Overview
Our project will be implemented by Engineered E.coli. The design consists of four primary functional modules: the “ Quorum sensing detect” module, the “Elimination” module, the “Aptamer detection” module, and the “Safety” module. Both the “Quorum sensing detect” and the “Aptamer detect” Module function as detective mechanism aimed at targeting Staphylococcus aureus (S.aureus). While the “Quorum sensing detect” Module functions inside the human digestive tract, where it triggers the release of self-lysis protein to initiate the functioning endolysins and amtimicrobial peptide (AMPs) in the human lumne, the “Aptamer detect” Module functions in everyday external circumstances. In addition, the “Safety” module contains a designed kill switch to avoid the potential release of engineered E.coli and its possible impact on the equilibrium of the bacterial flora within the human lumen. In conclusion, our project intends to develop a comprehensive cycle for elimination and confirmation of S. aureus.
The “Quorum Sensing detect” Module
Quorum Sensing System (QS System) is a pathway that bacteria use to sense their population density. Gram-positive and gram-negative bacteria use autoinducing peptides (AIPs) and (A) N-acyl-homoserine lactones (AHLs) as their QS molecules, respectively. In the case of Gram-positive bacteria like S. aureus, the primary regulatory element responsible for detecting AIPs and controlling downstream gene expression is the accessory gene regulatory (agr) operon. The agr operon consists of two transcription units, RNAII and RNA III, which are transcribed from divergent promoters, P2 and P3, respectively.
Figure 1. The quorum-sensing (QS) circuits. Autoinducing peptide (AIP) QS in Gram-positive bacteria by two-component signaling.
The P2 and P3 promotors are both agr-sensitive. Agr A and Agr C are necessary for the transcriptional activation of the agr locus, but Agr B and Agr D are only partially needed. The Agr operon is comprised of Agr A, C, D, and B. (1995; Novick et al.) The QS system, which includes AgrA, the master transcription activator of the agr operon, is encoded by RNAII. However, the effector RNA molecule that controls the expression of virulence genes is RNAIII. (Jonathan & Sivaramesh, 2011) The P3 promoter and its related operon are removed from our design to avoid the potential virulence that might have a pathogenic effect in this bacteria, leaving just the P2 promoter as the activation factor and components of the agr operon (Agr A and Agr C) in our design.
Figure 2(a) illustrates the S.aureus Agr QS circuit; Figure 2(b) The utilization of the S.aureus Agr QS circuit in our project's design.
Once the P2 promoter is triggered by the AIPs generated due to the high concentration of S.aureus in the human lumen, the downstream endolysin complex Spn1s_LysRZ will be released. This release of the endolysin complex leads to the lysis of E.coli, resulting in the functions of the endolysins and AMPs introduced in the "Elimination" Module.
The “Elimination” Module
1. Endolysin
Endolysin is a type of enzyme originated from bacteriophages that could cleave the covalent bonds on the cell wall that hold the host bacterial cell together. It has two critical domains: the Cell Binding Domain (CBD) which recognizes specific cell wall peptidoglycan structure, and the Enzymatically Active Domain (EAD) which subsequently breaks the cell (some types of endolysin also have an additional domain that assists its function). Exogenously, endolysin is a useful antibacterial agent while lysing the bacteria and displays high specificity due to the specificity of CBD, which indicates that it will merely target the harmful bacteria instead of the beneficial ones in the human body. Besides, all research shows that endolysin is non-resistible for bacteria, making it a promising substitution for antibiotics. Due to those reasons, endolysin can serve as a potential application for treating multidrug-resistant bacterial infections.
In our project, for gram-positive bacteria, lysins have a bactericidal effect by exteranlly destroying the bacteral cell wall peptideoglygans, which leads to osmotic lysis of the bacteria. We have selected three varieties of endolysins: LysGH15, ClyC, and LysDZ25, each of which exhibits distinct optimal operating salinity and pH levels. The three endolysins ensures the effectiveness for the various conditions in the human digestive tract.
2. LysDZ25
LysDZ25 exhibits remarkable resilience to serum and NaCl solutions. Even at a temperature of 37 degrees Celsius, LysDZ25 retains a high level of lytic activity. Research findings indicate that the impact of NaCl on the lytic activity of LysDZ25 remains consistent across both high and low salinity levels. Notably, the optimal lytic effects are observed at a NaCl concentration of 450 mM. The bactericidal efficiency of this endolysin is notably high, with a visible decline in bacterial OD value within the first five minutes of treatment. By the fifteenth minute, the OD has decreased by more than fifty percent. (chang et al., 2023)
Table 1. Lysis Effect of LysDZ25 on S.aureus and Gram-Negative Bacteria
3. ClyC
ClyC demonstrates remarkably rapid responsiveness in terms of its bactericidal effects. When a sample is treated with a ClyC concentration of 300nM, there is a substantial decline in its optical density (OD) within the initial 30 minutes. Notably, ClyC exhibits optimal lytic activity within a temperature range of 30-40 degrees Celsius and a pH range spanning from 6.5 to 10.0, which encompasses the environmental conditions found in the human lumen. These results strongly suggest that ClyC is a highly promising candidate as an antibacterial agent suitable for use in low-salt content foods. In fact, ClyC can be effectively applied in neutral to alkaline environments. (Lee et al., 2021)
Table 2. Lysis Effect of ClyC on S.aureus and other Bacteria
4. LysGH15
The lytic activity of LysGH15 is significantly influenced by the binding capacity of its binding domain, and this capacity exhibits a positive correlation with the concentration of NaCl. When the NaCl concentration is at 10mM, LysGH15 achieves its peak lytic activity. Conversely, the functional efficacy of LysGH15 diminishes to zero when the NaCl concentration reaches 150mM. LysGH15 operates optimally at a temperature of 35 degrees Celsius and exhibits its best performance at an approximate pH of 9.
5. Release of endolysin
The S. aureus targeting endolysin used in our project has a molecular mass varied from 35 to 55 kDa, making both passive and active cross-membrane transport hard to achieve as release methods. Therefore, our team chose endolysin complex Spn1s_LysRZ, a complex of holin, endolysin, and two Rz/Rz1-like proteins that specifically lyse E. coli and S. Typhimurium, to assist the release. (Lim et al., 2012) By inserting the ORF of Spn1s_LysRZ downstream of promoter P2 (Fig. 3), E. coli could automatically self-lyse when exposed to S. aureus quorum sensing signals (AIPs), releasing the S. aureus targeting endolysin from cytoplasm to environment. Although this release method results in the need to replenish the engineered E. coli after each round of successful defense, it is efficient, accurate, and error-tolerable (even if some E. coli failed to lyse itself, Spn1s_LysRZ released by its neighbors could also lyse it externally), making it comparingly suitable for practical use.
Figure 3. The gene circuit of the QS-release system
6. AMPs
Antimicrobial peptides (AMPs) are parts of innate immune responses that exist in most multi-cellular organisms. These peptides are demonstrated to kill bacteria, viruses, and even cancer cells, including our targeted pathogen, S. aureus. AMPs mainly kill cells by conducting pore formation in cytoplasmic membranes or interfering with intracellular molecules (DNA, RNA, protein synthesis, cell wall synthesis). (Brogden, 2005) (Nguyen et al., 2011)
The transmembrane pore-forming killing can be explained by the toroidal pore model, the barrel-stave model, or the carpet-like model. (Huan et al., 2020) In these models, the cationic portion of AMPs can bind with the anionic phospholipid headgroups in the inner membrane of bacteria, and then the hydrophobic parts of AMPs insert into the hydrophobic core, leading to the formation of pores. (Corrêa et al., 2019) After that, pore-forming disturbs membrane potentials and causes cell lysis. During this process, AMPs can affect the cell in a few seconds after contact with the cell membrane, having relatively rapid killing effects. (Bahar & Ren, 2013)
Intracellular killing usually occurs when AMP concentration is relatively low to disturb membrane integrity, which uses the same mechanism to bind membranes but directly penetrate into intracellular space. Interrupting intracellular molecules causes the death of the cell. Additionally, it can induce human immune system responses and facilitate the release of immune cells (Lymphocytes and Monocytes), which helps them to kill targeted cells. This ability also reduces the effect of antimicrobial resistance in bacteria. (Hancock & Sahl, 2006) AMPs have an essential advantage over other sterilizing molecules like endolysins: they can affect S.aureus without breaking the targeted cell.
Besides, owing to the specificity of AMPs toward bacteria, it won't affect the cells in the human body, so it's safe to use AMPs in our project. As described in the killing mechanism, AMPs rely on associations with anionic molecules in order to bind with the membrane. The bacterial inner membrane exhibits many negative phospholipids like cardiolipin and phosphatidylglycerol, while mammalian cells (like human body cells) are relatively neutrally charged. Thus, AMPs exhibit relatively low activity toward mammalian cells and target more bacteria. (Corrêa et al., 2019) (Huan et al., 2020)
7. LL-37
In our project, LL-37 is selected as targeted AMPs. The mature LL-37 has 37 amino acids, which are cleaved from protein hCAP-18. The first 31 amino acids form an α-helical structure, and the last 6 amino acids form the loop structure at the terminus.
Figure 4. The protein structure of LL-37
LL-37 has similar lysing mechanism to AMP's. The main approach is to disturb membrane activities. Since LL-37 has positive charge of +6, LL-37 can bind with negatively charge bacteria membranes and induces the formation of pores, which interrupts cell integrity and cause cell death.
Besides, LL-37 has the ability to penetrate cell membranes and bind intracellular receptors such as acyl carrier proteins to obstruct cellular transports. In addition to modulating cell migration and proliferation and inducing pro- and anti-inflammatory responses, LL-37 also kills bacteria indirectly by regulating cell migration and proliferation. (Ridyard & Overhage, 2021)
8. S. aureus in vivo elimination device
To achieve the goal of our project, the “Quorum Sensing Detect” module and the “Elimination” module should be connected, yielding the gene circuit shown below. This device could constitutively express S. aureus targeting endolysins and detect the presence of S. aureus QS signal. If the QS signal molecule is detected, the self-lysing enzyme composite (Spn1s_LysRZ) will be activated, and the engineered cell will lyse itself to release the endolysin that eliminates pathogenic S. aureus.
Figure 5. An abbreviated demonstration of the single cell gene circuit of S. aureus in vivo elimination device. Pro 1 and Pro 3 are both constitutive promoters. RBS and terminator are omitted for the sake of space.
Initially, we plan to assemble the whole circuit into one plasmid and use E. coli as the host organism, as presented in part BBa_K4593020. However, as each module is tested separately, the P2 promoter was found to have a very high background expression level in E. coli, making it unable to be a tight regulator for the release system. Thus, we modified the design to suit B. subtilis, as previous research showed that the same design works in closely related gram-positive bacteria (Bacillus megaterium) (Marchand, N., & Collins, C. H. (2013)), the detail of the new design is presented in part BBa_K4593021.
The design for expression in B. subtilis, however, is still flawed primarily due to the fact that no experimental data could support the lytic activity of Spn1s_LysRZ on B. subtilis. Thus, changing it to another endolysin that's known to target B. subtilis could make the design more practical.
To further eliminate the uncertainty of endolysin expression efficiency in B. subtilis, we proposed a novel design that separates the QS detection system and Elimination system into B. subtilis and E. coli respectively. After detecting the QS signal, B. subtilis will start to synthesize molecules that are able to passively transport cross membrane (such as DAPG), and these molecules will be received by E.coli, causing it to self-lyse and release the S. aureus targeting endolysin. Using DAPG as an example, the gene circuit is shown below. The feasibility of this method is being demonstrated by the work of Du et al. (Du et al., 2020)
Figure 6. An abbreviated demonstration of the double cell gene circuit of S. aureus in vivo elimination device. Pro 1, Pro 3, and Pro 4 are all constitutive promoters. RBS and terminator are omitted for the sake of space.
The “Aptamer detect” Module
1. Aptamer
The aptamer is a kind of single-strand DNA that can form the secondary structure and bind with a specific protein. As the ssDNA sequence is unique, the protien can be specifically identified. Generally, an aptamer typically consists of 25-90 bases, without coding functions, and the different combinations of nucleotides could significantly affect the properties, including the form conditions, affinities, and final physical structures they can form. (Rahimizadeh et al., 2017)
Aptamers have several advantages over conventional monoclonal antibodies. First, while antibodies typically have a molecular size of roughly 150 kDa, aptamers have a molecular size of less than 20 kDa, allowing them to penetrate tissues and reach their target more easily. (Dunn et al., 2017) The selection of high-affinity aptamer can be achieved through cycles of selection and replication. A combinatorial library with all possible sequences of aptamers will be incubated with the target molecule, leading to the binding and finding of the aptamer with the highest specificity. (Fesseha, 2020) No immunological response or cell processing is needed in the process. Therefore, the production of antibodies is expensive and time-consuming, usually taking months, while selected aptamers can be cheaply synthesized in several weeks. This unique chemical vitro selection process also allows aptamers to target any protein, including toxins or non-immunogenic targets. (Keefe et al., 2010)
Besides, while being used therapeutically, aptamers can also be deactivated through antisense oligonucleotides that can base pair with their binding domain. (Zhou & Rossi, 2016) Additionally, while antibodies easily and irreversibly denature under high temperatures and pH changes, aptamers can refold to their functional state through a simple annealing process. (So et al., 2005)
In our project, the Aptamer PA#2/8 is selected to target S. aureus due to its high affinity and specificity with native and recombinant Protein A. It is noticeable that PA#2/8 can distinguish various strains of S. aureus through different Protein A content. The 5' biotin is added to the aptamer to increase its binding affinity with Protein A. The sequence region of the 5'-end can form a secondary stem-loop motif (Figure. 5), with 5-7 nt forming the imperfect stem and 1-5 nt forming the loop. Removing this sequence motif will lead to a complete loss of function. (Stoltenburg et al., 2016)
Figure 7. The secondary stem-loop motif of PA#2/8
To test the affinity of the Aptamer PA#2/8, the three truncated aptamers are designed based on its secondary structure. The sequences of truncated ssDNA are listed below.
Table 3. Sequenecs of truancated PA#2/8
Figure 8. Secondary structure prediction of the aptamers using mfold program
(a) PA#2/8-a, (b) PA#2/8-b, (c) PA#2/8-c
The “Safety” Module
1. Kill Switch
To address the potential leakage of engineered E. coli, we have designed a cold-inducible kill switch that activates the self-destruction of E. coli. Specifically, this cold-inducible kill switch is highly sensitive to rapid temperature changes in the environment. The temperature is approximately 37 degree Celsius in the human lumen, and any sudden exposure to lower temperatures in the in vitro environment will trigger the switch. The switch will respond swiftly to temperature variation, which relies on the TEV ts-18 and TFts-2 proteins, which have corresponding temperature-transition points at 36.5 degree Celsius.
In this system, TFts and TEVts mutually inhibit each other, and the expression of TEVts is regulated by the TFts-repressed PR promoter. At lower temperatures, the constitutively expressed TFts is cleaved and inactive by TEVts. Consequently, the cold-inactivated TFts loses its ability to tightly repress the downstream suicide endolysin and TEVts. This allows TEVts to further cleave TFts, leading to the expression of the suicide endolysin and ultimately triggering the self-lysis of E. coli (Zheng et al., 2019).
Figure 9. The cold-inducible circuit design
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