UC Santa Cruz

This dissertation describes the development and application of a high-throughput, image-based screening platform for the discovery of small molecules with activity against Vibrio cholerae. Like many bacterial species, V. cholerae is capable of forming three-dimensional biofilms. Biofilm-mediated infections are prevalent in the clinic, and bacteria in the biofilm state are 10-10,000 fold less susceptible to antibiotic treatment than bacteria in the planktonic state. Despite the pervasiveness of these diseases there are no existing therapeutics for combatting these infections, nor is there a complete understanding of the molecular mechanisms that control biofilm formation. In order to address this problem, a wide range of techniques were utilized including high-throughput screening (UCSC Chemical Screening Center), image analysis tools, and structure elucidation methods.

Chapter 1 provides a history of bacterial biofilms, the role of biofilms in human disease, and the existing small molecules with reported biofilm inhibitory activity. The second chapter describes the development of an image-based, 384-well format platform for investigating the effects of small molecules upon a biofilm forming strain of V. cholerae. The chapter closes with the validation of this assay through the screening of a 5,080 member commercial library. Chapter 3 reports the results from a large scale screening campaign investigating both commercial and marine natural product (NP) libraries. The final half of the chapter details the development of two secondary screening platforms designed for the evaluation of small scale quantities of compound, allowing for characterization of pure natural products produced in low titer beyond the primary screening stage. Chapter 4 describes the discovery, isolation, structure elucidation, and biological characterization of a novel biofilm inhibitor. This compound, previously reported as the chromophore of auromomycin, displays a unique biofilm inhibitory phenotype indicating that the mechanism through which this molecule disrupts biofilm formation is unique compared to the previous compounds discovered using this screening platform.

The final chapter departs from the subject of biofilm inhibition, and concentrates on the effects of a known antibacterial training set and our natural product extract libraries on V. cholerae morphology. The development of a novel image analysis platform allowed for the quantification of cellular morphologies caused by compound treatment. The results from screening a dilution series of a set of known antibiotics indicated that the compounds can be binned based on their broad modes of action. This is the first example of a high-content bacterial screen, and has allowed for the mechanism-based profiling of NP extracts with unknown chemical constitution.