Date of Award
Doctor of Philosophy (PhD)
Luk, Yan-Yeung Y. L.
Castañeda, Carlos A.
Chemistry | Physical Sciences and Mathematics
The fight against bacterial infections and innovations in antibiotic therapy has never halted throughout human history. However, bacteria have evolved smartly and resistance against practically all conventional antibiotics has been developed. Furthermore, bacteria can form biofilms, which are surface-attached multicellular colonies. Biofilms shield bacteria against antibiotics and makes it harder to entirely eradicate infections . Under antibiotics stress, bacteria evolve into different phenotypes like hyper motile, hyper adherent and hyper virulent which are tolerant and persistent to antibiotic treatment. The use of antibiotic therapy to combat such resilient bacterial phenotypes is extremely tough. As a result, the scientific community is always looking for novel ways to defeat these resilient phenotypes. It important to notice that all antibiotic induce tolerant and persistent phenotypes and there is no direct remedy for eradication of these phenotypes. On other hands, resistance to one antibiotic can be tackled by alternative antibiotic usage.Here, we demonstrate a class of small molecules that inhibits a wide range of phenotypes of Pseudomonas aeruginosa and enables the antibiotics to kill tolerant bacteria and to prevent formation of new persistent bacteria. We identified two proteins, type IV pili and lectin LecA as receptors for our molecules by different methods, including a new label-free assay based on bacterial motility sensing the environment, chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure-activity relationship studies reveal a molecule that inhibits only pili appendage, and a class of chimeric ligands that inhibit both LecA and pili, with important structural elements of the ligand identified for each protein. This selective control of two proteins makes the correlation between the protein receptor to their controlled phenotypes. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and correlates with killing tolerant bacteria and preventing the development of new persistent bacteria. Inhibiting pili appendages impedes the swarming and twitching motilities, and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously control the multiple signaling mechanisms, by which bacteria elude antibiotic treatments. Glycolipid, ganglio-N-tetraosylceramide (asialo-GM1), on the mammalian cells are known to be recognized by type IV pili of Pseudomonas aeruginosa. In this work, we show that asialo-GM1 can also be recognized by Lectin A (LecA), another adhesin protein of the P. aeruginosa, by a fluorescent polarization assay, a label-free bacterial motility enabled binding assay, and bacterial mutant studies. On hydrated semi-solid gel surfaces, asialo-GM1 enables swarming and twitching motilities, while on solid surfaces facilitates the bacterial adherence of P. aeruginosa. These results indicate that asialo-GM1 can modulate bioactivities, adherence, and motilities, that are controlled by opposite signaling pathways. We demonstrate that when a solution of pilin monomers or LecA proteins are spread on hydrated gel surfaces, the asialo-GM1 mediated swarming motility is inhibited. Treatment of artificial liposomes containing asialo-GM1 as a component of lipid bilayer with pilin monomers or LecA proteins caused transient leakage of encapsulated dye from liposomes. These results suggest that pili and LecA proteins not only bind to asialo-GM1 but can also cause asialo-GM1 mediated leakage. We also show that both pili and LecA mutants of P. aeruginosa adhere to asialo-GM1 coated solid surfaces, and that a class of synthetic ligands for pili and LecA inhibits both pili and LecA-mediated adherence of P. aeruginosa on asialo-GM1-coated surfaces. The inspired by adjuvant molecule's control over different P. aeruginosa phenotypes, we here designed new class of antibiotics which showed broad spectrum activity against different bacteria. This class of antibiotics inhibits pili and lecA and hence control multiple phenotypes in P. aeruginosa. This antibiotic also did not show emergence any tolerant population. We also did not develop any resistance against this class of antibiotics after repeated exposure. Silver therapy is most commonly used against burn wounds to treat P. aeruginosa and S. aureus infections. These bacteria develop cooperative resistance against silver with the help of pyocyanin, a virulence factor of P. aeruginosa. Here, we showed Farnesol as adjuvant molecule with silver therapy against P. aeruginosa and S. aureus. Farnesol eradicates resistance against silver by inhibition of pyocyanin. Farnesol also potentiated silver against dual biofilms of P. aeruginosa and S. aureus.
Patil, Pankaj Dinkar, "A New Class of Antibiotic That Prevents Drug Tolerance, Persistence, and Resistance By Controlling Emergence of Phenotypes." (2022). Dissertations - ALL. 1566.