Part:BBa_K4607004
LysCSA13
Description
The biobrick consists of the endolysin Lys from the Staphylococcus aureus virulent bacteriophage CSA13, and it is composed of two domains: the CHAP domain, with an excellent catalytic activity of up to 90%, degrading almost 15 strains of Staphylococcus, including methicillin-resistant strains (MRSA) [1], and the SH3 domain, which recognizes and binds to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus peptidoglycan, activating the catalytic domain [2]. The enzyme has a length of 250 amino acids and a molecular weight of 28.36 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9, and it has intracellular protein expression. The average endolysin lifetime is about 20 minutes [3]. The part is adapted to the Golden Gate cloning method.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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 [4]. Our team has conducted an extensive investigation to find an alternative treatment for bovine mastitis without risking the environment.
The principle behind the mechanism of the endolysin from the Staphylococcus aureus virulent bacteriophage CSA13 comes from the original activity of the 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]. This endolysin is composed of two main domains: the N-terminal, which represents the catalytic domain, and the C-terminal, which is a cell wall binding domain that 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 [6] [3].
The endolysin Lys from the Staphylococcus aureus virulent bacteriophage CSA13 is composed of two domains that make it capable to lysate the bacteria. The bacteriophage CSA13 CHAP domain has excellent catalytic activity, up to 90%, degrading almost 15 strains of Staphylococcus including methicillin-resistant strains (MRSA) [1], which is a real advantage considering that the World Health Organization (WHO) called Methicillin-resistant strains a priority pathogen [4]. The catalytic activity of the protein also works perfectly on polystyrene, glass and stainless steel.
As with many of the endolysins, it cleaves to the cell wall by disrupting the peptidoglycan that composes the bacterial cell; for this to be possible, the bacteriophage CSA13 SH3 domain recognizes and binds to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus peptidoglycan, which makes it completely safe for the host [1] [3], and does not affect the organoleptic characteristics of the milk produced [7].
The enzyme has a length of 250 amino acids and a molecular weight of 28.36 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9. The lifetime of the endolysin is about 20.5 minutes [6]. Other characteristics of the endolysin are that it requires the presence of calcium and manganese to reach its maximum catalytic activity.
References
[1] Cha, Y., Son, B., & Ryu, S. (2019). Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. Food Microbiology, 84, 103245. https://doi.org/10.1016/j.fm.2019.103245
[2] Lade, H., & Kim, J.-S. (2021). Bacterial Targets of Antibiotics in Methicillin-Resistant Staphylococcus aureus. Antibiotics, 10(4), 398. https://doi.org/10.3390/antibiotics10040398
[3] Resch, G., Moreillon, P., & Fischetti, V. A. (2011). PEGylating a bacteriophage endolysin inhibits its bactericidal activity. AMB Express, 1(1), 29. https://doi.org/10.1186/2191-0855-1-29
[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] Połaska, M., & Sokołowska, B. (2019). Bacteriophages-a new hope or a huge problem in the food industry. AIMS Microbiology, 5(4), 324–346. https://doi.org/10.3934/microbiol.2019.4.324| None |