Elucidation of the spatially regulated macrophage-induced transcriptional response of Yersinia pseudotuberculosis growing within synthetic microcolonies

Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. In the case of Yersinia pseudotuberculosis (Yptb), disease progression is modulated by interbacterial interactions as well as bacterial-host cell interactions resulting in microbial growth, phagocytic attack and secretion of host antimicrobial factors. To overcome the limited ability to manipulate these infection sites, we established a system for Yptb growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues. To dissect the transcriptional response based on the locale within a microcolony, we used this cell culture strategy to isolate the subpopulations based on florescence of the hmp reporter and performed RNA-seq analysis. High Phmp::mcherry Yptb upregulate genes involved in combating nitrosative stress, while downregulating genes involved in translation, indicating that the high Phmp::mcherry bacteria are stressed, and less metabolically active than the low Phmp::mcherry population of bacteria. Furthermore, high Phmp::mcherry Yptb isolated from droplets incubated with activated BMDMs upregulate a three gene operon, ccl-ich-ict, which encodes the enzymes responsible for degrading the activated macrophage secreted metabolite, itaconate. Strikingly, Yptb lacking this operon have reduced fitness during infection, demonstrating that itaconate degradation contributes to survival, and that itaconate is part of the tissue microenvironment. Altogether, these findings provide insight into the growth dynamics of the host-driven Yptb subpopulations and additional macrophage secreted factors that target Yptb during infection.