Insight into the Antifungal Mechanism of Action of Human RNase N-terminus Derived Peptides
Abstract
:1. Introduction
2. Results
2.1. Peptides Design and Physicochemical Characterization
2.2. Antifungal Activity against Planktonic Cells of hRNases Is Retained at the N-terminus
2.3. The Antifungal Mechanism of hRNase-Derived Peptides Relies on Membrane and Cell Wall Interactions
2.4. Nucleic Acids Binding of RN3 and RN7 the Potent Anticandidal Peptides
2.5. Evaluation of the Activities of RN3 and RN7 Peptides against C. albicans Biofilms
2.6. Modulation of the Gene Expression Profile Related to Cell Wall Synthesis and Biofilm Formation in Planktonic Cells
3. Discussion
4. Materials and Methods
4.1. Peptide Synthesis
4.2. C. albicans Growth Conditions
4.3. Minimum Fungicidal Concentration
4.4. Minimal Agglutination Activity (MAC)
4.5. Cell Viability Assay
4.6. Cell Survival Assay
4.7. Cell Membrane Depolarization Assay
4.8. Cell Membrane Permeabilization Activity
4.9. Cell Binding Assay
4.10. DNA Binding Assay
4.11. Gene Expression Analysis
4.12. Biofilm Growth
4.13. Biofilm Viability Test
4.14. Biofilm Mass Eradication
4.15. Confocal Microscopy
4.16. Cytotoxicity Assay
4.17. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
CFU | Colony forming units |
EDTA | Ethylenediaminetetraacetic acid |
SDS-PAGE | Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
ATP | Adenosine triphosphate |
DiSC3 | 3,3′-Dipropylthiadicarbocyanine Iodide |
IC | Inhibition Concentration |
MFC | Minimum Fungicidal Concentration |
MAC | Minimum Agglutination Concentration |
PI | Propidium iodide |
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Peptide † | Sequence | Hydrophobicity | pI | K/R | Protein Antimicrobial Activity * | Cytotoxicity (THP1) # GIC50 (μM) |
---|---|---|---|---|---|---|
RN1 | ---KESRAKKFQRQHMDSDSSPSSSSTYSNQMMRRRNMTQGRSKPVNTFVH | −1.575 | 11.40 | 4/6 | n.r | >50 |
RN2 | KPPQFTWAQWFETQHINMTSQ------QSTNAMQVINNYQRRSKNQNTFLL | −1.069 | 10.28 | 2/2 | - | >50 |
RN3 | RPPQFTRAQWFAIQHISLNPP------RSTIAMRAINNYRWRSKNQNTFLR | −0.764 | 11.88 | 1/7 | +++ Ŧ | >50 |
RN3K | KPPQFTKAQWFAIQHISLNPP------KSTIAMKAINNYKWKSKNQNTFLK | −0.824 | 11.02 | 8/- | +++ | >50 |
RN4 | --QDGMY-QRFLRQHVHPEET-GGSDRYSNLMMQRRKMTLYHSKRFNTFIH | −1.228 | 10.15 | 2/6 | n.r | >50 |
RN5 | --QDNSRYTHFLTQHYDAKPQ-GRDDRYSESIMRRRGLTS-PSKDINTFIH | −1.430 | 9.40 | 2/6 | - | >50 |
RN6 | WPKRLTKAHWFEIQHIQPSPL------QSNRAMSGINNYTQHSKHQNTFLH | −1.096 | 10.45 | 3/2 | +++ | >50 |
RN7 | KPKGMTSSQWFKIQHMQPSPQ------ASNSAMKNINKHTKRSKDLNTFLH | −1.209 | 10.75 | 6/1 | +++ Ŧ | >50 |
RN7R | RPRGMTSSQWFRIQHMQPSPQ------ASNSAMRNINRHTRRSRDLNTFLH | −1.302 | 12.88 | -/8 | +++ | >50 |
RN8 | KPKDMTSSQWFKTQHVQPSPQ------ASNSAMSIINKYTERSKDLNTFLH | −1.044 | 9.70 | 5/1 | + | >50 |
hRNase Peptide | MFC100 (μM) | IC50 (µM) | |
---|---|---|---|
Sabouraud Broth | PBS | ||
RN1 | 3.75 ± 0.50 | 3.75 ± 0.30 | 2.10 ± 0.20 |
RN2 | >20 | >20 | >20 |
RN3 | 0.62 ± 0.20 | 0.62 ± 0.10 | 0.22 ± 0.05 |
RN4 | 4.00 ± 0.50 | 3.75 ± 0.20 | 1.80 ± 0.30 |
RN5 | >20 | >20 | >20 |
RN6 | 2.00 ± 0.50 | 1.75 ± 0.30 | 1.80 ± 0.20 |
RN7 | 0.93 ± 0.20 | 0.93 ± 0.10 | 0.45 ± 0.10 |
RN8 | >10 | >10 | >10 |
hRNase Peptide | Membrane Depolarization | Membrane Permeabilization | Cell Survival | ||
---|---|---|---|---|---|
ED50 (µM) | Depolmax * | Max. Perm. (AU) 1 | Perm. (%) 2 | % ¥ | |
RN1 | 4.63 ± 0.70 | 65.2 ± 4.6 | 26.1 ± 0.4 | 8.3 ± 0.4 | 67 ± 1 |
RN2 | 3.63 ± 1.20 | 28.6 ± 5.7 | 45.2 ± 0.8 | 12.4 ± 0.2 | 72 ± 0.5 |
RN3 | 0.60 ± 0.40 | 100 ± 3.2 | 166 ± 2 | 73.2 ± 1 | 15 ± 2 |
RN4 | 3.58 ± 1.60 | 76.2 ± 4.8 | 117 ± 2 | 38.7 ± 0.1 | 65 ± 10 |
RN5 | 1.51 ± 0.10 | 39.1 ± 5.1 | 66.5 ± 0.6 | 21.8 ± 0.4 | 39 ± 0.5 |
RN6 | 0.62 ± 0.10 | 86.2 ± 5.2 | 135 ± 1 | 48.4 ± 0.8 | 26 ± 2 |
RN7 | 0.53 ± 0.18 | 71.5 ± 3.7 | 136 ± 0.4 | 53.9 ± 0.5 | 12 ± 1 |
RN8 | 0.15 ± 0.20 | 36.1 ± 4.4 | 33.4 ± 0.4 | 11.2 ± 0.2 | 28 ± 1 |
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Salazar, V.A.; Arranz-Trullén, J.; Prats-Ejarque, G.; Torrent, M.; Andreu, D.; Pulido, D.; Boix, E. Insight into the Antifungal Mechanism of Action of Human RNase N-terminus Derived Peptides. Int. J. Mol. Sci. 2019, 20, 4558. https://doi.org/10.3390/ijms20184558
Salazar VA, Arranz-Trullén J, Prats-Ejarque G, Torrent M, Andreu D, Pulido D, Boix E. Insight into the Antifungal Mechanism of Action of Human RNase N-terminus Derived Peptides. International Journal of Molecular Sciences. 2019; 20(18):4558. https://doi.org/10.3390/ijms20184558
Chicago/Turabian StyleSalazar, Vivian A., Javier Arranz-Trullén, Guillem Prats-Ejarque, Marc Torrent, David Andreu, David Pulido, and Ester Boix. 2019. "Insight into the Antifungal Mechanism of Action of Human RNase N-terminus Derived Peptides" International Journal of Molecular Sciences 20, no. 18: 4558. https://doi.org/10.3390/ijms20184558