The transcriptional response of Yersinia pseudotuberculosis to macrophage-released metabolites during growth within synthetic microcolonies

Abstract

Yersinia pseudotuberculosis (Yptb) replicates in immune cell-encompassed microcolonies within tissues. Bacterial replication is controlled by protection against neutrophil attack and by macrophage-released antimicrobial factors, such as nitric oxide (NO). During these attacks, bacteria located on the microcolony periphery encounter extracellular signals that differ from those in the interior. To dissect individual microbial populations, {gamma} interferon-activated macrophages were used to challenge microdroplet-grown Yptb harboring an NO-responsive mCherry reporter. Subsequently, bacterial subpopulations that hyperactivated the reporter were isolated from droplets composed of a reversible polymer matrix. RNA-seq analysis indicated that induction of nitrosative stress-associated genes was the primary determinant distinguishing peripheral bacteria from the remaining population. In addition, a secondary stress response that induced prophage-associated genes was detected, which could not be traced to either DNA damage or nitrosative stress responses. Activated macrophages also induced the expression of the Yptb itaconate degradation enzyme-encoding transcript throughout the entire colony. To determine if itaconate production by the interferon-activated Irg1 protein played a role in restricting Yptb, bacteria harboring an itaconate-responsive reporter and Yptb mutants defective for itaconate degradation were analyzed during bacterial colonization of the murine spleen. Only a subset of colonies appeared to be exposed to itaconate, which may explain the very small defects exhibited by mutants unable to degrade the interferon-induced macrophage product. These results indicate that the primary response of bacteria to macrophage-elicited factors is likely associated with protection against NO-derived metabolites.