Journal Article

Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages

Joris van der Heijden, Else S. Bosman, B. Brett Finlay, Lisa A. Reynolds

2015

Overview

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. Significance To date, inadequate analytical methods have severely limited our ability to understand virulence strategies used by intracellular bacterial pathogens to evade reactive oxygen species (ROS) during infection. We have developed a system that is based on redox-sensitive GFP for real-time and nondisruptive measurement of redox stress experienced by Salmonella inside phagocytic cells. Using this system, we directly report intrabacterial redox dynamics during Salmonella infection of macrophages. This biosensor, which can easily be applied to other bacteria, enables a detailed study of ROS/reactive nitrogen species (RNS) evasion strategies, even detecting differences between individual bacteria. Additionally, this technique can be used for directly measuring redox responses in different environmental conditions for many bacteria.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: analytical methods / Animals / Bacteria / Biological Sciences / biosensors / Cell Line / Cells / environmental factors / Green Fluorescent Proteins - genetics / Green Fluorescent Proteins - metabolism / HeLa Cells / Humans / macrophages / Macrophages - metabolism / Macrophages - microbiology / Mice / Molecules / Nitrogen / Oxidative Stress / Pathogens / Reactive Nitrogen Species - metabolism / reactive oxygen species / Reactive Oxygen Species - metabolism / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / Respiratory Burst / Salmonella / Salmonella enterica typhimurium / Salmonella typhimurium - genetics / Salmonella typhimurium - metabolism / Salmonella typhimurium - pathogenicity / salmonellosis / Vacuoles - metabolism / Vacuoles - microbiology / virulence