KW01-A phage was inactivated when heated at 75oC for 10 min to reveal the lytic activity of endolysin in the precipitated protein samples. Apart from that, both the KW01-A phage and its endolysin have broad pH stability, ranging from pH 4.0 to 10.0.
Bacteriophage
- History of Bacteriophage
- Nature of Bacteriophages
- Life Cycle of Bacteriophage
- Applications of Bacteriophage
There are 14 different professional families that have been organized according to this approach (Elbreki, et al., 2014). Successful penetration of the phages is important for the survival and continuation of the life cycle of the phages (Rakhuba, et al., 2010).
Advantages and Disadvantages of Bacteriophage Therapy
In addition, repeated exposures of the same phage strain to the body will activate the adaptive immune response, resulting in antibody production. Therefore, the use of phage therapy should be reconsidered due to the risk of resistance development.
Endolysin
Nature of Endolysin
Due to the various disadvantages and limitations of phage therapy and the lack of currently recognized international human clinical trials, phage therapy for human use has not been approved in most countries.
Structure and Classification of Endolysin
They can be classified into at least five different groups, namely (I) N-acetyl-β-d-muramidases or lysozymes; (II) lytic transglycosylases; (III) N-acetyl-β-d-glucosaminidases or glycosidases; (IV) N-acetylmuramoyl-l-alanine amidases and (V) endopeptidases (Barrera-Rivas, et al., 2017). The N-acetylglucosaminil-β-1,4-N-acetylmuramine bonds were cleaved by both the N-acetyl-β-d-muramidases and lytic transglycosylases, while the other glycosidic bond in the sugar strand was hydrolyzed by N-acetyl- β-d-glucosaminidases.
Characteristics of Endolysin .1 Endolysin Specificity
Endolysin Immunogenicity
However, the lytic activity of the endolysin was found to be delayed but not inactivated in an in vitro assay (Loeffler, Djurkovicand Fischetti, 2003). Therefore, these results suggested that the antibodies raised were not neutralizing antibodies, thus would not deactivate the lytic activity of endolysins (Lobocka and Szybalski, 2012).
Endolysin Resistance
Some of the studies hypothesized that the strong binding affinity of some CBDs is greater than the affinity of endolysin-specific antibodies. Furthermore, external application of endolysin also prevents the bacterial host from developing resistance as most of the classical resistance mechanisms develop from the inside of the cells (Donovan, 2009).
Applications of Endolysin
According to Zhang et al. 2012), the Listeria bacteriophage endolysin LysZ5 has high and specific lytic activity against L. For example, a recombinant endolysin, LysBPS13, has been shown to exhibit high lytic activity under various conditions, which include a wide range of temperatures and ionic strengths. This was evident in a study by Mao et al. 2013), where chimeric Ply187 endolysin showed higher lytic activity against S.
Therefore, a recombinant endolysin or chimeolysin can be engineered to have an improved lytic activity compared to its parent endolysin. On the other hand, endolysin can synergize with other antimicrobial agents to enhance the lytic activity. Nisin enhanced the lytic activity of LysH5 up to 8-fold and successfully cleared the S.aeurus in the contaminated milk (García, et al., 2010).
Bacteriophage
Reagents, Chemicals and Equipment
Preparation of Buffers and Media
Growing and Amplification of Bacteriophage .1 Obtaining Single Bacterial Colony
- Preparation of Overnight Culture and Log Phase Bacterial Culture
- Amplification of Bacteriophage from Single Plaque
- Small Scale of Bacteriophage Amplification
- Large Scale of Bacteriophage Amplification
After the bacterial culture reached OD, μL of log-phase bacterial culture and 500 μL of the dialyzed single plate were added to 3 mL of upper agar and immediately mixed using a vortex mixer. A negative control plate was prepared by adding 4 mL of topagar to 200 μL of log phase host cells and 500 μL of TBS. Meanwhile, two tubes containing 40 ml log phase of bacterial culture were prepared as described in Section 3.4.2.
The supernatant was transferred to the tubes containing the log phase growth host cells and incubated at 37oC with a. Five hundred milliliters of log-phase growing host was prepared as described in the Section 3.4.2. After the OD600 of the culture was reached at 0.4-0.6, the lysate was added to the log phase growing host and incubated until total lysis was observed.
Precipitation of Bacteriophage
Precipitation of Bacteriophage with 20% (w/v) PEG 8000
Enumeration of Bacteriophage
Extraction of Endolysin
- Preparation of Crude Phage Lysate
- Ammonium Sulfate Precipitation
- Dialysis
- Ultrafiltration of Precipitated Protein Samples
The supernatant was kept until 20-40% ammonium sulfate precipitation, whereas the pellet was resuspended in an appropriate volume of TBS buffer solution and stored at 4oC. After the completion of the ammonium sulfate precipitation, all the protein samples were subjected to dialysis as described in Section 3.5.3 to remove excess ammonium sulfate in the samples. Then the dialysis tubing was placed in 500 ml of TBS solution and dialyzed at 4oC with gentle agitation.
The next day, the dialyzed samples were removed from the tube and SDS-PAGE was performed to analyze the precipitated proteins. Ultrafiltration was performed using Microsep Advance centrifugal equipment with Omega 10k membrane to further concentrate the precipitated protein samples. After centrifugation, the sample in the sample reservoir was collected and stored at 4oC while the sample in the filtrate receiver was discarded.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
- Preparation of 4X Resolving Gel Buffer Solution (pH 8.8)
- Preparation of 4X Stacking Gel Buffer Solution (pH 6.8)
- Preparation of 10X and 1X Running Buffer Solution
- Preparation of Staining and Destaining Solution
- Casting of SDS-PAGE Gel
- SDS-PAGE Analysis
Stacking gel buffer solution (0.5 M Tris base, 0.4% SDS) was prepared by adding 6.057 g of Tris base and 4 ml of 10% SDS solution to approx. 60 ml sterile deionized water and mixed thoroughly. After the desired pH was achieved, the solution was made up to 100 ml with sterile deionized water. To run SDS-PAGE, 10X running buffer was diluted to 1X running buffer by mixing 100 mL of 10X running buffer with 900 mL of sterile deionized water.
Two hundred milliliters of staining solution (0.1% Coomassie Brilliant Blue R-250, 1% acetic acid, 40% methanol) was prepared by adding 100 mg of Coomassie Brilliant Blue R-250 to 1 ml of acetic acid. The stacking gel solution was then loaded into the gap between the glass plates and the comb carefully inserted. After the stacking gel solidified, the entire gel was transferred to the gel reservoir and 1X liquid buffer was added to the reservoir.
Spot Lytic Assay
- Spot Lytic Assay for Non-EDTA-treated Bacterial Cells
- Spot Lytic Assay for EDTA-treated Bacterial Cells
- Heat Treatment of Bacteriophage Sample
- Heat Treatment for Precipitated Protein Samples
- pH Treatment for Bacteriophage Sample
- Determination of pH Stability of the Precipitated Endolysin
- Host Range Test for PEG-Precipitated Phage and Endolysin
Five microliters of each precipitated protein sample was spotted on the agar, along with 25 μg/mL lysozyme (positive control) and TBS (negative control) were spotted on the agar. Next, 5 μL of each heat-treated phage sample was spotted onto the agar and the plates were allowed to air dry before incubation at 37oC for 16-18 hours. Heat treatment was performed on the precipitated protein samples to inactivate co-precipitated phages present in the samples.
A sample of the precipitated protein containing endolysin was treated with different pH (pH 2, pH 4, pH 6, pH 8 and pH 10). A sample of the precipitated protein containing endolysin was first heat treated to remove coprecipitated phage. Next, 5 μl of the heat-treated endolysin-containing precipitated protein sample was plated on both agar plates, while the PEG-precipitated KW01-A phage was plated on non-EDTA-treated cells.
Extraction of Endolysin by Ammonium Sulfate Precipitation
protein marker; Lane 1: 0-20% ammonium sulfate saturation precipitated protein sample; Lane 2: 20-40% ammonium sulfate saturation-precipitated protein sample; Lane 3: 40-60% ammonium sulfate saturation precipitated protein sample; Lane 4: 60-80% ammonium sulfate saturation-precipitated protein sample.
Spot Lytic Assay
- Spot Lytic Assay for Bacterial Culture with and without EDTA Treatment
- Determination of Deactivation Temperature for KW01-A Phage
- Spot Lytic Assay for Bacterial Culture with and without EDTA Treatment using Heat-treated Protein Samples
- Determination of Deactivation pH of KW01-A Phage
- Spot Lytic Assay for Bacterial Culture with and without EDTA Treatment using pH-treated Protein Samples containing
- Comparison of Different Host Susceptibility against Phage KW01-A and Its Endolysin
In addition, the precipitated protein samples also formed clear zones on non-EDTA-treated cells (Figure 4.2B). Non-EDTA-treated bacterial lawn stained with 5 μL (I) PEG-precipitated KW01-A phage as positive control; (II) TBS buffer as negative control; (III) 0-20% ammonium sulfate saturation precipitated protein;. B & D) EDTA-treated bacterial lawn stained with 5 μL of similar samples as (A & C) except (I) 0.025 μg/mL lysozyme as a positive control.
Bacterial pellet untreated with EDTA, contaminated with 5 μL (I) phage KW01-A precipitated with PEG as positive control; (II) TBS buffer as negative control; (III) 0-20% protein precipitated by ammonium sulfate saturation; (IV) 20-40%. Lysis of all precipitated protein samples was still observed in both EDTA and non-EDTA treated samples. Based on Figure 4.7, the stained areas of precipitated protein samples that were heated at 80oC for 5 and 10 minutes in bacterial cells not treated with EDTA could not produce any clear areas.
Extraction of Endolysin by Ammonium Sulphate Precipitation
Additionally, another group of researchers performed double ammonium sulfate precipitation to precipitate endolysins from S. In addition, Gupta and Prasad (2011) also used the ammonium sulfate method to concentrate lysine P-27/HP (a lysine from a phage infecting S. aureus ). However, most studies have preferred the ammonium sulfate method as it does not denature proteins.
After precipitation, excess ammonium sulfate in the protein sample was removed by dialysis prior to SDS-PAGE analysis (Thermo Fisher Scientific, 2018). The presence of ammonium sulfate may interfere with the subsequent purification step or its enzymatic activity (Dako et al., 2012). Therefore, a spot lytic assay was performed to determine the presence of endolysin in the precipitated protein samples.
Spot Lytic Assay
- Spot Lytic Assay for Bacterial Culture with and without EDTA Treatment
- Determination of Deactivation Temperature for KW01-A Phage
- Spot Lytic Assay for Bacterial Culture with and without EDTA Treatment using Heat-treated Protein Samples
- Determination of Deactivation pH of KW01-A Phage
- Spot Lytic Assay for Bacterial Culture with and without EDTA Treatment using pH-treated Protein Samples containing
- Comparison of Different Hosts Susceptibility against KW01-A Phage and Its Endolysin
Therefore, these studies showed that the phage was precipitated with the proteins using ammonium sulfate. Therefore, in this study, heat inactivation method was used to deactivate the phage lytic activity. This may be one of the reasons why the phage was still stable even after heat treatment at 75oC.
According to Chandra, et al. 2011), prolonged exposure to high temperatures will gradually reduce phage lytic activity. Therefore, it was hypothesized that the phage present in the precipitated proteins can be deactivated at a lower temperature. Since the PEG-precipitated KW01-A phage was only inactivated for 10 minutes at 75°C, this clearly showed that heat treatment at these specific temperatures was insufficient to kill the phage in the precipitated protein samples.
Limitations of Study
According to Rakhuba, et al. 2010), the specificity of phage infection was determined by the success of viral adsorption, which depends on the presence of surface receptors on the bacterial surface. For example, phages that recognize smooth (S)-type lipopolysaccharides (LPS) of Gram-negative bacteria usually exhibit narrow host sensitivity (Rakhuba, et al., 2010). The spectrum of the lytic activity of the phage particles is thus always limited to intra-species or -genus.
As for the endolysin, CBD is responsible for the recognition specificity and this specificity is mostly the same across the bacterial genus (Loessner, et al., 2002).
Future studies
The lytic activity of endolysin was then revealed in the precipitated protein sample with 20-40% ammonium sulfate saturation. Bacteriophage therapy: an alternative for the treatment of Staphylococcus aureus infections in animals and animal models. Co-therapy using lytic bacteriophage and linezolid: effective treatment in eliminating methicillin-resistant Staphylococcus aureus (MRSA) from diabetic foot infections.
Synergy between the phage endolysin LysH5 and nisin to kill Staphylococcus aureus in pasteurized milk. Isolation, characterization and therapeutic potential assessment of bacteriophages virulent for Staphylococcus aureus associated with goat mastitis. Lytic activity of recombinant bacteriophage φ11 and φ12 endolysins on whole cells and biofilms of Staphylococcus aureus.
A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Bacteriophage or bacteriophage-derived lytic protein that is effective for treating Staphylococcus aureus biofilm.
