Doctoral Dissertations

Date of Award

5-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Shawn R. Campagna

Committee Members

Elizabeth M. Fozo, Micheal D. Best, Thanh D. Do

Abstract

The lipid membrane is the first component necessary to sustain life. To maintain homeostasis, segregate cellular machinery, provide protection from the environment, and reproduce, an organism must establish a boundary in which the processes can occur. Throughout the last two decades, research has propelled our knowledge of lipid membranes much beyond original hypotheses. Once thought of to be static and uniform, the understanding of the lipid membrane has evolved to encompass a structure that is responsive, unique, and intricately constructed by the organism itself. By chance or by choice, organisms adapt the lipid membrane according to the environment for which they are in. Many of these adaptations or alterations of the lipid membrane have been implicated to play a large role in human health and disease pathology; one example includes the ability of bacteria to circumvent antimicrobial drugs by altering cellular membrane components. In the chapters following, the unique link between fatty acid supplementation and antimicrobial drug tolerance will be investigated utilizing a variety of techniques including ultra-performance liquid chromatography high resolution mass spectrometry, genetic mutagenesis, and stable isotope tracing.

Daptomycin is a lipopeptide designed to target Gram-positive bacteria, yet since the discovery of daptomycin the 1980’s, instances of resistance or tolerance to the drug have been reported in several strains of Enterococci. Despite the efforts of many researchers, the mechanism by which resistance or tolerance occurs in Enterococcal strains is not yet understood although significant links to lipid membrane alterations have been reported. Chapter 1 demonstrates how daptomycin impacts the membrane of both susceptible populations and fatty acid protected populations. Susceptible and protected bacterial populations responded to daptomycin by altering percent of total lipid species present; therefore, a genetic approach was utilized to investigate the role of individual lipid species in Chapter 2. Mutagenesis studies carried out in Chapter 2 demonstrated the ability of E. faecalis to respond to genetic and environmental perturbation. Additionally, specific strains generated in this chapter had altered survival in the presence of membrane damaging agents. Chapter 3 focused on the design of an analytical technique capable of tracing basal and environmental nutrient fluxes using stable isotopes. Results from this study concluded E. faecalis is capable of altering its membrane in the presence of oleic acid by placing oleic acid onto acyl tails of lipids species as well as direct incorporation into the membrane as free fat.

Together, this body of work highlights the importance of cross discipline collaboration in efforts to improve the quality and longevity of human life. Yet at a fundamental level, this work reminds us to always consider parts of science others may rule out as lackluster.

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