University of Pennsylvania
University of Michigan, Ann Arbor
University of Michigan, Ann Arbor
It has been known for decades that bacterial populations are heterogeneous, but it has been difficult to develop therapeutics that take this into account. Individual bacteria within populations vary in their growth rates, leading to inherent differences in antimicrobial susceptibility. Additionally, many therapeutics have been designed to target specific virulence factors, which are required for bacterial growth in host organisms. However, it remains unclear when (temporally) these factors are required over the course of infection, and if these factors are produced by the entire population, or only a subset of bacteria. The extent of bacterial heterogeneity within host tissues has been largely unexplored, making this an important under-studied aspect of the disease process. Our lab explores the extent of heterogeneity within bacterial populations using transcriptional fluorescent reporters, to visualize gene expression at the single cell-level during growth within mouse tissue sites. Bacterial gene expression profiles may be specifically driven by interactions with distinct subsets of host immune cells, and we also use immunofluorescence staining to visualize heterogeneity within the host cell population. The overall goals of our lab are to: 1) probe the extent of heterogeneity within bacterial populations to identify which individual cells express given genes during the disease process, 2) determine how individual subpopulations impact disease outcome through interactions with the host immune system, and 3) to ultimately better define potential drug targets for therapeutics.
Using Yersinia pseudotuberculosis as a model system for bacterial replication and tissue lesion formation, we have found that multiple subpopulations of bacteria develop during growth within the spleen. Y. pseudotuberculosis replicates to form clonal clusters, called microcolonies, which are surrounded by a layer of neutrophils, which are in turn circumscribed by a layer of macrophages. Our previous studies have shown that peripheral bacteria respond to neutrophil contact through heightened expression of the type-III secretion system, which inhibits phagocytosis and the release of ROS by neutrophils. The peripheral bacteria also respond to nitric oxide (NO) diffusing from macrophages, and detoxify NO at the periphery of microcolonies to prevent diffusion into the center of microcolonies, thus protecting interior bacteria. Simultaneous responses to multiple host stresses may indicate that peripheral bacteria are growing more slowly than bacteria present in the interior of microcolonies, which could lead to differential antibiotic susceptibility across microcolonies. Current studies are focused on testing this hypothesis: by developing tools to detect growth rate differences amongst individual bacteria replicating within host tissues, determining whether stress responses are impacting growth rate, and determining if this impacts the antibiotic susceptibility of subpopulations of bacteria. We are also taking microfluidics and droplet-based fluidics approaches to kinetically model bacterial growth within tissues. Ultimately, the tools we generate to study the growth of Y. pseudotuberculosis can also be applied to many other microbial systems, and we plan to expand our future studies to additional medically-relevant bacterial pathogens, such as Staphylococcus aureus, which causes infections of public health importance.