Optical Sequencing to Link Quantitative Bacterial Phenotypes to Human Mutations - Numerous bacterial pathogens, such as Legionella pneumophila, grow within host cells during disease. Pathogen growth within cells allows them to grow in a protected site, sequestered from attack by inflammatory cells and protected from antibody attack. In spite of these advantages, the host cell has a number of defenses that can attack the intracellular pathogen. These include degradative processes, such as autophagy which can sequester pathogens in antimicrobial compartments, as well as innate immune sensing systems that directly attack and kill the pathogen within the host cell cytoplasm. One strategy for avoiding these attacks in the host cytoplasm is for a pathogen to construct a membrane-bound compartment that provides a niche for replication. These organisms are called intravacuolar pathogens. To understand how intravacuolar pathogens such as L. pneumophila establish their niche and continue to grow in this site, the host proteins that are involved in promoting and restricting growth must be identified. The usual strategy for identification of these proteins is through mutational analysis. Unfortunately, current strategies for evaluating the effects of host mutations on pathogen growth have significant shortcomings. These shortcomings include an inability to identify important host proteins as well as a lack of quantitative approaches to measure the alteration of pathogen properties within mutant cells. This application proposes to overcome these problems by linking quantitative measures of pathogen traits, such as fitness, to host cell mutations. The experiments described here propose to quantitatively link L. pneumophila intracellular traits to host CRISPR/Cas9 guide mutations using optical sequencing. As outlined, two different L. pneumophila traits will be measured by low-power fluorescence microscopy. The host mutations that cause the changed property of the pathogen will be identified by performing DNA sequencing on the guide, followed by microscopic identification of each sequencing run. This will allow the gene targeted by the mutation to be linked to the behavior of the bacterium. In the first set of experiments, the trait followed will be iron starvation. Mutations will be identified by optical sequencing that prevent the host cell from restricting iron access to Legionella pneumophila, using a fluorescent reporter system for iron starvation. The second set of experiments will link pathogen fitness directly to the host mutation, by measuring accumulation of fluorescent bacteria. To this end, CRISPR/Cas9 editing of genes that alter host mitochondrial function will be targeted. By successful completion of these goals, a generalizable strategy will be developed that can be applied to all intracellular pathogens, allowing linkage of quantitative pathogen phenotypes to host mutations.
