Genome-Wide Mutant Fitness Profiling Identifies Nutritional Requirements for Optimal Growth of Yersinia pestis in Deep Tissue

ABSTRACT Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposonmutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injec-tion. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fit-ness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complemen-tation effects. We also identified more than 30 genes with roles in nutrient acquisition andmetabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes—encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)—both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes. IMPORTANCE Our understanding of the functions required by bacteria to grow in deep tissues is limited, in part because mos