Difference between revisions of "Part:BBa K4607008"

The biobrick consists of a polycationic nonapeptide (PCNP) which is capable of destabilizing the lipopolysaccharide and binding to gram-negative bateria. 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. The peptide has a length of nine amino acids and is adapted with an additional flexible linker sequence in order to increase its possibility of being incorporated into new codifying sequences without affecting the functionality of other parts or their domains' requirements [1].

Sequence and Features

Usage and Biology

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 [2]. 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 [3]. 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. However, the average endolysin lifetime is 20 minutes [4] [3].

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 [1] [5].

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 [1][5][6][7]. This part sequence has been incorporated into our fusion protein codifying sequence for the biobrick BBa K4607012.

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] 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

[3] 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

[4] 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

[5] 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

[6] 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 ‌

[7] 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 ‌