Colleen M. Courtney

Sequence-Specific Peptide Nucleic Acid-Based Antisense Inhibitors of TEM-1 β-Lactamase and Mechanism of Adaptive Resistance

The recent surge of drug-resistant superbugs and shrinking antibiotic pipeline are serious challenges to global health. In particular, the emergence of β-lactamases has caused extensive resistance against the most frequently prescribed class of β-lactam antibiotics. Here, we develop novel synthetic peptide nucleic acid-based antisense inhibitors that target the start codon and ribosomal binding site of the TEM-1 β-lactamase transcript and act via translation inhibition mechanism. We show that these antisense inhibitors are capable of resensitizing drug-resistant Escherichia coli to β-lactam antibiotics exhibiting 10-fold reduction in the minimum inhibitory concentration (MIC). To study the mechanism of resistance, we adapted E. coli at MIC levels of the β-lactam/antisense inhibitor combination and observed a nonmutational, bet-hedging based adaptive antibiotic resistance response as evidenced by phenotypic heterogeneity as well as heterogeneous expression of key stress response genes. Our data show that both the development of new antimicrobials and an understanding of cellular response during the development of tolerance could aid in mitigating the impending antibiotic crisis.

Potentiating antibiotics in drug-resistant clinical isolates via stimuli-activated superoxide generation

Engineered nanoparticle for controlled superoxide flux potentiates antibiotics in MDR clinical isolates. The rise of multidrug-resistant (MDR) bacteria is a growing concern to global health and is exacerbated by the lack of new antibiotics. To treat already pervasive MDR infections, new classes of antibiotics or antibiotic adjuvants are needed. Reactive oxygen species (ROS) have been shown to play a role during antibacterial action; however, it is not yet understood whether ROS contribute directly to or are an outcome of bacterial lethality caused by antibiotics. We show that a light-activated nanoparticle, designed to produce tunable flux of specific ROS, superoxide, potentiates the activity of antibiotics in clinical MDR isolates of Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Despite the high degree of antibiotic resistance in these isolates, we observed a synergistic interaction between both bactericidal and bacteriostatic antibiotics with varied mechanisms of action and our superoxide-producing nanoparticles in more than 75% of combinations. As a result of this potentiation, the effective antibiotic concentration of the clinical isolates was reduced up to 1000-fold below their respective sensitive/resistant breakpoint. Further, superoxide-generating nanoparticles in combination with ciprofloxacin reduced bacterial load in epithelial cells infected with S. enterica serovar Typhimurium and increased Caenorhabditis elegans survival upon infection with S. enterica serovar Enteriditis, compared to antibiotic alone. This demonstration highlights the ability to engineer superoxide generation to potentiate antibiotic activity and combat highly drug-resistant bacterial pathogens.

ROS mediated selection for increased NADPH availability in Escherichia coli

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli.

Designing Superoxide-Generating Quantum Dots for Selective Light-Activated Nanotherapy

The rapid emergence of superbugs, or multi-drug resistant (MDR) organisms, has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy, while also potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence band (CB-VB) positions and alignment with different redox half-reactions—and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well-matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band (CB) position at −0.5 V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and electrochemical impedance spectroscopy (EIS). Using these findings, we demonstrate the rational design of QDs as selective therapeutic to kill more than 99% of a priority class I pathogen, thus providing an effective therapy against MDR superbugs.

Design of a de novo aggregating antimicrobial peptide and bacterial conjugation delivery system

Traditional antibiotics are reaching obsolescence as a consequence of antibiotic resistance; therefore novel antibiotic approaches are needed. A recent non-traditional approach involves formation of protein aggregates as antimicrobials to disrupt bacterial homeostasis. Previous work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding the first de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein aggregator Lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decreases. Further, to deliver this artificial gene, we developed a probiotic approach using RK2, a broad host range conjugative plasmid, to transfer opaL from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying the opaL gene by adding the RK2 origin of transfer. We show that opaL is non-toxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we observe selective growth repression in T7 polymerase expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control opaL expression. This system provides a basis for the design and delivery of novel antimicrobial peptides. Importance The growing threat of antibiotic resistance necessitates new treatment options for bacterial infections that are recalcitrant to traditional antimicrobials. Existing methods usually involve small-molecule compounds which interfere with essential processes in bacterial cells. By contrast, protein aggregates operate by causing widespread disruption of bacterial homeostasis and may provide a new method for combating infections. We used rational design to create and test an aggregating de novo antimicrobial peptide, OpaL. In addition, we employed bacterial conjugation to deliver the opaL gene from donor bacteria to recipient bacteria while using a strain-specific promoter to ensure that OpaL was only expressed in targeted recipients. To the best of our knowledge, this represents the first design for a de novo peptide with aggregation-mediated antimicrobial activity. We envision that OpaL’s design parameters could be used in developing a new class of antimicrobial peptides to help treat antibiotic resistant infections.