Part:BBa_K4607013
Expression cassette for PCNP-CecA-LysSS protein
Description
This part contains the linear sequence of the PCNP-CecA-LysSS (BBa_K4607012) nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described below: the T7 promoter with LacO regulations (BBa_J435350), the medium strength RBS (BBa_Z0262), and the triple terminator (BBa_J435371). For our plasmid construction, we used the high copy pUC ori /Kan R backbone (BBa_J435330). It also contains the BBa_K4607012 that consists of a fusion protein based on three main parts: the polycationic nonapeptide (PCNP), Cecropin A peptide, and the LysSS protein, all integrated with flexible linkers that assure its functionality. The first part, PCNP is capable of destabilizing the lipopolysaccharide and binding to gram-negative bacteria. The principle of the PCNP is related to the ionic interactions between the phosphate groups, divalent cations, and hydrophobic lipids' stacking, where the PCNP acts as a destabilizing agent. This part has been fused with endolysins in order to increase their capability to lyse gram-negative bacteria, demonstrating that the addition of the PCNP allows the endolysin introduction into the bacteria's cell membrane. It has been evaluated in Escherichia coli [1]. The second part is cecropin A (CecA), which was selected for its ability as an antimicrobial peptide. CecA has demonstrated excellent capacity for improving the endolysins antibacterial activity against gram-negative bacteria when it's incorporated in the N-terminal region. The principle behind CecA's antibacterial potential resides in its composition, which includes a cationic region that facilitates lipid interactions, favors a stronger ionic interaction, and finally degrades the cell wall by damaging bacterial inner membranes. CecA peptide has been evaluated in gram-negative bacteria as Escherichia coli [2]. And finally, the endolysin LysSS from the bacteriophage SS3e from Salmonella that has demonstrated antibacterial activity against gram-negative bacteria such as Escherichia coli, and gram-negative bacteria such as Staphylococcus aureus including methicillin-resistant strains. In comparison with other endolysins, LysSS contains positive charges at the C-terminal region that destabilize the gram-negative cell membrane. LysSS has intracellular and insoluble protein expression. The fusion protein PCNP-CecA-LysSS has a lenght of 227 amino acids and a molecular weight of 24.359 kDa [3].
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 833
Illegal XbaI site found at 96
Illegal SpeI site found at 127
Illegal SpeI site found at 814
Illegal PstI site found at 505 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 833
Illegal SpeI site found at 127
Illegal SpeI site found at 814
Illegal PstI site found at 505
Illegal NotI site found at 975 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 833
Illegal BglII site found at 30
Illegal BamHI site found at 827
Illegal XhoI site found at 984 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 833
Illegal XbaI site found at 96
Illegal SpeI site found at 127
Illegal SpeI site found at 814
Illegal PstI site found at 505 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 833
Illegal XbaI site found at 96
Illegal SpeI site found at 127
Illegal SpeI site found at 814
Illegal PstI site found at 505 - 1000COMPATIBLE WITH RFC[1000]
Biology and usage
As a brief contextualization, bovine mastitis is the result of the infection of the bovine mammary glands caused by pathogenic microorganisms, mainly gram-positive and negative bacteria. This disease reduces milk quality production to a great extent and produces painful damage to the bovine. The main treatment for mastitis is the use of diverse antibiotics, therefore the overuse and misuse of them have caused a real problem in the development of multidrug-resistant pathogens [4]. Our team has conducted an extensive investigation to find an alternative treatment for bovine mastitis without risking the environment.
The principle behind our proposal is the use of fused proteins based on efficient bacteriophage endolysins. The function of a bacteriophage is to infect bacteria in order to kill them. Once the bacteria are infected and the virions are mature, they release holins, which are enzymes that create pores in the inner cell membrane. Endolysins now have access to the cell wall, so they can degrade it. Endolysins have lytic activity for the purpose of setting free the phage progeny to continue infecting other cells [5]. Endolysins are composed of two main domains: the N-terminal, which represents the catalytic domain, and the C-terminal, which is a cell wall binding domain, which interacts by binding itself to the bacterium's cell wall, activating the catalytic region, and causing cell wall lysis. [5] [4].
The main purpose of the PCNP is to counteract the endolysins' disadvantages to penetrate the Gram-negative cell membrane. The PCNP brings the opportunity to introduce our endolysins into the Gram-negative cell membrane through a principle related to the PCNP's ability to perform an ionic interaction between the phosphate groups, divalent cations, and hydrophobic lipids' stacking, resulting in the outter membrane permabilization, inducing impairments in the membrane's surface and pores formation which enables the bacterial peptidoglycan exposition as a result of its destabilization, favoring its posterior lysis [6] [7].
One of the most notable advantages of using polycationic peptides is their non-necessity to cross the cell membrane to affect bacteria, in comparison with the principle of endolysins which need to interact with the peptidoglycan layer to make a real impact on bacteria. Considering the PCNP's benefits, is possible to design codifying sequences against Gram-negative bacteria. For this to be possible, it's necessary to take care of the endolysin's functionality, adding a flexible linker between them to be assured of the domains' activity. In the same way, it's better to design intein-fusion proteins where the PCPN is located in the N-terminal region, followed by the flexible linker and the endolysin sequence. Its part has been evaluated in Escherichia coli [6][7][8][9].
Endolysins need to interact with the peptidoglycan layer to identify and lyse the bacterial cell wall, however, gram-negative bacterial structure makes the process more complicated. To overcome our drawbacks, we incorporated the CecA as a potential solution. The addition of CecA to the N-terminal of a fusion protein improves its antibacterial activity against gram-negative bacteria; this is possible because of its cationic regions, which facilitate the interaction with gram-negative bacterial lipids. Consequently, the amphipathic helix interacts with the lipid phosphate groups of the outer membrane, at the same time, the amphipathic helix may induce the endolysin's introduction through the membrane. Finally, the fusion protein is capable of degrading the inner membranes of the gram-negative bacteria. CecA has been evaluated in gram-negative bacteria as E. coli successfully [2][10][11][12].
The endolysin LysSS from the bacteriophage SS3e from Salmonella, as many of the endolysins, is composed by two domains that confers it the ability to lysate bacteria. The bacteriophage SS3e has a broad host gram-negative spectrum including Salmonella enteritidis, Salmonella typhimurium, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae. It also recognizes gram-positive bacteria as Staphylococcus aureus along with methicillin-resistant strains which is a real advantage considering that the World Health Organization (WHO) called Methicillin-resistant strains a priority pathogen [3][10][13]. The enzyme has a length of 173 amino acids and a molecular weight of 18.56 kDa. LysSS has intracellular and insoluble protein expression. The average endolysin LysSS lifetime is about 30 hours. The part is adapted to the Golden Gate cloning method. The PCNP-CecA-LysSS protein-fusion has the ability to recognize and lyse efficiently, gram-negative bacteria as E. coli.
Plasmid Construction and Analysis
To design this coding sequence, we have been evaluated different protein sequences through a protein structural model analysis tool, Alpha Fold 2, in order to identify the original functional protein structures and compare them with our final designs, ensuring their enzymatic activity. (Figure 3) LysSS demonstrated that using short flexible linkers, any of the original peptide sequences (a), (b), or LysSS original protein sequence (c) would have been affected, so the final design (d) conserved its enzymatic activity. Additionally, we assured that all our final intein-fusion proteins kept the original proteins’ distribution with functional domains.
Golden Gate Assembly and identification
From the iGem 2023 plate, BioBrick™ BBa_J435330 (plate 2, well A15) was resuspended, which was then transformed into E. coli 5-alpha using heat shock and subsequently extracted and purified with the assistance of the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760). This part was chosen as the backbone due to its high replication origin, dual antibiotic resistance (kanamycin and ampicillin), and compatibility with the Golden Gate assembly method. Once the BBa_J435330 backbone was extracted and purified, Golden Gate assembly reactions were performed using the BsaI enzyme (NEB E1601, Lot #10182427) to insert each of the transcriptional units into the BBa_J435330 backbone.
After the Golden Gate assembly reaction, a bacterial transformation into E. coli 5-alpha was performed, followed by plating on LB agar with kanamycin (50 µg/mL). However, colonies were obtained both on the plates seeded with the transformation and in the negative controls. This led us to suspect that the plates from the Golden Gate reaction might contain a mixture of colonies transformed with the target construct and others transformed with the backbone alone, making them indistinguishable by visual inspection.
To confirm this, re-plating of several colonies from the same plate was conducted onto other plates with kanamycin and ampicillin. This was done to take advantage of the dual antibiotic resistance feature present in the utilized backbone. A colony from the reaction-seeded plate was selected and then plated onto two separate plates: one with kanamycin (50 µg/mL) and another with ampicillin (100 µg/mL). The same identifying number was assigned to both antibiotics.
The following day, observation revealed that some colonies grew on both antibiotics, while specific colonies only grew on kanamycin (50 µg/mL) and not on ampicillin (100 µg/mL). This suggested that these colonies might have been transformed with a construct carrying resistance only to kanamycin (50 µg/mL) and not to ampicillin. This led us to suspect that our construct might indeed be present in these colonies.
To verify this, a colony that exclusively exhibited kanamycin resistance (50 µg/mL) was selected. DNA plasmid extraction and purification were performed using the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760). Subsequently, enzymatic digestion was carried out to linearize the DNA, followed by agarose gel electrophoresis for result visualization.
Using SnapGene® software, a simulation of enzymatic digestion was conducted on the constructs to identify potential enzymes for linearization. Subsequently, in the laboratory, the digestion reaction PCNP-CecA-LysSS was performed using the EcoRI restriction enzyme (Figure 3).
References
[1] Briers, Y., Walmagh, M., Van Puyenbroeck, V., Cornelissen, A., Cenens, W., Aertsen, A., Oliveira, H., Azeredo, J., Verween, G., Pirnay, J.-P., Miller, S., Volckaert, G., & Lavigne, R. (2014). Engineered endolysin-based “Artilysins” to combat multidrug-resistant gram-negative pathogens. MBio, 5(4), e01379-01314. https://doi.org/10.1128/mBio.01379-14
[2] Jeong, T.-H., Hong, H.-W., Kim, M. S., Song, M., & Myung, H. (2023). Characterization of Three Different Endolysins Effective against Gram-Negative Bacteria. Viruses, 15(3), 679. https://doi.org/10.3390/v15030679
[3] Kim, S., Lee, D.-W., Jin, J.-S., & Kim, J. (2020). Antimicrobial activity of LysSS, a novel phage endolysin, against Acinetobacter baumannii and Pseudomonas aeruginosa. Journal of Global Antimicrobial Resistance. https://doi.org/10.1016/j.jgar.2020.01.005
[4] World Health Organization. (2021, November 17). Antimicrobial resistance. Who.int; World Health Organization: WHO. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance
[5] Gutiérrez, D., Fernández, L., Rodríguez, A., & García, P. (2018). Are phage lytic proteins the secret weapon to kill Staphylococcus aureus?. MBio, 9(1), 10-1128. https://doi.org/10.1128/mbio.01923-17
[6] Fernández, L., González, S., Campelo, A. B., Martínez, B., Rodríguez, A., & García, P. (2017). Downregulation of Autolysin-Encoding Genes by Phage-Derived Lytic Proteins Inhibits Biofilm Formation in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 61(5), e02724-16. https://doi.org/10.1128/AAC.02724-16
[7] Alfei, S., & Schito, A. M. (2020). Positively Charged Polymers as Promising Devices against Multidrug Resistant Gram-Negative Bacteria: A Review. Polymers, 12(5), 1195. https://doi.org/10.3390/polym12051195
[8] Söylemez, Ü. G., Yousef, M., Kesmen, Z., Büyükkiraz, M. E., & Bakir-Gungor, B. (2022). Prediction of Linear Cationic Antimicrobial Peptides Active against Gram-Negative and Gram-Positive Bacteria Based on Machine Learning Models. Applied Sciences, 12(7), 3631. https://doi.org/10.3390/app12073631
[9] Gutiérrez, D., & Briers, Y. (2021). Lysins breaking down the walls of Gram-negative bacteria, no longer a no-go. Current Opinion in Biotechnology, 68, 15–22. https://doi.org/10.1016/j.copbio.2020.08.014
[10] Schmelcher, M., Powell, A. M., Becker, S. C., Camp, M. J., & Donovan, D. M. (2012). Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands. Applied and Environmental Microbiology, 78(7), 2297–2305. https://doi.org/10.1128/aem.07050-11
[11] Heselpoth, R. D., Euler, C. W., Schuch, R., & Fischetti, V. A. (2019). Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria. Antimicrobial Agents and Chemotherapy, 63(6). https://doi.org/10.1128/aac.00342-19
[12] Kim, S., Patel, D. S., Park, S., Slusky, J., Klauda, J. B., Widmalm, G., & Im, W. (2016). Bilayer Properties of Lipid A from Various Gram-Negative Bacteria. Biophysical Journal, 111(8), 1750–1760. https://doi.org/10.1016/j.bpj.2016.09.001
[13] Kim, S., Kim, S.-H., Rahman, M., & Kim, J. (2018). Characterization of a Salmonella Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria. Journal of Microbiology, 56(12), 917–925. https://doi.org/10.1007/s12275-018-8310-1
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