Natural and Synthetic Ligand-Binding Induce Different Pilin Assemblies in vitro and Control P. aeruginosa Bioactivities in vivo, and Development of Bacterial-Motility Enabled Binding Assays
Date of Award
Doctor of Philosophy (PhD)
Olga V. Makhlynets
chemical biology, pili, Pseudomonas aeruginosa, rhamnolipids, swarming motility
Physical Sciences and Mathematics
Proteins found on bacterial cells surfaces are capable of sensing and transducing signals from the environment to elicit a biological response. Fibrous appendages formed by assembly of pilin proteins on P. aeruginosa are surface proteins that are necessary for host colonization, adhesion on abiotic surfaces, controlling motilities, formation of biofilm, horizontal gene transfer and virulence production of the bacterium. Upon contact with stimuli, pili appendages extend and retract on the cell surface, driven by the assembly and disassembly of pili at inner membrane of the bacteria cells. The dynamic response of this protein assembly is likely caused by a conformational change in the pilin monomers at the tip of the pili appendage upon making contact with a ligand or surface, as well as caused by chemical signals within the bacterium. Despite all the functions of pili identified, the natural and synthetic ligands specific for pilin proteins remain elusive. In this research, we report natural and synthetic ligands of pilin that control P. aeruginosa bioactivities.
For biochemical and structural studies on pilin protein, we used a common technique of recombinantly expressing truncated pilin in E. coli. The truncation of pilin from the N’-terminal α-helix retains the perceived binding region within the disulfide loop and yields a soluble pilin. Our approach of expressing truncated variants of the P. aeruginosa PA1244N3(pPAC46) and single amino acid mutants have demonstrated that pilin binds to the natural and synthetic ligands and that the disulfide loops plays an important role for this function.
Using a novel bacterial motility-enabled binding assay, we demonstrated that spreading expressed pilin monomers on the hydrated gel surface can inhibit the swarming motilities of the wild-type P. aeruginosa by binding and sequestering rhamnolipids secreted by the bacterium, or reactivate the swarming motility of the bacteria by sequestering the synthetic inhibitor added in the hydrated gel. Separating the components of rhamnolipids reveal that monorhamnolipid is more active than dirhamnolipid at controlling the swarming motility of P. aeruginosa.
Ligand-induced changes to pilin structures were detected by circular dichroism, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. Pilin monomers bind to the rhamnolipids at picomolar ranges and induce pilin proteins to form linear nano-assemblies. About one pico-molar of the ligands causes the transition of fluorescence signal to plateau for 100 nM of pilin monomers. This 10-5 equivalence effect is likely due to tight ligand-receptor binding rather that a catalytic effect based on titration studies. The mechanism of the assembly appears to be isodesmic and does not require a critical aggregation concentration to form linear assemblies. A class of synthetic ligands consists of saturated farnesol tethered with disaccharide also binds directly to pilin proteins at picomolar range by intrinsic fluorescence, and to dominate rhamnolipids resulting in complete inhibition of swarming motilities, and to induce the pilin proteins to form an amorphous assembly.
The nonamphiphilic chromonic salt, disodium cromoglycate (5’DSCG) was used to conduct preliminary crystallization studies on pilin from P. aeruginosa. We demonstrated that the 5’DSCG molecules demix in the presence of peptides and form isodesmic assemblies. This demixing phenomenon was then further explored to precipitate and crystallize pilin proteins. The resulting precipitates include radial precipitates for the native 1244 pilin and needle-like crystals for the truncated pilin. These results, along with past findings, suggest that 5’DSCG isodesmic assemblies has the potential to assist in protein purification and crystallization.
This work presents the use of a label-free bacterial motility enabled assay, together with biophysical techniques to provide a mechanistic understanding of the ligand binding between natural and synthetic ligands to pilin. The findings and methods in this study have potential use for the development and screening of therapeutics targeting the protein receptor that control the bioactivites of P. aeruginosa.
Ibañez, Arizza Chiara Siwa, "Natural and Synthetic Ligand-Binding Induce Different Pilin Assemblies in vitro and Control P. aeruginosa Bioactivities in vivo, and Development of Bacterial-Motility Enabled Binding Assays" (2020). Dissertations - ALL. 1222.