Modulating pathogenesis with Mobile-CRISPRi

Abstract

Pathogens express a set of proteins required for establishing and maintaining an infection, termed virulence life-style genes (VLGs). Due to their outsized importance in pathogenesis, VLG products are attractive targets for the next generation of antimicrobials. However, precise manipulation of VLG expression in the context of infection is technically challenging, limiting our ability to understand the roles of VLGs in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of gene knockdown tools that are transferred by conjugation and stably integrate into pathogen genomes that we call \"Mobile-CRISPRi\". Here we show the efficacy of Mobile-CRISPRi in controlling VLG expression in a murine infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid non-specific toxicity. As a proof of principle, we demonstrate that knockdown of a VLG encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of VLGs across many bacterial species and pathogenesis models.\n\nImportanceAntibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically these genetic drivers have been difficult to manipulate, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study virulence life-style genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.