Image-based transposon screening reveals a flavin reductase that restrains intracellular <i>Salmonella</i> replication in macrophages

<jats:p> Intracellular bacterial pathogens can survive and replicate within host cells, yet an isogenic population follows divergent fates: some bacteria are killed, some arrest growth, and others replicate to high numbers. Identifying the bacterial functions behind each fate requires recovering mutants from within the host cells displaying it. But systematic approaches, such as transposon insertion analysis, can only estimate fitness of mutants from the whole infected population. Here, we developed an approach that couples a genome-wide mutagenesis library to image-enabled cell sorting (ICS), sorting infected cells by the number of bacteria they contain and assigning mutants to defined replication outcomes. We applied this approach to <jats:italic>Salmonella enterica</jats:italic> serovar <jats:italic>Typhimurium</jats:italic> ( <jats:italic>S.</jats:italic> Tm) in macrophages, and revealed genes required for replication from genes that restrain it, whose disruption increased replication. Among the latter we identified the cytosolic flavin reductase Fre, which supplies reduced flavins to a broad range of bacterial processes. We uncovered a mechanism whereby loss of <jats:italic>fre</jats:italic> protected <jats:italic>S.</jats:italic> Tm from oxidative and nitrosative damage and increased bacterial numbers. Inside macrophages this advantage was mediated by the upregulation of the iron-sulfur-independent cytochrome bd-I oxidase. By resolving a mutant library into phenotypically defined subpopulations, this framework can be applied to characterize bacterial or host genes that drive infection phenotypes in any infection model. </jats:p>