Assessing Permeability Across Bacterial Membranes

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Holsinger, Kadie, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Pires, Marcos, AS-Chemistry (CHEM), University of Virginia

Bacterial infections and the continual rise of antibiotic resistance pose a serious threat to healthcare worldwide. According to the Centers for Disease Control and Prevention’s (CDC) 2019 Antibiotic Resistance Threat Report, more than 2.8 million cases of antibiotic resistance are reported each year in the United States alone, causing more than 35,000 deaths. While scientists had previously made progress in combatting antibiotic resistance, the CDC states that the emergence of COVID-19 in 2020 caused a serious setback. The clear threat of antibiotic resistance calls for the development of new antibiotics. However, in order for drugs to be most effective, they must permeate across the bacterial cell membrane. Thus, determining drug modifications to make them more permeable and being able to monitor this permeability are of utmost importance in this fight against antibiotic resistance.

In this work, we studied modifications that eliminate a hydrogen-bond donor from peptides to improve their permeability into mycobacteria, which is the class of bacteria responsible for tuberculosis. By eliminating a hydrogen, the peptide forms fewer hydrogen bonds with the solvent, owing to a smaller desolvation penalty when the peptide enters the cell. The main modification studied here to eliminate the hydrogen bond donor was N-methylation. The nitrogen atoms in the backbone of peptides were systematically N-methylated and their permeability was tested using a click-chemistry based fluorescent assay. This study showed that increasing the methylation degree of a peptide increases the permeability across the mycomembrane to a certain extent. However, the addition of several methyl groups could potentially make the peptide too lipophilic, causing the peptide to interact with the mycomembrane and not permeate across. Another method studied here to eliminate hydrogen-bond donors was peptoid substitutions. Since peptoids are amino acid analogs with the side chain attached to the backbone nitrogen, the replacement of amino acids with their peptoid analogs allows for the elimination of a hydrogen-bond donor without the addition of any group to the peptide. Preliminary results with peptoid substitutions showed that this method also has the ability to modulate permeability of the molecules across the mycomembrane.

Testing permeability into Gram-negative bacteria is also an important step in drug development for treating antibiotic-resistant infections. Current methods for assessing permeability into E. coli cells are limited since they tend to have large modifications, high background, and/or low throughput. Thus, we proposed a novel assay for monitoring molecule permeability into Gram-negative bacteria to address these limitations. This assay involves the expression of the enzyme luciferase in the cytosol of E. coli cells. Then, the cells are incubated with two molecules that only undergo a click-chemistry reaction in the cytosol to form the substrate of luciferase, allowing this assay to report on permeability to the cytosol. To develop this assay, we determined the optimum induction time for protein expression. We also determined the best variant of CBT and the appropriate concentration to use. We then tested the assay on D-cystine and a molecule that is known to permeate into Gram-negative bacteria. The results from these two molecules showed the ability of this assay to monitor permeability of molecules into the cytosol of E. coli in a quick, high throughput method.

MS (Master of Science)
Permeability, Mycomembrane, Luciferase, Gram-negative bacteria, Luminescence, Peptidoglycan
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