The clinical arsenal of antibiotics is losing effectiveness due to the spread of resistance. To counter this, our lab is working to discover new antibiotics. This comes in two parts, both of which we are working on, 1) finding new active compounds and 2) characterizing the compounds’ target and mechanism of action. To find new compounds, we are in active collaboration with Dr.Mindi Yan and other synthetic chemists. To characterize the target and mechanism of action of novel antimicrobial molecules we use chemogenomic approaches followed by focused validation of putative targets. Identifying the targets of new antimicrobials is critical to further rational drug optimization approaches.
Using the cystic fibrosis pathogen Burkholderia cenocepacia as a model, we are working to uncover the molecular details of new and approved antibiotics. This includes both mechanisms of action (how the compounds inhibit cell processes) and resistance (how cells are able to grow in the presence of the compounds).
Our platform harnesses the power of next-generation sequencing with high-throughput target inquiry. Using transposon mutagenesis to systematically inactivate every non-essential gene in the genome, we gain a genome-wide perspective on how each gene contributes to resistance or susceptibility. Hundreds of thousands of mutants are grown in a pool with a small molecule. Illumina sequencing is then used to determine the abundance of each mutant. We have recently modified the mutants to each contain a unique DNA barcode, greatly increasing the throughput of our platform.
Molecular biology investigations
Genome-wide approaches generate many hypotheses that need to be validated. Our lab uses a variety of molecular biology techniques to gain detailed insight into antibiotic mechanisms. Below are some that we routinely use:
Microscopy for morphology changes: The regulation of cell shape and size is remarkably sensitive to perturbation by small molecules. These changes can be used to pinpoint compounds that affect cell envelope biogenesis. For example, the cephalosporins induce filamentation by inhibiting septal peptidoglycan synthesis, whereas the carbapenems rapidly induce lysis and bulging by inhibiting lateral peptidoglycan synthesis. Our department is equipped with a state-of-the-art microscopy facility.
Fluorescent dyes for membrane permeability and redox potential: Small molecules often induce changes in cell physiology that can be probed with specific reporters. For example, membrane permeability can be assayed with propidium iodide or N-phenylnaphthylamine. Cellular redox state can be probed with resazurin and ROS generation can be detected with dichlorofluorescein diacetate using a plate reader.
Synthetic biology toolkits: We routinely create genetic mutants to investigate the effects of antibiotics on entire pathways or processes. We have techniques to delete genes/operons and carefully titrate gene expression, even of essential genes, using CRISPR-interference.
Clinical pathogen panels: Our lab maintains active collaborations with clinical microbiology labs and strain collections and boasts a wide array of Gram-positive and Gram-negative pathogens. We use these panels to improve the real-world translation of our research.
• Chemical interactions with large antibiotic screens: Small molecules are specific inhibitors of cellular processes. Patterns of interactions (drug synergy and antagonism) can be used to show how uncharacterized compounds affect certain processes. For example, the beta-lactamase inhibitor avibactam synergizes with multiple beta-lactams, and the membrane permeabilizer chlorhexidine synergizes with large scaffold compounds excluded by the outer membrane.