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Papers by Casey Pope
Journal of Bacteriology, 2004
Gene products required for in vivo growth and survival of microbial pathogens comprise a unique f... more Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were r...
Biochemistry, 2010
Typically, biochemical screens that employ pure macromolecular components focus on single targets... more Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction contains up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, where inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential. Typically, in vitro high-throughput screening (HTS) 1 assays target single proteins or protein pairs. This approach has enabled significant success. To exploit all of the targets available in complex pathways or molecular machines, researchers often resort to cellular screens, to ensure the availability of all relevant targets. Using these approaches, novel targets have been revealed that have led to the discovery of new interactions, validating the power of forward chemical genetics (1). However, cellular screens have the drawback of missing compounds that cannot achieve suitable intracellular concentrations due to low permeability, unfavorable metabolism or efflux. These issues could be overcome if all of the machinery involved in a complex process could be reconstituted in vitro, enabling a biochemical screen. This approach would permit identification of inhibitors that could subsequently be optimized for potency and permeability in parallel with other favorable pharmacological properties.
Journal of Bacteriology, 2004
Gene products required for in vivo growth and survival of microbial pathogens comprise a unique f... more Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were r...
Biochemistry, 2010
Typically, biochemical screens that employ pure macromolecular components focus on single targets... more Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction contains up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, where inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential. Typically, in vitro high-throughput screening (HTS) 1 assays target single proteins or protein pairs. This approach has enabled significant success. To exploit all of the targets available in complex pathways or molecular machines, researchers often resort to cellular screens, to ensure the availability of all relevant targets. Using these approaches, novel targets have been revealed that have led to the discovery of new interactions, validating the power of forward chemical genetics (1). However, cellular screens have the drawback of missing compounds that cannot achieve suitable intracellular concentrations due to low permeability, unfavorable metabolism or efflux. These issues could be overcome if all of the machinery involved in a complex process could be reconstituted in vitro, enabling a biochemical screen. This approach would permit identification of inhibitors that could subsequently be optimized for potency and permeability in parallel with other favorable pharmacological properties.