Part:BBa_K4607005
CHAPK from K bacteriophage
Description
The biobrick consists of the CHAPk domain from the Lys of the bacteriophage K, with the ability to degrade the cell wall of antibiotic-resistant strains of Staphylococcus aureus [1] [2]. The domain expresses in a soluble fraction and has twice the catalytic activity of the original enzyme. The principle of its catalytic activity is the cleavage between the first glycine of the pentaglycine cross-bridge and D-alanine. The CHAPk domain contains 125 amino acids and a molecular weight of 18.6 kDa. It loses its activity at a temperature of 42.5 °C, meaning it denatures, and works at a pH of 6.5 to 7.5 [3].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 196
- 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 has 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.
To design our proposal of a novel non-antibiotic treatment against the losses of milk and bovine, capable of decreasing the effects on the milk industry and their consequences for the nutrition of the Mexican population, we took into account the CHAPk domain from the LysK of the bacteriophage K. This domain has an efficient catalytic activity against S. aureus strains, including the Methicillin-Resistant strains [3].
The principle behind the endolysin mechanism relies on the K bacteriophage. It is composed of three domains. For the design of a novel antimicrobial enzyme, the CHAPk domain from the K bacteriophage was selected for its ability to cleave between the D-alanine and the first glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the peptidoglycan, with high efficiency. When the CHAPk domain is cloned as a truncated enzyme, which means that the endolysin is cloned without its other 2 domains: amidase-2 and cell-wall binding domain SH3b, it overexpresses as a soluble protein and has twice the activity of the native protein [5].
The endolysin LysK from the bacteriophage K has a CHAPk region with the ability to degrade the cell wall of antibiotic-resistant strains of Staphylococcus aureus [1] [2]. The function of a bacteriophage is to infect specific bacteria, in this case S. aureus, in order to kill them. Once the bacteria is 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 a lytic activity for the purpose of setting free the phage progeny to continue infecting other cells [6].
References
[1] Haddad Kashani, H., Schmelcher, M., Sabzalipoor, H., Seyed Hosseini, E., & Moniri, R. (2018). Recombinant endolysins as potential therapeutics against antibiotic-resistant Staphylococcus aureus: current status of research and novel delivery strategies. Clinical microbiology reviews, 31(1), 10-1128. https://doi.org/10.1128/cmr.00071-17
[2] Filatova, L. Y., Donovan, D. M., Ishnazarova, N. T., Foster-Frey, J. A., Becker, S. C., Pugachev, V. G., Dmitrieva, N. F., & Klyachko, N. L. (2016). A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: a study of both kinetics of inactivation and specifics of interaction with anionic polymers. Applied biochemistry and biotechnology, 180, 544-557. https://doi.org/10.1007/s12010-016-2115-7
[3] Sanz-Gaitero, M., Keary, R., Garcia-Doval, C., Coffey, A., & van Raaij, M. J. (2013). Crystallization of the CHAP domain of the endolysin from Staphylococcus aureus bacteriophage K. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 69(12), 1393–1396. https://doi.org/10.1107/s1744309113030133
[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] Sanz-Gaitero, M., Keary, R., Garcia-Doval, C., Coffey, A., & van Raaij, M. J. (2013). Crystallization of the CHAP domain of the endolysin from Staphylococcus aureus bacteriophage K. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 69(12), 1393–1396. https://doi.org/10.1107/s1744309113030133
[6] 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
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