Part:BBa_K4607013
Expression cassette for PCNP-CecA-LysSS protein
Description
This biobrick 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 [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].
Tomar como ejemplo:
The main purpose of the albumin binding domain (ABD) is to counteract the problems related to the brief in vivo time life of the endolysins. These domains have the capacity to increase the lifetime of antibodies, proteins, and enzymes through the incorporation of their sequences into the fusion protein. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part consists of an affinity-maturated variant of the streptococcal protein G which has been used for LysK expression, with results of up to 34 hours in increasing the lifetime of the protein in mice [1]. The best results have been achieved with the following conformation: CHAP domain-ABD-SH3 domain [5]. The domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9. The average ABD-endolysin lifetime is about 30 hours [1].
References
[1] Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.02927
[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] 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
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