Part:BBa_K4607000

LysCSA13-ABD

Description

The biobrick consists of a fusion protein based on the endolysin Lys from Staphylococcus aureus virulent bacteriophage CSA13 which is composed of three domains: the CHAP domain, with excellent catalytic activity, up to 90%, degrading almost 15 strains of Staphylococcus, including methicillin-resistant strains (MRSA) [1]; 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] [Figure 1].

Figure 1. LysCSA13-ABD protein diagram.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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 S. 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.

Plasmid Construction and Analysis

To design this coding sequence, we have been evaluated different protein sequences through a protein structural model analysis tool, Alpha Fold 2, in order to identify the original functional protein structures and compare them with our final designs, ensuring their enzymatic activity. In Figure 2, is possible to analyze the components of each protein coding sequence. For it case, we realized that the presence of 6X HisTag (c) affects the functional protein domains, as can be seen in Figure 2, in comparison to the original LysCSA13 (a) and LysCSA13-ABD (b) structures. To solve this problem, we have added an TEV site to its posterior removal (d)

Figure 2. Alpha Fold 2’s LysCSA13-ABD protein folding analysis. (a) Original folding from LysCSA13. (b) Folding of LysCSA13-ABD (c) LysCSA13-ABD-6X HisTag’s folding.(d) LysCSA13-ABD-TEV Site-6X HisTag’s folding

Golden Gate Assembly and identification

From the iGem 2023 plate, BioBrick™ BBa_J435330 (plate 2, well A15) was resuspended, which was then transformed into E. coli 5-alpha using heat shock and subsequently extracted and purified with the assistance of the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760). This part was chosen as the backbone due to its high replication origin, dual antibiotic resistance (kanamycin and ampicillin), and compatibility with the Golden Gate assembly method. Once the BBa_J435330 backbone was extracted and purified, Golden Gate assembly reactions were performed using the BsaI enzyme (NEB E1601, Lot #10182427) to insert each of the transcriptional units into the BBa_J435330 backbone.

After the Golden Gate assembly reaction, a bacterial transformation into E. coli 5-alpha was performed, followed by plating on LB agar with kanamycin (50 µg/mL). However, colonies were obtained both on the plates seeded with the transformation and in the negative controls. This led us to suspect that the plates from the Golden Gate reaction might contain a mixture of colonies transformed with the target construct and others transformed with the backbone alone, making them indistinguishable by visual inspection.

To confirm this, re-plating of several colonies from the same plate was conducted onto other plates with kanamycin and ampicillin. This was done to take advantage of the dual antibiotic resistance feature present in the utilized backbone. A colony from the reaction-seeded plate was selected and then plated onto two separate plates: one with kanamycin (50 µg/mL) and another with ampicillin (100 µg/mL). The same identifying number was assigned to both antibiotics.

The following day, observation revealed that some colonies grew on both antibiotics, while specific colonies only grew on kanamycin (50 µg/mL) and not on ampicillin (100 µg/mL). This suggested that these colonies might have been transformed with a construct carrying resistance only to kanamycin (50 µg/mL) and not to ampicillin. This led us to suspect that our construct might indeed be present in these colonies.

To verify this, a colony that exclusively exhibited kanamycin resistance (50 µg/mL) was selected. DNA plasmid extraction and purification were performed using the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760). Subsequently, enzymatic digestion was carried out to linearize the DNA, followed by agarose gel electrophoresis for result visualization.

Using SnapGene® software, a simulation of enzymatic digestion was conducted on the constructs to identify potential enzymes for linearization. Subsequently, in the laboratory, the digestion reaction LysCSA13 was performed using the EcoRI restriction enzyme (Figure 3).

Figure 3. pDNA agarose gel electrophoresis (1%). Lane 1. Quick-Load® Purple 1 kb Plus DNA Ladder. Lane 2. LysCSA13 digested with single-cut enzyme EcoRI. Lane 3. Negative control LysCSA13 digestion (3159 bp) pDNA.

Protein Induction Kinetic

To identify the best protein induction parameters, we performed protein induction kinetics. Based on previous studies, we identify that the best temperature and time conditions were about 6 hours at 37° C. Related to the IPTG concentration, the experiments were performed at 0.2 mM, 0.5 mM, and 1 mM, for all of our transformants. The inductions were performed with the E. coli BL21 (DE3) transformed with the expression plasmids obtained from the Golden Gate Assembly.

Protein induction kinetics were performed at 37° C, for six hours at maximum agitation in an orbital shaker. Five treatments were included: Negative control with E. coli BL21 (DE3), negative control with E. coli BL21 (DE3) transformed with LysCSA13 without IPTG induction, and three E. coli BL21 (DE3) transformed with the endolysin induced at three different concentrations of IPTG, 0.2 mM, 0.5 mM, and 1 mM. The samples were collected by hour, harvesting 200 μL, by each treatment.

To identify the best induction parameters, a SDS-PAGE was performed. Considering that the molecular weight of LysCSA13-ABD was 35.098 kDa, the concentrations of the SDS-PAGE phases were 12% and 6%, for separator and concentrator phases respectively. The best parameters were IPTG at 0.2 nM and 5 hours for LysCSA13-ABD (Figure 4).

Figure 4. LysCSA13-ABD 0.2 mM IPTG protein induction kinetic. A) ColorBurst protein ladder. B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted, fourth hour. D) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, fourth hour. E) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted, fifth hour. F) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, fifth hour. G) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted, sixth hour. H) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, sixth hour. I) E. coli BL21 (DE3), first hour.

Massive Protein Induction

Once the best protein induction parameters were established, a new SDS-PAGE was performed to assess the protein expression site in the cell. Our endolysin were inducted at 0.2 mM IPTG, for five hours at 37° C. We obtained a massive protein expression after pellets were harvested. To evaluate the expression site, the reagent B-PER extracted the intracellular proteins, liberating the soluble and insoluble proteins. For the LysCSA13-ABD an insoluble fraction has been demonstrated (Figure 5).

Figure 5. LysCSA13-ABD soluble (A) and insoluble (B) fractions. Visualization through a polyacrilamide gel 12% (SDS-PAGE) results. Both gels present the same distribution of wells, each one corresponding to the fraction (soluble or insoluble) assigned. A) MWM (for gel A: Precision Plus Protein Dual Color Standards (10–250 kD), for gel B: PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa). B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted. D) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 2 μL. E) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 4 μL. F) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 6 μL. G) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 8 μL. H) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 10 μL. I) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 15 μL. J) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, 20 μL.

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