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Dissertation
The Activation of VanR/VanS, the Two-Component System that Regulates Vancomycin Resistance
Doctor of Philosophy (Ph.D.), Drexel University
Jul 2020
DOI:
https://doi.org/10.17918/00000263
Abstract
VanR/VanS is the two-component system that regulates the expression of vancomycin-resistance genes in vancomycin-resistant enterococci (VRE). VanR is a transcription factor that initiates the transcription of the resistance genes upon phosphorylation. VanS is a membrane-bound sensor histidine kinase that modulates the phosphorylation state of VanR. VanS detects vancomycin and transduces that signal to activate VanR. A critical question that has dominated the field centers on how VanS senses vancomycin. We investigated whether vancomycin can directly activate the VanS proteins from the two most clinically relevant VRE types (VRE-A & VRE-B) by first reconstituting VanS_A and VanS_B into nanodiscs and then testing the effect of vancomycin on their enzymatic activities. We demonstrate that vancomycin stimulates VanS_B's autophosphorylation activity and slightly decreases its dephosphorylation activity. These effects would combine to enhance VanR_B phosphorylation and subsequently increase the expression of the resistance genes. The antibiotic's activation of purified VanS_B implies that vancomycin interacts directly with the histidine kinase; consistent with this, we have demonstrated that vancomycin binds the periplasmic sensing domain of VanS_B with a K_D ~ 20 [mu]M. Interestingly, VanS_A's activities are not modulated by vancomycin, showing that not all VanS proteins are activated by the same mechanism. In addition to functionally characterizing two VanS proteins, we have also structurally characterized the VanR protein from Streptomyces coelicolor in its inactive and activated states. We demonstrate that phospho-induced dimerization is the primary mode of activation. Our research provides a deeper understanding of how the individual proteins within the VanR/VanS two-component systems are activated.
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Details
- Title
- The Activation of VanR/VanS, the Two-Component System that Regulates Vancomycin Resistance
- Creators
- Lina J. Maciunas
- Contributors
- Patrick J. Loll (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- ix, 144 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991014695233604721