Browsing by Subject "Antibiotics"

An essential property of microbial communities is the ability to survive a disturbance. This is readily observed in bacteria, which have developed the ability to survive every antibiotic treatment at an alarming rate, considering the timescale at which new antibiotics are developed. Thus, there is a critical need to use antibiotics more effectively, extend the shelf life of existing antibiotics and minimize their side effects. This requires understanding the mechanisms underlying bacterial drug responses. Past studies have focused on survival in the presence of antibiotics by individual cells, such as genetic mutants. Also important, however, is the fact that a population of bacterial cells can collectively survive antibiotic treatments lethal to individual cells. This tolerance can arise by diverse mechanisms, including resistance-conferring enzyme production, titration-mediated bistable growth inhibition, swarming and interpopulation interactions. These strategies can enable rapid population recovery after antibiotic treatment and provide a time window during which otherwise susceptible bacteria can acquire inheritable genetic resistance.

To further explore bacterial antibiotic responses, I focused on bacteria producing β-lactamase, an enzyme that has drastically limited the use of our most commonly prescribed antibiotics: β-lactams. Through the characterization of clinical isolates and a computational model, my Ph.D. thesis has three implications:

First, survival can be achieved through resistance, the ability to absorb effects of a disturbance without a significant change, or resilience, the ability to recover after being perturbed by a disturbance. Current practices for determining the antibiotic sensitivity of bacteria do not characterize a population as resistant and/or resilient, they only report whether the bacteria can survive the antibiotic exposure. As resistance and resilience often depend on different attributes, distinguishing between these two modes of survival could inform treatment strategies. These concepts have long been applied to the analysis of ecological systems, though their interpretations are often subject to debate. This framework readily lends itself to the dissection of the bacterial response to antibiotic treatment, where both terms can be unambiguously defined.

Second, the ability to tolerate the antibiotic treatment in the short term corresponds to resistance, which primarily depends on traits associated with individual cells. In contrast, the ability to recover after being perturbed by an antibiotic corresponds to resilience, which primarily depends on traits associated with the population.

And finally, understanding the temporal dynamics of an antibiotic response could guide the design of a dosing protocol to optimize treatment efficiency for any antibiotic-pathogen combination. Ultimately, optimized dosing protocols could allow reintroduction of a repertoire of first-line antibiotics with improved treatment outcomes and preserve last-resort antibiotics.

A series of experiments and mathematical models explore the response of the bacteria E. coli to stressors. Experimentally, the effect of L-homocysteine, a non-protein amino acid, is explored, and via math models, the effect of trimethoprim, a common antibiotic, is also explored. Previous work on L-homocysteine labels it a stressor, and this assertion is refined via the presented work. A mathematical model that improves on a previous work published by Kwon et al. (2008) explores the response of E. coli to various supplementations of amino acids when exposed to trimethoprim. New methods of developing antibiotics and therapeutic drug treatments are also explored.

Natural products belonging to the lipid II-binding family act as potent antimicrobial agents by disrupting cell wall biosynthesis via sequestering the late-stage intermediate lipid II. However, the emergence of resistance mechanisms and poor bioavailability have hindered the utility of these molecules as promising therapeutic intervention strategies to combat pathogenic bacterial infections. Gaining a deeper understanding of structural components and biosynthetic pathways can lead to the creation of second-generation derivatives to improve bioactivity and pharmacological properties. To explore this superfamily, we have used bioanalytical, biochemical, synthetic, computational, and enzymatic approaches that have been applied to three distinct projects. The first includes efforts to characterize the relationship between structural feature and bioactivity for the lipid II-binding CDA (calcium dependent antibiotic), malacidin. Through a series of minimally complex analogs, we determined non-proteinogenic amino acids and the N-acyl fatty acid moiety are essential for bioactivity. For the second project, we investigated a conserved mechanism of action for phylogenetically-related natural products within the lasso peptide subfamily. This work led to the discovery of a novel class I lasso peptide, arcumycin, and we validated a conserved mechanism of action for Actinobacteria-produced lasso peptides in targeting lipid II biosynthesis. Our last project sought to elucidate the mechanism of lipoinitiation for the ramoplanin family of molecules. Through a series of bioactivity assays, we found the transfer to the acyl carrier protein (ACP) in a fatty acyl-AMP ligase (FAAL)-dependent manner determined the specificity of lipids selected in the biosynthetic process. Collectively, through each project we have gained a deeper understanding of the structural elements and biosynthetic pathways of lipid II-binding antimicrobials.

New strategies are urgently needed to overcome the growing threat of antibacterial drug resistance. One mechanism of drug resistance commonly employed by bacterial pathogens is expression of β-lactamases, which confer resistance through hydrolysis of β-lactam antibiotics, an important class of antibacterial compounds that target cell wall biosynthesis. We have developed multiple new strategies to take advantage of β-lactamase expression to deliver antibacterial metal chelators to drug-resistant bacteria. First, we describe the copper-dependent activity of an antibacterial prodrug named PcephPT, which is based on the structure of a cephalosporin, a type of β-lactam antibiotic. Using antibacterial susceptibility assays and analytical techniques, we demonstrate that PcephPT disrupts the homeostasis of copper, an essential but toxic metal, by releasing the antimicrobial chelator pyrithione (PT) in response to β-lactamase-mediated hydrolysis. We found that, in addition to inhibiting bacterial growth in a copper-dependent manner, PcephPT leads to inhibition of NDM-1, a β-lactamase that catalyzes hydrolysis using enzyme-bound zinc ions. Bacterial pathogens expressing metallo-β-lactamases such as NDM-1 are difficult to overcome with antibacterial treatment, and the success of PcephPT in inhibiting NDM-1 is a promising indication that chelator-releasing prodrugs (“prochelators”) are a useful strategy for metallo-β-lactamase inhibitor development. Enzymatic and spectroscopic assays reveal important information about the mechanism of PcephPT-mediated NDM-1 inhibition, which involves formation of a ternary complex with the zinc-containing active site and PT, one of the products of PcephPT hydrolysis. We next present progress on synthesizing and evaluating new prochelators that are designed to have increased specificity for drug-resistant strains. These compounds have core structures based on β-lactamase inhibitors, which are not inherently antibacterial, but may still release chelators for both antibacterial activity and metallo-β-lactamase inhibition. Finally, we discover a new class of metallo-β-lactamase inhibitors based on benzimidazole and benzoxazole zinc-chelating agents that form a ternary complex in the active site of NDM-1 and have the potential to be further optimized for inhibitory properties and targeting of drug-resistant bacteria.