Part:BBa_K4607010
LysSS
Description
This biobrick consists of the endolysin LysSS from the bacteriophage SS3e from Salmonella that has demonstrated antibacterial activity against gram-negative bacteria 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. This part 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 lifetime is about 30 hours. The part is adapted to the Golden Gate cloning method [1].
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 206
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 206
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 206
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 206
- 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].
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 [1][5][6]. 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.
References
[1] 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
[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
[6] 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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