UC Irvine

Antibiotic resistant bacteria are an increasing threat to global human health. These drug resistant infections are challenging to treat and contain, and the antibiotic discovery pipeline does not match the pace of the growing number of antibiotic resistant bacteria. In Chapter 1, I summarize the outlook of antibiotic resistance and how the new antibiotic, teixobactin, serves as a promising template to combat drug resistant infections. Teixobactin is a peptide antibiotic that kills Gram-positive bacteria without detectable resistance and is able to kill pathogens that are considered as urgent and serious threats by the CDC. I provide a summary of the mechanism of action of teixobactin, structure-relationship studies of its pharmacophore, and various synthentic methods to make the antibiotic. In the last section of this chapter, I review how fluorescent antibiotics are versatile probes to understand the mode of action of antibiotics, elucidate off-target effects (toxicity studies), detect antibiotic resistance, and serve as diagnostics for detecting infections in-vivo or ex-vivo.

Chapter 2 describes the first synthesis and application of a fluorescent teixobactin analogue that exhibits antibiotic activity and binds to the cell walls of Gram-positive bacteria. The teixobactin analogue, Lys(Rhod)9,Arg10-teixobactin, has a fluorescent tag at position 9 and an arginine in place of the natural allo-enduracididine residue at position 10. The fluorescent teixobactin analogue retains partial antibiotic activity, with minimum inhibitory concentrations of 4–8 µg/mL across a panel of Gram-positive bacteria, as compared to 1–4 µg/mL for the unlabeled Arg10-teixobactin analogue. Lys(Rhod)9,Arg10-teixobactin is prepared by a regioselective labeling strategy that labels Lys9 with an amine-reactive rhodamine fluorophore during solid-phase peptide synthesis, with the resulting conjugate tolerating subsequent solid-phase peptide synthesis reactions. Treatment of Gram-positive bacteria with Lys(Rhod)9,Arg10-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)9,Arg10-teixobactin and BODIPY FL vancomycin results in septal co-localization, providing further evidence that Lys(Rhod)9,Arg10-teixobactin binds to cell wall precursors. Controls with either Gram-negative bacteria, or an inactive fluorescent homologue with Gram-positive bacteria, showed little or no staining in fluorescence micrographic studies. Lys(Rhod)9,Arg10-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.

Chapter 3 describes a new approach to selectively label Lys9,Arg10-teixobactin and Lys10-teixobactin with a variety of N-Hydroxysuccinimide (NHS) ester fluorophores. The reaction affords regioselective labeling of the lysine sidechain amines of either Lys10- or Lys9,Arg10-teixobactin. Using this labeling method, we were able to generate four fluorescent teixobactin analogues, bearing different fluorophores, that retain antibiotic activity and stain the cell walls of Gram-positive bacteria. This approach also enabled us to determine that position 10 tolerates fluorophores better than position 9, using MIC assays and fluorescence microscopy as readouts. Structured illumination microscopy of the fluorescent teixobactin analogues in live B. subtilis cells enabled further study of the aggregation of teixobactin in bacterial membranes, with observation of both clusters and aggregates of the antibiotic in bacterial membranes. To further understand the aggregation of teixobactin in live bacteria, we used fluorescence lifetime imaging Förster resonance energy transfer (FLIM-FRET) microscopy, which demonstrated that fluorescent teixobactin analogues are in intimately interacting with each other in live cells. Lastly, we treated NRK-52E rat kidney cells with our fluorescent teixobactin analogues to determine if teixobactin had any nephrotoxic effects due to its unfavorable aggregation propensity. Taken together, we demonstrate useful applications of fluorescent teixobactin analogues that enabled elucidation of its on-target and off-target effects in bacterial and kidney cells, respectively.

Chapter 4 reports the biological activities of a toxic, fluorescent peptide derived from Aβ15-36 (peptide 1*) using the LDH release assay as well as confocal laser scanning microscopy (CLSM). The fluorescent L (7-hydroxycoumarin-4-yl) ethyl glycine (7-HC) amino acid was synthesized and incorporated into coumarin-QK15-36 (peptide 1*), thereby making it suitable for localization studies. Peptide 1* was shown to be toxic to human neuroblastoma SH-SY5Y cells over a range of concentrations using the LDH release assay. Furthermore, peptide 1* was uniformly internalized in the cytosol of SH-SY5Y cells, suggesting that it mediates toxicity by disrupting intracellular homeostasis. This is the first time the Nowick lab has characterized the cellular localization of a toxic peptide and the results from this study may further our understanding of Aβ neurotoxicity.

Chapter 5 summarizes my teaching and chemical education research experiences at UC Irvine. I provide reflections of my experiences in two pedagogical training programs offered at UC Irvine and summarize my experiences as an instructor of record for Chemistry 51LB and Chemistry 101W. This chapter concludes with a summary of my involvement in two chemical education research studies, where I learned about research survey design and data analysis.