Part:BBa_K4607006
Expression cassette for LysCSA13-ABD protein
Expression cassette for LysCSA13-ABD protein: Fusion endolysin against S. aureus bacteria who causes bovine mastitis
This part contains the linear sequence of the biobrick BBa_K4607000 optimized for Escherichia coli. It incorporates some of the most efficient biobricks as described below: the T7 promoter with LacO regulations BBa_J435350, the medium strength RBS BBa_Z0262, the triple terminator BBa_J435371, and the high copy pUC ori /Kan R backbone BBa_J435330. It also contains the BBa_K4607000 that codifies for a fusion protein based on the endolysin Lys from Staphylococcus aureus virulent bacteriophage CSA13 which is composed of two domains: the CHAP domain, with excellent catalytic activity, 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]; and the albumin binding domain (ABD) from streptococcal protein G. The ABD is capable of increasing the antibody, protein, and enzyme lifetimes. 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 section consists of an affinity-maturated variant of the streptococcal protein G which has been used for the expression of LysK in mice, with results of up to 34 hours in increasing the lifetime of the protein [3]. The part is adapted to the Golden Gate cloning method. This part also contains a TEV cleavage site for the removal of the x6 HisTag after the protein purification process. The enzyme has a length of 316 amino acids and a molecular weight of 35.098 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9, and it has extracellular protein expression. Other characteristics of the endolysin are that it requires the presence of calcium and manganese to reach its maximum catalytic activity. The average endolysin lifetime is about 30 hours [3].
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1100
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1100
Illegal SpeI site found at 127
Illegal NotI site found at 1242 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1100
Illegal BglII site found at 30
Illegal BamHI site found at 1094
Illegal XhoI site found at 1251 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1100
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1100
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 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 Lys 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, 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 [6] [7].
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] [7], and does not affect the organoleptic characteristics of the milk produced [8].
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 [3]. The best results have been achieved with the following conformation: CHAP domain-ABD-SH3 domain [9].
The fusion of both proteins and the ABD generates a new basic part capable of lysating the S. aureus and MRSA, which are important parts of the pathogenic microbiota that cause bovine mastitis, without damaging the beneficial organisms. Our team has proposed a probable 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.
Results
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] 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
[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] 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
[8] 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
[9] 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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