Development of novel therapeutic strategies is urgently required to counter the growing public health threat of antimicrobial resistance (AMR). While antibiotics/antimicrobials target essential processes, antivirulence is an emerging concept aiming to disrupt virulence, potentially reducing the selective pressure for AMR development. Pathogen-associated genes (PAGs), whose conservation only in bacterial pathogens indicates potential role in virulence, may be suitable candidates for antivirulence drug targets. In this thesis, I developed a computational workflow, with select experimental validation, for 1) characterizing and prioritizing PAGs as potential antivirulence drug targets, 2) analyzing the structure-activity relationship of the approved drug raloxifene as an antivirulence agent, and 3) assessing the application of PAGs towards metagenomics-based pathogen surveillance. First, I refined a previously developed PAG prediction algorithm to generate an updated PAG dataset. In addition, a genus-specific PAG analysis was performed, focusing on the antibiotic-resistant, priority pathogen Pseudomonas aeruginosa and the related Pseudomonas species. Seventeen PAGs identified from P. aeruginosa PA14 were subsequently analyzed in silico (predicting gene mobility, evolutionary selection, and protein subcellular localization using a newly expanded database), and in vivo (virulence assay), identifying novel targets, including two within a genomic island found in several multi-drug resistant P. aeruginosa isolates. Expanding a previous drug-repurposing study of raloxifene as an anti-pseudomonal antivirulence agent, I compared the properties of raloxifene analogs in a quantitative pyocyanin assay, a growth curve assay and a standardized C. elegans infection model, which revealed that at least one hydroxyl group of raloxifene likely contributed to its antivirulence activity. Lastly, to evaluate the potential application of PAGs in pathogen surveillance, I examined the prevalence of PAGs in lung microbiomes and watershed microbiomes. Cystic fibrosis lungs, relative to healthy lungs, were disproportionately enriched in PAGs, but interpretation of watershed analyses was affected by the abundance of uncharacterized bacterial species in freshwater. The analyses suggested that the application of PAGs in pathogen surveillance warrants further study but may be limited to well characterized microbial environments. Collectively, this work expands knowledge of PAGs and an associated proposed antivirulence drug, revealing new avenues for PAG research in surveillance and expanding potential drug targets for antivirulence therapies.
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Thesis advisor: Brinkman, Fiona S. L.
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