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The topic of this research was initiated by the discovery, in part by Dr. David Giles, that Vibrio cholerae, the enteric bacteria responsible for the disease cholera, has the ability to uptake and utilize long chain polyunsaturated fatty acids up to 22 carbon atoms long. Before this discovery was made, conventional belief was that fatty acids of greater than 18 carbons long were toxic to bacteria. Further investigations have revealed that this uptake is not unique to Vibrio cholerae, and a range of bacteria such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and even Escherichia coli have been found to uptake and use these longer chain fatty acids to varying degrees. This uptake has been linked to increased antimicrobial resistance, motility, biofilm formation, and virulence phenotypes. This research was a collaboration between Dr. David Giles and Dr. Bradley Harris to computationally investigate the uptake mechanism for the uptake of long chain fatty acids, as well as the effects these long-chain fatty acid integrated phospholipids have on antimicrobial resistance. The investigation was performed in two parts; the first involving the uptake of fatty acids by the long chain fatty acid transport protein FadL; the second a membrane system interacting with cationic antimicrobial peptides. The FadL uptake study involved the atomistic level modelling of various FadL homologs found in virulent V. cholerae serotypes. Docking of equilibrated FadL homologs presented information on the binding energies and binding locations of each fatty acid tested for a wide range of conformations. The summation of the binding locations revealed preferred binding sites and oftentimes the channels between them. To investigate cationic resistance, atomistic membrane systems were generated with varying fatty acids replacing the phospholipid tail groups. For each of the test membranes, various cationic antimicrobial peptides were pulled through the membrane systems using steered molecular dynamics. The membrane permeability for each fatty acid was analyzed from the energy required to move the cationic peptide through the membrane as well as the response of the membrane structure as the peptide passed through. Future suggested studies would include steered molecular dynamics for fatty acid transport through each FadL homolog. This would verify the found channels and investigate any conformational shifts in structure to accommodate the passage of the fatty acid. For the membrane permeability study, additional peptides could be tested as well as systems using the outer membrane of gram-negative bacteria. Additional studies using less complex membrane leaflets may also be insightful.

Gram-negative bacterial infections are a worldwide problem and antibiotics capable of clearing such infections are an unmet clinical need. The polymyxins are highly potent drugs against Gram-negative bacteria but suffer from dose-limiting toxicity. Although the exact mechanism for polymyxin activity is unclear, it is known that polymyxin binds lipopolysaccharide. Lipopolysaccharide is a complex glycolipid that is synthesized inside the cell and transported through three cellular compartments to the cell surface by the seven essential lipopolysaccharide transport (Lpt) proteins. We discovered that novobiocin, a gyrase inhibitor, also binds the inner membrane Lpt complex LptB2FGC and stimulates lipopolysaccharide transport. The central finding of this dissertation is that stimulation of lipopolysaccharide transport by novobiocin derivatives sensitize bacteria to the polymyxins. The thesis presents preliminary evidence that polymyxin therapy can be safer when used in combination with a novobiocin derivative. We demonstrated that novobiocin derivatives that bind LptB2FGC and stimulate lipopolysaccharide transport can synergize with polymyxin B independent of gyrase inhibition. In Chapter 2, a semi-synthetic route was used to modify the novobiocin scaffold at the sugar and benzamide positions. An NMR binding assay was combined with a previously published cellular assay for measuring lipopolysaccharide transport to assess the ability of novobiocin derivatives to maintain their target engagement on LptB2FGC. In Chapter 3, we showed that novobiocin derivatives that maintained their ability to stimulate lipopolysaccharide transport synergized with polymyxin. By adding these two drugs together in a thigh infection model, we demonstrated that combination therapy is a therapeutically viable strategy for making polymyxin efficacious at a lower dose and therefore safer. Previously published work suggests that active lipopolysaccharide transport is important in the mechanism of action of the polymyxin antibiotics and ongoing work suggests that polymyxin acts at the inner membrane. In Chapter 4, we tethered novobiocin and polymyxin to leverage the co-localization of these molecules and their targets in the inner membrane, and we describe an efficient strategy to synthesize chimeric molecules in a modular fashion. This dissertation validates a novel therapeutic modality and describes the development of new chemical matter to treat Gram-negative infections by targeting active lipopolysaccharide transport.

Abstract: Polymyxins are cationic lipopeptide antibiotics, and are considered to be drugs of last resort to fight against multidrug resistant Gram negative bacteria. In this study the gene cluster encoding the non-ribosomal peptide synthetase which catalyses polymyxin biosynthesis was identified and sequenced in Paenibacillus polymyxa PKB1. The gene cluster consists of three biosynthetic genes and two ABC transporter-like genes. Disruption of a biosynthetic gene resulted in complete loss of antibiotic production whereas deletion of one or both transporter genes resulted in reduced antibiotic production. Two antibacterial compounds were identified in the wild type culture supernatants by HPLC analysis and agar diffusion bioassay. LC-MS and bioinformatic analysis suggested these compounds to be analogs of polymyxin B2 and B1, containing a D-DAB instead of L-DAB residue in position three of the molecules.