Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens
Abstract
:1. Introduction
2. Results
2.1. Bioinformatic Screen of Crocodylian Genomes Identified 18 Novel crocCATHs
2.2. Mature crocCATHs Displayed Characteristic Properties of α-Helical Cathelicidins
2.3. As-CATH8 and Gg-CATH5 Exhibited Broad In Vitro Activities against Planktonic and Biofilm Bacteria
2.4. As-CATH8 and Gg-CATH5 Completely Eradicated S. aureus Biofilms in a Human Organoid Skin Model
2.5. As-CATH8 Showed a Strong Anti-Infective Effect in a Murine Abscess Model
2.6. As-CATH8 Was More Bactericidal and Killed Faster Than Antibiotics
2.7. Potential Role of Interaction with Bacterial Membranes and DNA in the Antibacterial Activity of As-CATH8
3. Discussion
4. Material and Methods
4.1. Identification of Cathelicidin Sequences Using HMMs
4.2. Phylogenetic Analysis of the Cathelicidin Sequences
4.3. Prediction of Physicochemical and Structural Characteristics of Mature crocCATHs
4.4. Peptides, Reagents and Culture Media
4.5. Determination of the Secondary Structure of Synthetic Cathelicidins
4.6. Bacterial Strains and Culture Conditions
4.7. Antimicrobial Activity and Biofilm Inhibition Assays
4.8. Cell Lines and Peripheral Blood Mononuclear Cells (PBMCs)
4.9. Lactate Dehydrogenase (LDH) Release Assays
4.10. Skin Model Experiments
4.11. Bacterial Abscess Formation and Peptide Treatment
4.12. Planktonic and Biofilm-Killing Assays
4.13. Membrane Depolarization and Permeabilization Assays
4.14. Agarose Gel Electrophoretic Mobility Shift Assay
4.15. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Sequence | Length | MW | Charge | HI | HM |
---|---|---|---|---|---|---|
As-CATH7 | KRVNWRKVGRNTALGASYVLSFLG | 24 | 2693 | 4.76 | −0.15 | 0.25 |
As-CATH8 | KRVNWAKVGRTALKLLPYIFG | 21 | 2431 | 4.76 | 0.06 | 0.29 |
Gg-CATH5 | TRRKWWKKVLNGAIKIAPYILD | 22 | 2670 | 4.76 | −0.39 | 0.40 |
Gg-CATH7 | KRVNWRKVGLGASYVMSWLG | 20 | 2308 | 3.76 | −0.11 | 0.23 |
LL-37 | LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES | 37 | 4493 | 5.76 | −0.72 | 0.56 |
Bacteria | MIC in μM | |||||
---|---|---|---|---|---|---|
As-CATH7 | As-CATH8 | Gg-CATH5 | Gg-CATH7 | Polymyxin B | Vancomycin | |
E. cloacae | 0.5 | 0.5 | 0.5 | 2 | 0.5 | n.d. |
S. aureus | 4 | 0.5 | 1 | 16 | n.d. | 0.5 |
K. pneumoniae | 1 | 0.5 | 0.5 | 4 | 0.5 | n.d. |
A. baumannii | 0.25 | 0.25 | 0.5 | 1 | 0.5 | n.d. |
P. aeruginosa | 4 | 1 | 1 | 8 | 0.5 | n.d. |
E. faecium | >64 | >64 | >64 | 64 | n.d. | >64 |
E. coli | 2 | 1 | 4 | 4 | 0.5 | n.d. |
S. Typhimurium | 2 | 1 | 0.5 | 4 | 1 | n.d. |
P. vulgaris | >64 | 4 | 8 | >64 | >64 | n.d. |
Bacteria | MBIC95 in μM | |||
---|---|---|---|---|
As-CATH7 | As-CATH8 | Gg-CATH5 | Gg-CATH7 | |
E. cloacae | 32 | 4 | 4 | 16 |
S. aureus | 4 | 1 | 1 | 4 |
A. baumannii | 1 | 0.5 | 0.5 | 1 |
P. aeruginosa | >64 | 64 | 32 | >64 |
E. coli | 32 | 1 | 32 | 32 |
S. Typhimurium | 1 | 1 | 1 | 4 |
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Santana, F.L.; Estrada, K.; Alford, M.A.; Wu, B.C.; Dostert, M.; Pedraz, L.; Akhoundsadegh, N.; Kalsi, P.; Haney, E.F.; Straus, S.K.; et al. Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens. Antibiotics 2022, 11, 1603. https://doi.org/10.3390/antibiotics11111603
Santana FL, Estrada K, Alford MA, Wu BC, Dostert M, Pedraz L, Akhoundsadegh N, Kalsi P, Haney EF, Straus SK, et al. Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens. Antibiotics. 2022; 11(11):1603. https://doi.org/10.3390/antibiotics11111603
Chicago/Turabian StyleSantana, Felix L., Karel Estrada, Morgan A. Alford, Bing C. Wu, Melanie Dostert, Lucas Pedraz, Noushin Akhoundsadegh, Pavneet Kalsi, Evan F. Haney, Suzana K. Straus, and et al. 2022. "Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens" Antibiotics 11, no. 11: 1603. https://doi.org/10.3390/antibiotics11111603