Profiling of salmonella transcriptional kinetics during macrophage infection using a comprehensive reporter library

Pathogenic bacteria can cause a wide range of diseases, and the factors contributing to the variations in disease outcomes include pathogenicity-associated genetic elements, transcriptional responses, and proteomic composition. Salmonella enterica subspecies enterica is comprised of 2,500 Salmonella serotypes that vary widely in terms of virulence, host range, and geographic distribution. Of these S. enterica subsp. enterica serovars, common serovars associated with human disease include serovars Newport, Typhi, Enteritidis, and Typhimurium. Understanding the determinants of their diversity in bacterial-derived disease would reveal strategies for reducing the health impact of these pathogens. The objective of this work is to profile the transcriptome of Salmonella enterica serovar Typhimurium (S. Typhimurium) during RAW 246.7 macrophage infection and identify the molecular determinants of S. Typhimurium pathogenicity. The transcriptome of S. Typhimurium dynamically responds to the rapid environmental changes intrinsic to its lifestyle, such as entry into the Salmonella containing vacuole (SCV) within macrophages. Intracellular S. Typhimurium must respond to the acidity of the SCV, accumulation of reactive oxygen/nitrogen species, and fluctuations in nutrient availability. In Chapter 1, we describe what is known on S. Typhimurium pathogenicity during human infection and previous molecular approaches for profiling the genetic and transcriptomic determinants of disease. Chapter 2 introduces my novel approach for profiling the transcriptome of intracellular S. Typhimurium during macrophage infection using a comprehensive library of GFP-reporter strains representing ~3,000 computationally identified S. Typhimurium promoters. To begin, we describe in silico and in vitro construction of the S. Typhimurium GFP-reporter library to study the dynamics of transcriptional regulation. Our approach for using time-lapse fluorescence microscopy to comprehensively profile intracellular promoter activity is introduced, as are the methods for quality control of the library. Chapter 2 also provides an overview of the dynamic promoter activity of S. Typhimurium during intracellular infection of RAW 246.7 macrophages. Using bulk measurements and single-cell imaging, we uncover macrophage-specific transcriptional regulation of virulence-related promoters, and previously unidentified transcriptional activity of metabolic genes, prophage genes, and canonical pathogenicity islands during infection. In Chapter 3, we describe the metabolic response of intracellular S. Typhimurium during late-stage macrophage infection, specifically, increased activity of promoters-associated with the Entner-Doudoroff pathway. In addition, we provide a brief description of the inter-serovar variability of pathogenicity-related promoters, thus revealing the influence of transcriptional dynamics in determining serovar-specific regulation of pathogenicity-associated genetic elements. Finally, Chapter 4 discusses the how this dataset should collectively provide a powerful resource for systems-level quantification of Salmonella transcriptional dynamics and integration with an in vitro dataset profiling the S. Typhimurium GFP-reporter library in defined and complex media conditions

Keywords: Medicine.
Microbiology and Immunology.
Advisor: Monack Denise M.
Amieva Manuel
Huang Kerwyn Casey
Spormann Alfred M.