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Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages.
Pulsifer, Amanda R; VanCleave, Tiva T; Lawrenz, Matthew B.
Afiliação
  • Pulsifer AR; Department of Microbiology and Immunology, Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville School of Medicine, Louisville, KY, USA.
  • VanCleave TT; Department of Microbiology and Immunology, Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville School of Medicine, Louisville, KY, USA.
  • Lawrenz MB; Department of Microbiology and Immunology, Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville School of Medicine, Louisville, KY, USA. matt.lawrenz@louisville.edu.
Methods Mol Biol ; 2010: 181-196, 2019.
Article em En | MEDLINE | ID: mdl-31177439
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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Peste / Yersinia pestis / Macrófagos Limite: Animals / Humans Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Peste / Yersinia pestis / Macrófagos Limite: Animals / Humans Idioma: En Ano de publicação: 2019 Tipo de documento: Article