Date of Award

December 2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Yan-Yeung Luk

Second Advisor

Anthony Garza

Keywords

Biofilm, Pseudomonas aruginosa, Swarming, Synthesis

Subject Categories

Physical Sciences and Mathematics

Abstract

Throughout the human history, the fight against bacterial infections had never stopped but the remedies for bacterial infections were often insufficient and for many infectious diseases, there was no treatment available. The revolution in antimicrobial infection therapy began with the discovery of penicillin by Alexander Fleming in 1928. However, since the first introduction of antibiotics, bacteria over time have evolved sophisticated resistant strains against almost all the available antibiotics which cause selection pressure on the bacteria to evolve their genetic makeup and develop resistance against such agents. Furthermore, bacteria can form surface attached multicellular communities known as biofilms. Bacteria residing within biofilms are protected by biofilms which renders the bacteria more difficult to eliminate because of the low permeability of antibiotics through outer membranes. Combating such resistant bacteria is an extremely difficult task if using antibiotics alone. Hence scientific community continuously seeks new strategies to overpower these resistant bacteria.

The focus of the research work presented here is to develop a class of chimera ligands that can bind to both pili and LecA protein of Pseudomonas aeruginosa to inhibit both swarming motility and biofilm formation. The potential adjuvant agents of these chimera ligands that can increase the effectiveness of antibiotics were demonstrated. In addition, the ability of our adjuvant molecules to eliminate drug-tolerant bacteria and to reduce persisters, in combination with antibiotics was demonstrated.

The binding property of chimera ligands was demonstrated by competitive fluorescence polarization assay (LecA) and by adding a functional group to a ligand that can covalently attach to the receptor protein only when the physical ligand-receptor binding takes place (Pili). In addition, the effect of externally added pili on the swarming motility of Pseudomonas aeruginosa was tested to support the mechanistic study of the pili as the receptor (or one of the receptors) that will bind to rhamnolipids and our synthetic agents, and upon binding, causing the bacterial activities.

For quantification of polysaccharides, two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide were described. Both approaches provide efficient methods for monitoring alginate production by mucoid Pseudomonas aeruginosa.

The effect of a class of synthetic analogs of rhamnolipids at controlling (promoting and inhibiting) the biofilm formation activities of a non-rhamnolipid-producing strain – rhlA – of Pseudomonas aeruginosa was demonstrated. The bioactive synthetic analogs of rhamnolipids promote biofilm formation by rhlA mutant at low concentrations but inhibit the biofilm formation at high concentrations. To explore the internal structures formed by the biofilms, the wild-type biofilms formed with substantial topography (hills and valleys) when the sample is under shaking conditions were observed by confocal microscope. Using this observation as a comparison, the effect of synthetic analogs of rhamnolipids on promoting structured (porous) biofilm of rhlA mutant, at intermediate concentrations between the low ones that promoted biofilm formation and the high ones that inhibited biofilm formation was demonstrated. This study suggests a potential chemical signaling approach to control multiple bacterial activities.

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