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.oxidative stress | reactive oxygen species | Salmonella | bacterial pathogenesis | redox-sensitive GFP

Hoodwinking the Big-Eater to Prosper: The <b><i>Salmonella</i></b>-Macrophage Paradigm

“…The integrity of the SCV is also crucial to evade ROS. Cytosolic population of Salmonella has been reported to face enhanced ROS levels in both human and murine macrophages [32].…”

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

“…While substantial work has illuminated bacterial strategies to evade host antibacterial activities in macrophages that are exerted by the phagocyte NADPH oxidase and inducible nitric oxide synthase (iNOS) (26,27,28,29), much less is known about Salmonella adaptation mechanisms in host epithelial cells. Given that Salmonella encodes ϳ4,500 proteins, adequate proteome sampling would be crucial to our understanding of Salmonella infection biology.…”

In vitro Real-time Measurement of the Intra-bacterial Redox Potential

“…This calculation has previously been described (Gutscher et al, 2008;van der Heijden et al, 2015). In addition to the R, R red and R ox we need the fluorescence intensity at 480 nm under reduced or oxidized conditions (I480max and I480min respectively).…”

“…Previously, inadequate analytical methods prevented appropriate analysis of the intra-bacterial redox potential. Herein, we describe a method for the measurement of real-time changes to the intra-bacterial redox potential using redoxsensitive GFP (roGFP2) (van der Heijden et al, 2015). The roGFP2 protein is engineered to contain specific cysteine residues that form an internal disulfide bridge upon oxidation which results in a slight shift in protein conformation (Hanson et al, 2004).…”