The dramatic rise of antimicrobial resistance has created the need for new approaches in the design of novel drug systems. Amphotericin B (AmB) is an antifungal drug that has been used clinically for over 50 years with only limited cases of clinical resistance reported. Due to the toxic nature of AmB's side effects, alternative antifungals have been developed, but treatment with these alternatives is already generating increasing numbers of resistant strains despite a much shorter period of use. This has led to a renewed interest in the study of AmB as a better understanding of its activity could inform on how to design more effective antimicrobials with lower occurrences of resistance. A wide range of investigations has resulted in three, very different, proposed models for AmB's mechanism of action. We are proposing a series of experiments based on transient absorption microscopy which will image label-free AmB as it interacts with both living and model systems. Our hypotheses are (1) that our proposed methodology will be able to distinguish between the different proposed models of AmB activity and (2) that the mechanism of action by which AmB interacts with a membrane exists as an environmentally driven interplay between the proposed models of ergosterol sequestration, ion channel formation, and oxidative damage. In the first specific aim of this project we will monitor the distribution and orientation of AmB in giant unilamellar vesicles and S. cerevisiae and C. albicans cells. The second specific aim will elucidate the role of aggregation in the mechanism of AmB's interaction. This aggregation is dependent on the environmental conditions making it a likely driver for which model of action AmB employs while also having important implications for the delivery of the drug. Overall, these innovative studies fit well within NIGMS's mission to lay the foundation for advancements in disease treatment and will provide hands-on training and education with cutting-edge imaging instrumentation for four students, including those at both the undergraduate and graduate levels.

Public Health Relevance

Amphotericin B is an important antifungal therapeutic, often used as a last line of defense for systemic fungal infections that has developed limited cases of clinical resistance despite decades of use. A better understanding of how this drug operates within cells could inform on what design principles may be necessary to incorporate into novel drug systems to reduce the occurrences of antimicrobial resistance. We are using transient absorption microscopy to directly image how amphotericin B acts in both model and living systems to achieve its effective behavior.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM119111-01
Application #
9099045
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
2016-08-01
Project End
2019-07-31
Budget Start
2016-08-01
Budget End
2019-07-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Tennessee Knoxville
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003387891
City
Knoxville
State
TN
Country
United States
Zip Code
37996