Date Published: February 8, 2019
Publisher: Public Library of Science
Author(s): William S. Sawyer, Lisha Wang, Tsuyoshi Uehara, Pramila Tamrakar, Ramadevi Prathapam, Mina Mostafavi, Louis E. Metzger, Brian Feng, Christopher M. Baxter Rath, Charles Michael Greenlief.
Lipopolysacharride (LPS) forms the outer leaflet of the outer membrane in Gram-negative bacteria and contributes to the permeability barrier and immune response. In this study, we established a method for monitoring the LPS biosynthetic intermediates of the Raetz pathway (lpxA-lpxK) in Escherichia coli. Metabolites from compound-treated cells and genetically-perturbed cells were extracted from whole cells and concentrated by mixed-mode weak anion exchange (WAX) solid-phase extraction (SPE) prior to analysis by normal phase (NP)LC-MS/MS. Data was normalized to cell density and an internal standard prior to comparison against untreated cells in order to determine fold accumulation and depletion for affected metabolites. Using this LC-MS/MS method, we were able to reliably monitor changes in levels of the LPS intermediates in response to compound-treatment and genetic modification. In addition, we found that deletion of periplasmic CDP-diacylglycerol pyrophosphatase dramatically increased levels of the UDP-containing LPS intermediates, suggesting the enzymatic breakdown during sample preparation. This assay allows for probing a key essential pathway in Gram-negative bacteria in an effort to discover antibacterial agents that inhibit enzymes in the LPS biosynthetic pathway.
Due to the increasing rate of infections by multi-drug-resistant (MDR) Gram-negative bacterial pathogens, an urgent need for new antibiotics has become apparent. Patients infected by these MDR bacterial strains, such as carbapenem-resistant Enterobacteriaceae, cannot be treated with available antibiotics. Despite the dramatic reduction in effective treatment options for patients, there unfortunately has not been a substantial increase in discovery and development of novel antibiotics. One of the biggest obstacles to overcome during target-based drug discovery is bridging the gap between in vitro biochemical and cellular activity. For example, enzyme inhibitors may be discovered rapidly with modern high-throughput screening and a good biochemical assay, but is often difficult to optimize them for cellular activity. This disconnect between in vitro and cellular activities is particularly true for MDR Gram-negative bacteria, where the outer membrane serves as a permeability barrier that limits influx of large, hydrophobic antibiotics into the cell. It is thought that the chemical properties to get in and stay in bacterial cells may be quite different for antibiotics versus molecules typically encountered in pharmaceutical screening libraries. In addition, Gram-negative pathogens possess multidrug efflux pumps, which can reduce the intracellular concentration of antibiotics. Thus, a novel antibiotic requires an aggregate of biochemical potency, good permeability, and desirable efflux properties, all of which must be addressed for bacterial growth inhibition to be observed for drugs that inhibit growth via intracellular targets. To enter the periplasm of Gram-negative bacteria, some biologically-active compounds are thought to transit through protein channels or porins, which favor the passage of small polar molecules. However, the properties required to translocate through porins are at odds with those required to passively diffuse through the inner membrane. The difficulty of meeting these criteria cannot be overstated as a hurdle to the development of novel antibiotics. As well, current economic incentives are not thought to support the development of novel “drugs of last resort” for antibiotic resistance. In light of these challenges, new approaches to aid in understanding essential pathways in Gram-negative bacteria must be explored to aid in the scientific challenges of antibiotic discovery.
Here we present a robust method for monitoring the lipid IVA biosynthesis pathway and quantitative evaluation of inhibitors that target this pathway. At the time point examined in this assay, the concentration of these pathway intermediates varied depending on the step of the pathway, as indicated by our ability to detect certain analytes better than others despite similar limits of detection (S2 Fig). Metabolic profiling of chemical and genetic perturbations of the early LPS biosynthetic pathway revealed accumulation of substrates in response to chemical inhibition or genetic perturbation at specific points of the pathway. Additionally, we were able to observe unpredicted effects on intermediates in cells treated with ChemDiv C324-2728 (11). This assay allows for characterization of this Gram-negative pathway which is the source of the permeability barrier, LPS, presenting one of the largest challenges to antibiotic drug development. Furthermore, this assay provides a cellular context that can be used in conjunction with biochemical assays to perform target-based drug discovery and identify LPS inhibitors from phenotypic hits.