and nMag), VVDs variants engineered to heterodimerize through electrostatic interactions upon blue light exposure. The split Cpf1 fragments linked with magnets are constitutively expressed and thus their post-translational mechanism may make the activation process faster.

After the C-Cpf1-pMags and N-Cpf1-nMag heterodimerization, the functional Cpf1 will be guided by shorter crRNA to specific gene targets. One significant versatility of Cpf1 is its capability to process a single transcript crRNA array into multiple crRNAs. That is, we can design a cassette for multiple targets. Even though this light-activated split-Cpf1-Mag has been applied in mammalian cells, Cpf1 has shown high efficiency for multiple targets deletions in B. subtilis [3][4]. Finally, regarding our KS Module goals, we designed a multiplex crRNA to target several essential genes (and protein reporter for future tests) to trigger cell lysis.

sgRNA Design

The sgRNAs were designed to cleave essential genes and eliminate synthetic DNA. We selected candidate targets from the DEG15 database[11], a database for several essential genes. We set divIB (BSU15240), which plays an important role in the sporulation process; dnaA (BSU00010), necessary for chromosomal replication; and the reporter mCherry, for future experimental tests purposes. Then we used the web tool CHOPCHOP[5][6] to design the guide RNAs. The complementary target candidates were evaluated considering the off-targets, self-complementarity and efficiency, respectively. Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) sequence, which can improve the efficiency in B. subtilis[9]. Thus, we set PAM sequence = TTTN.

Future Challenges

In our project, GMOs releasing was was a concern that was studied from the beginning and highlighted in meetings with experts. Prof. Salomon, a tropical diseases specialist in Argentina and Prof. Margareth Capurro, a transgenic mosquitoes specialist in Brazil, warned us of the risks of implementing paratransgenesis. Horizontal gene transfer and recombinant expression hazardous in off-target organisms are some problems that can occur (and a considerable barrier for regulatory institutions!), even more with B. subtilis spores release risks.

Finally, we highlight some future challenges to improve our paratransgenesis biocontainment:

• Spore release control: Prevention of re-sporulation after germination in the vector's midgut;

• Physical biocontainment: Propose a sugar baits architecture regarding safety aspects;

• Kill Switch experiment: Evaluate the efficiency of the designed killswitch in Wet Lab;