Saved in:
| Main Authors: | , |
|---|---|
| Format: | Recurso digital |
| Language: | |
| Published: |
Zenodo
2025
|
| Online Access: | https://doi.org/10.5281/zenodo.15743861 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
Table of Contents:
- <p>The emergency of antibiotic resistance is serious. We must identify molecules that escape the self-defense mechanisms of microorganisms. In this study, we optimized a protocol for identifying Streptococcus mutans proteins impaired by manool, a metabolite from Salvia officinalis with antimicrobial activity. The research showed that S. mutans can evade antibiotics and grow. This study shows the potential of manool as a new antimicrobial agent and the importance of understanding the molecular mechanisms of plant-derived compounds in the fight against AMR. Samples were obtained using the Drug Affinity Responsive Target Stability (DART-S) assay. A new protocol was performed for bacterial cells that allows the DARTS assay to be applied to a prokaryotic model using a minimal medium. The DARTS assay was initially performed by incubating manool with a protein extract (pe-DARTS) from S. mutans, followed by limited proteolysis, SDS-PAGE analysis of digestion products, and proteomic identification of the separated proteins. The bc-DARTS assay involved the incubation of manool with Streptococcus mutans bacterial cells, followed by proteolysis, SDS-PAGE analysis, and LC-MS. The S. mutans overnight inoculum was centrifuged at 3000 rpm for 15 minutes, and the cells were resuspended in BHI medium to a density of 0.05 OD/ml. The cells were incubated at 37°C and cultured to the established optical densities (0.5 OD/ml and 1 OD/ml). Then cells were centrifuged at 3000 rpm for 15 minutes. After discarding the supernatant, the pellet was resuspended in M9 minimal medium 1X supplemented with 0.4% glucose for both control and treated samples, at a density of 0,5 OD/ml and 1 OD/mL. The treated samples were incubated with manool at 5 µM and 10 µM for 90 minutes.<br><br></p>