UCSF

Despite their extraordinary ability to cure infectious diseases, most antibiotics have dose-limiting, off-target toxicities that impede the clinical development of novel small molecule candidates targeting multi-drug resistant pathogens. For existing antibiotic targets, large dosages of antibiotics are needed to achieve the requisite cellular potency to clear the bacterial burden during infection. In this work we explore strategies to lower the required dosage by identifying genetic vulnerabilities of the pathogen Pseudomonas aeruginosa during lung infection and antibacterial therapy administration. We probe these genetic vulnerabilities with Mobile-CRISPRi, a genetic tool that enables partial genetic inhibition and detection of hypersensitivity to clearance by the immune system or predation by bacteriophage. Designing chemical inhibitors that mimic the genetic inhibition leading to loss of bacterial fitness during lung infection or phage therapy may provide an avenue for future antibacterial development efforts. Chapter 1 profiles antibiotics with Gram-negative activity that have been discontinued during clinical development over the last decade, largely due to toxicity issues in phase 1 clinical trials; Chapter 2 details the construction of a Mobile-CRISPRi system in Pseudomonas aeruginosa with constitutive promoters driving dCas9 activity and its implementation in a murine pneumonia model to recapitulate the attenuation of virulence through inhibition of the transcriptional activator exsA; Chapter 3 entails the construction of a pooled Mobile-CRISPRi library in Pseudomonas aeruginosa, where each strain has a distinct essential gene knocked down, and the implementation of this library in a murine pneumonia model to detect in vivo genetic vulnerabilities; Chapter 4 features genetic and proteomic efforts to identify Pseudomonas aeruginosa determinants of hypersensitization to killing by DMS bacteriophage.