Topologically correct synthetic reconstruction of pathogen social behavior found in deep tissue sites

Abstract

Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. Disease 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 Yersinia pseudotuberculosis (Yptb) growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues. Chemical generation of nitric oxide (NO) in the absence of immune cells was sufficient to reconstruct microbial social behavior, as witnessed by expression of the NO-inactivating protein Hmp on the extreme periphery of microcolonies, mimicking spatial regulation in tissues. Similarly, activated macrophages that expressed inducible NO synthase (iNOS) drove peripheral expression of Hmp, allowing regeneration of social behavior observed in tissues. These results argue that topologically correct microbial tissue growth and associated social behavior can be reconstructed in culture.

Modifying TIMER, a slow-folding DsRed derivative, for optimal use in quickly-dividing bacteria

“…In comparison with TIMER42, hmp reporter signal was significantly enriched at the periphery (Fig 7B). This hmp reporter signal pattern was consistent with our previously published results (22,26,37) but suggested that NO stress was not significantly slowing bacterial growth within host tissues, based on TIMER42 signal accumulation in some, but not all, peripheral cells. Representative images indicate there was substantial overlap between dps and TIMER42 signals, and that hmp and TIMER42 are detecting distinct subsets of bacteria (Fig 7C).…”

“…Cultures of the TIMER42 Phmp::gfp and TIMER42 Phmp::gfp-ssrA strains were exposed to 2.5mM DETA-NONOate for 4h, and fluorescence microscopy was used to detect fluorescent signals within individual bacterial cells. The 2.5mM dose was chosen for these experiments based on our previous studies, which showed this dose is sufficient to promote hmp reporter expression (22,26,37). Treatment with the NO donor compound resulted in high levels of hmp reporter expression with both strains (Fig 6A), and TIMER42 signal accumulation (Fig 6B).…”