While engineering L. plantarum for our PRESS treatment, we needed additional safety mechanisms that prevent any unintended risks, either environmental or human health-related, such as leakage to the environment, horizontal gene transfer (HGT), bacterial mouth remnants or translocation across the alveolar-capillary barrier into the bloodstream, causing septicemia. To overcome these issues, we created the PemK/I toxin-antitoxin (TA) system to guarantee the safe use of L. plantarum in PRESS.
We decided to develop a toxin-antitoxin (TA) system, specifically we chose the PemK/I system, as it is originally isolated from L. plantarum. The PemK toxin is an endoribonuclease that specifically recognizes and cleaves the tetrad sequence U↓AUU in target mRNA in a ribosome-independent manner [2], thus inhibiting protein synthesis and arresting bacterial growth due to degrading its genome. The PemI, which is the antitoxin, binds to PemK, neutralizing its endoribonuclease effect. This rPemI-rPemK complex becomes catalytically inactive when both proteins interact in a molar stoichiometry of 1:1 [2].
Function: Primary biocontainment through plasmid addiction
Mechanism: PemI (antitoxin) rapidly degraded by Lon protease; PemK (toxin) stable
Activation: Plasmid loss → antitoxin depletion → endoribonuclease activity → cell death
Fail-Safe: Cannot be disabled once plasmid is lost
In its native form, despite the PemI attains conformational stability upon rPemK interaction, it displays vulnerability to proteolysis and is rapidly degraded by the Lon protease, which is a cellular enzyme that targets specific proteins for breakdown [3]. This lability of PemI causes the post-segregational killing (PSK) mechanism, which is called "addiction module." If the plasmid that expresses the PemK-PemI system is lost during horizontal gene transfer, replication, or environmental stress, the stable PemK toxin persists due to its slower degradation rate and longer half-life, so it will take the upper hand due to imbalance between the more stable toxin and the unstable antitoxin, leading to bacterial cell death. We used this native characteristic to ensure that engineered L. plantarum cannot survive outside controlled conditions, mitigating unintended risks and enhancing safety.
The post-segregational killing (PSK) is known as the addiction module and enhances safety by ensuring that bacteria losing the plasmid either via HGT or during replication are rapidly eliminated [3]. This mechanism maintains a stable, pure, and homogeneous population of plasmid-containing L. plantarum, preventing the survival of both undesirable and non-functional bacteria in the lung, ensuring that the desired therapeutic functions and CO-BERA expression are consistently maintained in the same bacterial population, preventing loss of the TSLP-targeting siRNA function, preventing asthma from reoccur again [4]. The PSK system supports long-term stability by creating a dependency, as only the plasmid-containing L. plantarum, reducing the opportunity for HGT to occur in mixed bacterial populations where other species might acquire the plasmid, so by using this mechanism we are sure that the dose we decided will not be changed after a period by expressing CO-BERA by other bacteria in the lung, reducing the immunomodulation effects and the over-expression of CO-BERA.
To further enhance safety, we integrated the PemK/I system with both phoB promoter and heat-inducible Thermosensor RNA 2U, which collectively form an environmental sensing and response mechanism.
After the corticostreroid inhalation, the remnants in the mouth causes fungal infection, which is candidiasis, but in our treatment any baterial mouth remnants will be died by our toxin-antitoxin system by inactivating to the phoB promoter as the salivary inorganic phosphate level is 1.52±0.63 mmol/l [29] that prevents oral microbiome disruption and this feature ensures that our approach promises safer, more effective treatments, paving the way for personalized medicine with minimal side effects.
If the bacteria cross the alveolar-capillary barrier into the bloodstream, we will be ready for this condition by the phoB promoter, which enhances the system's specificity by responding to high phosphate levels, which differ significantly between the human bloodstream and the lung.
The PhoB promoter is activated by the PhoB kinase, which is part of the Pho regulon, upon the phosphate level:
1. When phosphate levels are low, PhoR activates PhoB, boosting the production of genes for both the toxin and antitoxin in our PemK-PemI system.
2. When phosphate is abundant, PhoR will dephosphorylate PhoB, and therefore inactivating it, repressing the PemK/I expression, leaving PemI to be rapidly degraded without enough supply, the more stable PemK toxin dominates, and finally leads to bacterial cell death.
We discovered that normal blood phosphate levels in adults vary between 2.5 and 4.5 mg/dL (0.81 and 1.45 mmol/L), with reference intervals indicating a range of 3.0 to 4.5 mg/dL [5]. It is sensitive to phosphate values ranging from 0 to 1000 µM, especially over 50 µM [28]. Then, our phoB promoter will be activated in low-phosphate conditions, such as those found outside the blood, such as in the lab or the lung; in contrast, it will be repressed in the high-phosphate environment of human blood, preventing bacterial survival in the bloodstream and lowering the risk of translocation and subsequent septicemia, which is a severe condition caused by bacterial infection in the blood.
Additionally, we used Thermosensor RNA 2U to eliminate our L. plantarum if it is outside its normal niche, such as in cases of laboratory leakage, release through exhalation, coughing, improper handling (e.g., by children), or residues in disposed unsterilized inhalers.
It is known that RNA thermosensors (RNATs) present in non-coding regions of certain mRNAs, that enable rapid upregulation of proteins' translation when the temperature of the bacterium rises after entering a mammalian host.
We decided to integrate this system into our bacteria. So, we used Thermosensor RNA 2U, which is a heat-inducible non-coding RNA that regulates TA system expression based on temperature.
When L. plantarum is outside its normal niche, the ambient temperature typically drops below the physiological range (37°C). This temperature decrease triggers the Thermosensor RNA 2U to halt the transcription of both PemK and PemI. In vitro melting studies showed conformational transitions of the ROSE element leading to its opening up with increasing temperatures, with the SD sequence occluded at 25°C. And due to the rapid degradation of PemI, the more persistent PemK toxin dominates, leading to bacterial cell death.
By using both the phoB promoter and the thermosensor RNA 2U, we can confidently say that our system forces L. plantarum to diminish both the toxin and antitoxin synthesis under non-physiological and non-intended conditions, allowing the more stable PemK toxin to eliminate the bacteria. Then, our safety system provides a way to prevent any probable risks associated with our L. plantarum bacteria, such as the risks associated with environmental release, horizontal gene transfer, and unintended survival in the bloodstream, ensuring both patient safety and ecological protection.
| Condition | Lung | Blood circulation | Lab leakage/Outside of body | HGT/plasmid loss |
|---|---|---|---|---|
| [Phosphate] | Low (0.2-0.4 mM) | High (2.5-4.5 mg/dL / 0.81-1.45 mM) | Low (0.1-0.3 mM) |
—
|
| Temperature | 37°C | 37°C | ≤25°C |
—
|
| Toxin expression | Basal | Low | Low |
—
|
| Antitoxin expression | Basal | Low | Low |
—
|
| Net result | Neutralization | Stable toxin persists | Stable toxin persists |
Stable toxin persists
|
| Status of L. plantarum | Alive | Dead | Dead |
Dead
|
Our PRESS project has multilayered safeguards involving the PemK/I system and environmental sensors, which come together to mitigate different risks
The PemK/I system is a bacterial toxin-antitoxin system found in various bacteria, including L. plantarum [16]. Site-directed mutagenesis confirmed the role of His-59, as either proton donor or acceptor, and Glu-78, which is a proton acceptor, as an acid-base couple in mediating the ribonuclease activity; together, they facilitate the chemical reaction by managing proton transfers. This acid-base couple is essential for the ribonuclease function, enabling the cleavage of RNA molecules by hydrolyzing the phosphodiester bonds in the RNA backbone [25].
In vitro studies validate PemK's specific RNA cleavage with preference for cleavage between U and A residues of sequences (U↓ACU)and (U↓ACG) and PemI's neutralization through direct binding, while in silico modeling of thermosensor RNA 2U and PhoB dynamics predicts robust containment [1,4,5].
We are pleased to share our approach to preserve both bacterial viability and Thermosensor RNA 2U function during freeze-drying preparation for our PRESS asthma treatment. We have focused on merging modern technology with reliable safety to ensure L. plantarum delivers its therapeutic effects without any problem.
We managed to develop a comprehensive freeze-drying protocol for our engineered L. plantarum that combines thermal equilibration of thermosensor RNA at 37°C, trehalose vitrification for structural protection, EDTA chelation to prevent premature toxin activation during processing, followed by controlled EDTA removal and low-temperature storage to create a stable dry powder inhaler formulation while maintaining bacterial viability, therapeutic efficacy and biocontainment functionality.
When it comes to engineering L. plantarum for something as critical as our PRESS asthma treatment, we focus on balancing exceptional efficiency and absolute safety. Sure, L. plantarum is a friendly microbe [2], but we're not taking chances with risks. We've used Thermo-sensitive RNA 2U that keeps our bacteria confined to the lung environment, prevents horizontal gene transfer (HGT), any environmental accidents, and if these bacteria exist at low temperature it will definitely be killed, So it’s critical to ensure that our bacteria stay alive and the thermosensor RNA 2U stays ready to act after freeze-drying.
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