title: Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages.
abstract: Many significant bacterial pathogens have evolved virulence mechanisms to evade degradation and exposure to reactive oxygen (ROS) and reactive nitrogen species (RNS), allowing them to survive and replicate inside their hosts. Due to the highly reactive and short-lived nature of ROS and RNS, combined with limitations of conventional detection agents, the mechanisms underlying these evasion strategies remain poorly understood. In this study, we describe a system that uses redox-sensitive GFP to nondisruptively measure real-time fluctuations in the intrabacterial redox environment. Using this system coupled with high-throughput microscopy, we report the intrabacterial redox dynamics of Salmonella enterica Typhimurium (S. Typhimurium) residing inside macrophages. We found that the bacterial SPI-2 type III secretion system is required for ROS evasion strategies and this evasion relies on an intact Salmonella-containing vacuole (SCV) within which the bacteria reside during infection. Additionally, we found that cytosolic bacteria that escape the SCV experience increased redox stress in human and murine macrophages. These results highlight the existence of specialized evasion strategies used by intracellular pathogens that either reside inside a vacuole or "escape" into the cytosol. Taken together, the use of redox-sensitive GFP inside Salmonella significantly advances our understanding of ROS and RNS evasion strategies during infection. This technology can also be applied to measuring bacterial oxidative and nitrosative stress dynamics under different conditions in a wide variety of bacteria.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; and Departments of Microbiology and Immunology, and.
authors.forename: Joris
authors.initials: J
authors.lastname: van der Heijden
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; and.
authors.forename: Else S
authors.initials: ES
authors.lastname: Bosman
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; and.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Reynolds
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; and Departments of Microbiology and Immunology, and Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 bfinlay@interchange.ubc.ca.
authors.forename: B Brett
authors.initials: BB
authors.lastname: Finlay
mesh_terms: D000818:Animals; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology*; D051379:Mice; D018384:Oxidative Stress; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016897:Respiratory Burst; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology
chemical_list: D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
keywords: Salmonella; bacterial pathogenesis; oxidative stress; reactive oxygen species; redox-sensitive GFP