Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages
Yersinia pestis is able to survive and replicate within macrophages, while also being able to live in the extracellular milieu of the host. Assays that facilitate better understanding of how Y. pestis survives intracellularly and subverts normal host antimicrobial defenses require the ability to monitor intracellular Y. pestis survival and replication. In this chapter three different assays for monitoring intracellular survival and replication will be described, along with the formulas and methods to quantify and present the acquired data. These assays are fundamental to answering a multitude of questions pertaining to which bacterial factors are important for intracellular survival. Additionally, these assays can be used, with modifications, for other intracellular pathogens of interest. The first assay discussed will be the conventional bacterial enumeration assay, which quantifies bacterial numbers directly through a classic colony forming units (CFU) assay. Quantifying bacterial burden through CFU determination allows for differentiation between intracellular/cell-associated bacteria and extracellular bacteria. However, CFU determination is laborious, does not allow for direct kinetic monitoring of bacterial growth, and is difficult to adapt to high throughput assays. Bioluminescence bioreporters that use luciferase to monitor bacterial numbers allow for simple, plate reader-based, real-time kinetic monitoring of bacterial growth that is amendable to high throughput techniques. Finally, we will describe live cell microscopy using fluorescent bioreporters, which allows for monitoring of bacterial replication in individual cells and the possibility to visualize interactions between bacterial and host proteins during intracellular infection.
Key wordsIntracellular bacteria Macrophages Plague Live cell microscopy Bioluminescence
The authors acknowledge the James Graham Brown Cancer Center Confocal Imaging Core and Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases for resources and technical support. Funding support was from NIAID (AI119557, AI097608) and NIGMS (GM125504) to M.B.L.
- 8.Connor MG, Pulsifer AR, Chung D, Rouchka EC, Ceresa BK, Lawrenz MB (2018) Yersinia pestis targets the host endosome recycling pathway during the biogenesis of the Yersinia-containing vacuole to avoid killing by macrophages. MBio 9(1):e01800-17Google Scholar