Author ORCID Identifier

https://orcid.org/0000-0002-5774-6431

Date Available

4-29-2022

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department/School/Program

Chemistry

First Advisor

Dr. Yinan Wei

Abstract

The outer membrane of Gram-negative bacteria (GN) makes them distinct among superbugs that are associated with the development of antibiotic resistance. The outer membrane, and inner membrane, separated by the periplasm, form a double-membrane barrier to the entry of antibiotics into the cell. Several studies have been conducted to examine the role of outer membrane modifications such as porins, lipopolysaccharides, and efflux pumps on antibiotic resistance. However, there is a paucity of knowledge on how antibiotics behave in the periplasm, to gain access into their target region. My thesis focuses on understanding the mechanism of antibiotic permeability through the cellular envelope of Gram-negative bacteria.

I studied the distribution of fluoroquinolones in the two aqueous compartments (periplasm and cytoplasm) of Escherichia coli using fluorescence intensity measurement and minimum inhibitory concentration (MIC) test. We treated the bacteria cells with each antibiotic, allowed the antibiotic to accumulate in the cells, fractionated the cells and quantified the concentration of accumulated antibiotic through the measurement of its fluorescence intensity. The compound accumulation assay showed that the efflux-deficient strain (DtolC) accumulated more antibiotic than the wild type (WT) strain, for all nine fluoroquinolones tested. An analysis of the subfractions showed a greater accumulation of the antibiotic in the periplasm than in the cytoplasm. A positive correlation was observed between the MIC ratio (WT/DtolC) and the cytoplasmic accumulation ratio (DtolC/WT). This is an indication of the importance of measuring accumulation in the target rather than the whole cells.

I also studied the impact of osmo-regulated periplasm glucans (OPGs) on antibiotic susceptibility in GN. We created E. coli and Salmonella Typhimurium strains deficient in OPG production. We also created an E. coli strain that produced neutral OPGs. The drug susceptibility test showed that the strains that are either deficient in OPG production or produce neutral OPGs were less susceptible to some of the positively charged aminoglycosides compared to the WT strain. A similar response was observed when the bacteria strains were treated with the fluoroquinolones and tetracyclines. We speculate that this behavior is due to the net positive charge carried by these antibiotics from complexes formed with Mg2+. In contrast, the strains grew slower in the presence of negatively charged cefuroxime and neutral linezolid. The observed MIC changes were not due to a leaky membrane. The OPG deficient strains produced significantly reduced amount of OPGs compared to the parent strain. Our study demonstrated that charge plays a significant role in OPG-mediated susceptibility to antibiotic.

We also probed the role of OPGs on copper homeostasis in GN bacteria. We found that the disruption of the opgGH operon had an impact on the tolerance of GN bacteria to copper. Copper quenched the fluorescence of cytosolic GFP faster in the OPG- deficient strains compared to the WT strain. The GFP-quenching effect of copper ions was diminished with the increase of ionic strength, indicating that Cu2+ penetration into the cytoplasm slowed down under high salt condition.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.127

Funding Information

This study was funded by National Science Foundation (CHE-1709381), National Institutes of health/National Institute of Allergy and Infectious Diseases (AI137020 and AI142063), National Institutes of health/national Heart, Lung, and Blood Institute (HL142640), National Institutes of health/National Institute of General Medical Sciences (GM132443), and National Institutes of health (1R56AI137020, 1R21AI142063-01).

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