Title

Structure-activity Relationships Of Pili-binding Ligands Reveals Mechanisms For Controlling Phenotypes Of Pseudomonas Aeruginosa

Date of Award

Spring 5-22-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Yan-Yeung Luk

Subject Categories

Biology | Chemistry | Life Sciences | Organic Chemistry | Physical Sciences and Mathematics

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that particularly poses a threat to immuno-compromised individuals. The species can undergo multicellular events that enhance its pathogenesis and resistance including some forms of motility and the formation of biofilm communities. Under external stresses, such as antibiotic treatment, immune cells, starvation or low oxygen supply, the pathogen can survive by acquiring new phenotypes that involve genetic mutations or morphological adaptations. As a result, P. aeruginosa can demonstrate enhanced multi-drug resistance and in many cases can be very difficult to eradicate. The focus of this work is to explore the structure activity relationships of small molecule inhibitors of the pathogenic activities that contribute their virulence and survival, and to explore and discover the protein receptors for these molecules, which were not known. Alginate-overproducing mucoid is an example of a highly resistant clinical strain that is associated with chronic infections. Here we describe a class of small molecules, polar hydrocarbons, that delay and inhibit mucoid alginate production. Among these molecules, specific benzophenone-derivatives (BPDs) induce swarming motility on soft agar. By activating swarming motility, BPDs revert the mucoid phenotype to non-mucoid, reducing alginate production to the level of wild type bacteria. We also investigate the radical quenching nature of BPDs as a possible mechanism for their control. Structure-activity studies of the BPDs suggest that specific ligand-receptor binding interactions are required to induce phenotypic reversion. To identify their protein receptors, we designed a bacterial motility enabled binding (BMEB) assay to validate two receptors for these ligands, pilin and LecA. Luk lab has previously demonstrated that the class of polar hydrocarbons that inhibit mucoid alginate production also control other bacterial activities that are influenced by rhamnolipids - polar hydrocarbons that are naturally secreted by P. aeruginosa. We have synthesized a library of rhamnolipid analogues and discovered new compounds that dominate the activities of rhamnolipids. Organic synthesis provides an opportunity to optimize their structures. To this end, we make systematically branched hydrocarbons, and substitute them on an activity-validated lead compound – saturated farnesyl-β-maltose (SFβM). We establish a correlation between the structure and function of these polar hydrocarbons by studying their effects on the ability of P. aeruginosa to swarming and form biofilms. Another behavior that we study in P. aeruginosa is filamentous growth. In the presence of low levels of certain antibiotics, gram negative bacteria can switch to a filamentation phenotype characterized by elongation of cells, and changes in virulence of the pathogen. Filamentation is believed to be a surface-mediated phenomenon. Here we confirm that surface attachment does promote filamentation in P. aeruginosa. This discovery suggests that adhesins on the bacterial surface, specifically pili appendages, are essential components of the filamentation process. We present the pili as a new target for the control of virulence in antibiotic-induced filaments by demonstrating that the growth of filaments is pili-mediated and is also promoted by specific ligands that bind to pili on the bacterial surface. Collectively, this work provides a chemical approach to controlling bacterial phenotypes and bioactivities of P. aeruginosa and rigorously defines the structures that can mediate these processes.

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