UC San Diego

The appearance of multi-drug resistant microbes and the decrease in the number of new antibiotics to treat them coming through the clinical pipeline has created a great need for development of novel antibiotics. Although breakthroughs in sequencing technologies, mass spectrometry, and bioinformatics have revealed an almost unlimited potential for new compounds with antibiotic activity, there still remains a major bottleneck in the determination of mechanism of action (MOA) for these potential antibiotics. Thus although many new antimicrobial compounds are being isolated and structurally characterized, we lack MOA information for most. Determining the MOA is critical for understanding which new molecules will have the greatest potential as an antibiotic safe for clinical use. Current techniques to identify MOA are lengthy, low-throughput and require a large amount of compound. We have developed a rapid and precise method to determine the MOA of compounds in Bacillus subtilis utilizing fluorescence microscopy and viability data, termed cytological profiling. We also developed microculture techniques that allow testing of compounds utilizing less than a microgram of material. This method was used to demonstrate that the cannibalistic toxin sporulation delaying protein (SDP) kills the cell via PMF collapse, which was confirmed by PMF assays, and we have applied the technique to other natural products. All natural products and control antibiotics have shown a distinctive pattern in cell architecture that is unique to their MOA. Further development of cytological profiling by screening of a large library of control compounds, complemented by cytological profiling data obtained from E. coli, and paired with development of automated image analysis and microscopy technologies will make cytological profiling a high-throughput and accurate method of MOA determination sensitive enough to be used at sub-MIC levels and to identify the MOA of natural products prior to purification through screening of crude extracts