Part:BBa_K3078005

LL-37

2. Characterization

2.1 Validation of LL-37 construction

To verify the construction of pVE-LL-37 which we generated, the digestion by BglII/EcoRV was performed by a standard protocol followed by agarose gel electrophoresis (Figure 1).

Figure 1. Digestion and electrophoresis of pVE-LL-37.

2.2 Expression of pVE-LL-37

To detect the expression of pVE-LL-37 in E. coli, the constructs were transformed into BL21. Compared to the mock, there was a small size band shown in pVE-LL-37 (Figure 2).

Figure 2. Expression of pVE-LL-37. The bacteria were collected and ultrasonicated. The lysate was centrifuged and supernate was electrophoresed on the Tricine-SDS-PAGE gel, followed by Fast silver staining. The LL-37 peptide was synthesized as positive control.

2.3 Candidacidal effect of LL-37

To assess the candidacidal activity of LL-37, the spot assay was performed. As shown in Figure 13, C. albicans viability after LL-37 treatment was visually compared with that of control cells (no LL-37 treatment). We found that cell viability was more sensitive to LL-37.

Figure 3. Candidacidal activity of LL-37. C. albicans were collected and suspensions were resuspended to 105 cells/mL. 10 µL C. albicans suspension and 20 µL sterile BL21-transformed bacteria were gently mixed. After a 10-min incubation, the mixture was spotted onto the SDA agar plates. Cell viability was detected after incubation at 30 ℃ for 18 hrs.

To further validate the candidacidal effect of LL-37, C. albicans suspensions were incubated with it. After incubation, LL-37 caused an immediate increase in PI fluorescence indicates that LL-37 can kill C. albicans (Figure 4).

Figure 4. Candidacidal activity of LL-37. 1.5 µM PI (propidiumiodide) was added into C. albicans suspensions (106 cells/mL, 1 mL) and serially BL21-transformed bacteria supernatant (2 mL) mixture. The whole system was incubated at 30 ℃. During the 1 h incubation, the fluorescence intensity was measured every 5 min at the excitation wavelength λexc 544 nm and emission wavelength λem 620 nm. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test.

3. Conclusion

We successfully characterized LL-37 and demonstrated its ability to kill C. albicans from multiple angles.

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]

MIT_MAHE 2020

Summary

LL-37 is an antimicrobial peptide (AMP) from the cathelicidin family of antimicrobial peptides. It is a 37-residue helical peptide found throughout the human body, exhibiting a broad spectrum of antimicrobial activity as well as a variety of immunomodulating effects. LL-37 is able to form aggregates in solution and lipid bilayers and thus, unlike other antimicrobial peptides, is protected from proteolytic degradation. Exposure to LL-37 results in recruitment of inflammatory cells, induction of M1 macrophages and stimulation of inflammatory responses such as inflammasome activation and type I IFN production. LL-37 has strong anti-inflammatory effects such as neutralization of TLR4 activation by LPS, downmodulation of inflammatory cytokine responses and preventing invasion and inflammatory responses to pathogenic bacteria.

References

1. Kahlenberg, J. M., & Kaplan, M. J. (2013). Little peptide, big effects: the role of LL-37 in inflammation and autoimmune disease. Journal of immunology (Baltimore, Md. : 1950), 191(10), 4895–4901. https://doi.org/10.4049/jimmunol.1302005

2. Dürr, U. H., Sudheendra, U. S., & Ramamoorthy, A. (2006). LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochimica et biophysica acta, 1758(9), 1408–1425. https://doi.org/10.1016/j.bbamem.2006.03.030 "