Overview
Our team contributed to further investigating the LL-37's sterilizing effects. We both documented new findings of LL-37's effect on S.aureus, and the part was further characterized by sterilizing S. aureus under different LL-37 concentrations, and our team measured the bactericidal effects and the OD value of the bacteria solution with the change of time, adding new data to the part page. (BBa_K1162006)
Secondly, our team also contributed to the troubleshooting and documented the potential mistakes that need to be avoided. (EMSA) The protocols and the troubleshooting processes are listed below.
LL-37 characterization improvements
In our project, LL-37 is selected as targeted AMPs. The mature LL-37 has 37 amino acids, which are cleaved from protein hCAP-18. The first 31 amino acids form an α-helical structure, and the last 6 amino acids form the loop structure at the terminus.
Figure 1. The protein structure of LL-37
LL-37 has similar lysing mechanism to AMP's. The main approach is to disturb membrane activities. Since LL-37 has positive charge of +6, LL-37 can bind with negatively charge bacteria membranes and induces the formation of pores, which interrupts cell integrity and cause cell death. Besides, LL-37 has the ability to penetrate cell membranes and bind intracellular receptors such as acyl carrier proteins to obstruct cellular transports. In addition to modulating cell migration and proliferation and inducing pro- and anti-inflammatory responses, LL-37 also kills bacteria indirectly by regulating cell migration and proliferation. (Ridyard & Overhage, 2021)
Cell spreading under different concentrations of LL-37
The LL-37 was synthesized by Genescript company, with the sequence shown below,
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
Figure 2. The report of the synthesized protein was provided by Genescript
The protein's 0.8mg white lyophilized powder was dissolved by 800μL of Glacial Acetic Acid to 1mg/mL. Dissolved LL-37 was diluted into different concentrations (1.0mg/ml, 0.5mg/ml), and the S.aureus solution which OD600=0.48 was resuspended with the prepared LL-37 solution, water, and Glacial Acetic Acid with each of 200 μl (as controls), spreading to the four agar plates, respectively. The plates were cultivated under 37 degrees Celsius overnight.
Figure 3. Overnight S.aureus chromogenic plate of S.aureus treated with different dissolved LL-37 concentrations
(A)Glacial Acetic Acid (B)water (C) 0.5mg/ml LL-37 (D)1.0mg/ml LL-37
The result shows a success proof of LL-37's bactericidal effect.
LL-37 lysing effect towards S. aureus under different concentration
To gain a detailed view of the sterilization effect of LL-37 against S. aureus at different concentrations, overnight S. aureus culture was inoculated to fresh TSB medium their OD 600 value was tested. After the OD 600 value of cultures reaches 1.0, they are separated into 4 different sterilized tubes, 5 ml each. Then 100μl of Glacial Acetic Acid and dissolved LL-37(1mg/ml) are added to 4 tubes respectively, achieving different final concentrations (0.1mg/ml, 0.05mg/ml, 0.01mg/ml). The OD 600 values over time of all 4 tubes are recorded after 0.5min, 5min, 10min, 20min, and 30min being treated; each group repeated three times.
Figure 4. The OD600 value of S. aureus under different concentrations of LL-37 with respect to time
(error bars are too small to be visible)
The data clearly indicates the sterilization ability of LL-37. As the time increases, there is an obvious decrease in OD 600 values of S. aureus. At the beginning of the experiment, OD 600 values for all groups are close to 1.0, while the number drops to 0.8 for most groups and even 0.3 for the 0.1mg/ml group after 30 minutes. The effect of ll-37 at a concentration of 0.01mg/mL is not obvious since its concentration is too low for LL-37 to work normally.
Concluison
BNDS-China gains a different perspective on the sterilization effects of LL-37 targets S. aureus.
EMSA
In our project, we utilized Electrophoretic Mobility Shift Assay (EMSA) to assess the affinity capability of Protein A and PA#2/8, our target ssDNA. This technique enables us to examine the binding interactions between the probes and the target, which is reflected in the Gel-shift results.
In EMSA, complexes formed between proteins and nucleic acids maintain their integrity during electrophoresis. Consequently, they migrate more slowly through the gel compared to unbound nucleic acid probes. This differential migration allows for the separation and visualization of both complexes and free probes.
Generally speaking, followed the literature reviews, the samples are treated under the room temperature and incubated with EMSA reagents. An 6% polyacrylamide gel is used for electrophoresis step in EMSA. The protein a only and DNA only samples are set as control groups. The Gel running of EMSA is at 100V for 80 minutes with a pre-running of 10V for 10 minutes. Then, the gel is transferred to a pre-treated PVDF membrane under 160V for 60 minutes. The membrane is blocked to prevent non-specific binding, and then it is incubated with a strptavidin-HRP conjugate after the UV light for 15 minutes to achieve the highest conjugation effects. Finally, the membrane is developed after the chemiluminescent substrate treatment and displayed by X-ray film. See more information on the Result Page.
Figure 5. Results of EMSA. lane 1: only protein A
lane 2: protein A and PA#2/8
lane 3: PA#2/8
Troubleshooting Process:
1.Gel Preparation: Precise control of gel concentration during preparation is crucial. We observed that a high gel concentration can prevent probes from effectively entering the gel. After multiple rounds of troubleshooting using different percentages ranging from 4% to 12%, we determined that gel concentrations of 10% to 12% offered the cleanest resolution of complex and free probe bands for visualization.
Figure 6. The EMSA gel needs to be fixed
2. Sample Preparation: It is important to maintain the concentration of ssDNA within the range of 100ng/ul per sample. This may result in a situation where fluorescence is exceptionally high, which is typical for ssDNA.
3. Conjugation Time Adjustment: We found that extending the conjugation time was necessary under real-world conditions. The optimal conjugation time was determined to be within 15 minutes. Prolonging the conjugation time beyond this duration may lead to the drying of the sheet.
References
Ridyard, K. E., & Overhage, J. (2021, May 29). The potential of human peptide LL-37 as an antimicrobial and Anti-Biofilm agent. Antibiotics (Basel, Switzerland). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227053/