Breaching barriers : new antibiotic strategies for targeting pathogenic bacteria

The emergence of antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae (CRE), necessitates the development of new therapeutics. Antibiotic resistance can result in life-threatening infections, where resistant bacteria are able to proliferate in the presence of high concentrations of antibiotic due to mechanisms such as efflux and enzymatic deactivation. An additional phenomenon which can cause recalcitrant infection is antibiotic persistence. Persistent bacterial cells withstand antibiotic treatment by entering a dormant or slow-growing state in response to cellular stress and are often found within biofilms, which are communities of bacteria enmeshed in a self-produced extracellular matrix. Most clinically used antibiotics target metabolically active bacteria and are largely ineffective against biofilms and other persistent bacterial populations. Innovative therapeutic strategies are needed to treat both resistant and persistent bacteria, and this dissertation describes the development and characterization of new chemical approaches to target these pathogens. Our studies were inspired by the ability of uropathogenic E. coli (UPEC), the most common causative agent of urinary tract infection (UTI), to contribute to chronic infection via several mechanisms, including robust adhesion to bladder epithelial cells and formation of intracellular biofilms. We first investigated the molecular determinants of UPEC adhesion to bladder cells using a model mammalian cell culture system, enabling the identification and development of new strategies to prevent bacterial adhesion. We also investigated the ability of conventional antibiotics to treat intracellular UPEC, which motivated the use of cell-penetrating, guanidinium-rich molecular transporters (GR-MoTrs) to improve delivery of antibiotics to intracellular pathogens. We prepared and evaluated a series of antibiotic-GR-MoTr conjugates to treat UPEC, focusing on the glycopeptide class of antibiotics, which are unable to cross mammalian or bacterial cell membranes. While our antibiotic-transporter conjugates exhibited no activity against UPEC, we discovered a vancomycin-oligoarginine conjugate (V-r8) that exhibited extraordinary activity against MRSA biofilms and persister cells. We investigated its mechanism of action and observed rapid cell killing and enhanced cellular association as a result of covalent conjugation of vancomycin to octaarginine. Together with demonstrating its remarkable activity in eradicating biofilm-associated bacteria in a mouse wound infection model, we also generated testable hypotheses for the molecular-level mode of action of V-r8, consistent with all of our biochemical data. In parallel, we screened other glycopeptide conjugates in an effort to target UPEC, where we discovered a vancomycin-arginine conjugate (V-R) effective against UPEC and CRE. The V-R derivative appeared to access intracellular cell-wall targets and served to disrupt bacterial cell-wall synthesis. Our studies inform future antibiotic development efforts for difficult-to-treat bacterial populations and highlight the utility of conjugating antibiotics to GR-MoTrs to generate potent therapeutics with new activity and efficacy.

Keywords: Chemistry.
Advisor: Cegelski Lynette 
Cui Bianxiao 
Wender Paul A.