Antibiotics : Studies on the Biological Mechanisms of Antibiotics

Project Summary/Abstract Antibiotic-resistant Gram-negative infections pose a major threat to human health. Gram-negative pathogens are intrinsically resistant to most clinically used classes of antibiotics due to the presence of an outer membrane that prevents antibiotic entry. Many Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and E. coli, are now multi-drug resistant and can only be killed by colistin, an old antibiotic that was previously almost abandoned because it has dose-limiting toxicity. My lab played a large role in discovering the machines that assemble the Gram-negative outer membrane and has developed a comprehensive set of in vitro and in vivo tools to study outer-membrane assembly. Here, we will use our existing tools as well as new approaches to elucidate the mechanisms of action of two peptide antibiotics that target the lipopolysaccharide transport (Lpt) pathway, thanatin and murepavadin. The molecular mechanisms by which these compounds disrupt outer membrane assembly remain poorly understood. We hypothesize that a molecular understanding of how these compounds block lipopolysaccharide transport will enable the development of analogs with more promising properties as drugs. In Aims I and II, we will address the mechanism of action of thanatin using a range of biophysical and biochemical assays, including single molecule TIRF microscopy in living cells, in vitro biochemical assays that monitor different steps in LPS transport, and in vivo crosslinking assays that monitor LPS transport in cells. In Aim III, we will investigate the mechanism of action of murepavadin, and we will characterize the Pseudomonas aeruginosa multiprotein complex that we propose it targets. Using cyclic peptides, we have recently established that this multiprotein complex is a valid therapeutic target in Acinetobacter baumannii to treat Carbapenem-resistant Acinetobacter baumannii (CRAB) infections, so we will also use the Pseudomonas multiprotein complex in a screen to identify other antimicrobial peptides that may share a similar mechanism of action. In this way, the fundamental knowledge we obtain about how thanatin and murepavadin function could enable discovery of other classes of compounds to expand the spectrum of antibiotic-resistant Gram-negative pathogens that are susceptible to lipopolysaccharide transport inhibitors.