Abstract
Salmonella enterica serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the Salmonella-containing vacuole (SCV) within host cells. ROS damage cellular compounds, thereby impairing bacterial viability and inducing cell death. Proteins containing iron–sulfur (Fe–S) clusters are particularly sensitive and become non-functional upon oxidation. Comprising five enzymes with Fe–S clusters, the TCA cycle is a pathway most sensitive toward ROS. To test the impact of ROS-mediated metabolic perturbations on bacterial physiology, we analyzed the proteomic and metabolic profile of STM deficient in both cytosolic superoxide dismutases (ΔsodAB). Incapable of detoxifying superoxide anions (SOA), endogenously generated SOA accumulate during growth. ΔsodAB showed reduced abundance of aconitases, leading to a metabolic profile similar to that of an aconitase-deficient strain (ΔacnAB). Furthermore, we determined a decreased expression of acnA in STM ΔsodAB. While intracellular proliferation in RAW264.7 macrophages and survival of methyl viologen treatment were not reduced for STM ΔacnAB, proteomic profiling revealed enhanced stress response. We conclude that ROS-mediated reduced expression and damage of aconitase does not impair bacterial viability or virulence, but might increase ROS amounts in STM, which reinforces the bactericidal effects of antibiotic treatment and immune responses of the host.
Highlights
Pathogenic bacteria are continuously exposed to reactive oxygen species (ROS), which arise from three major sources. (i) During aerobic respiration, intrinsic ROS formation occurs by the electron transport chain (Messner and Imlay, 1999). (ii) ROS production is one of the antimicrobial mechanisms of bacterial clearance by professional phagocytes (Aussel et al, 2011). (iii) recent studies demonstrated increased concentration of intrinsic ROS induced by antibiotic treatment (Kohanski et al, 2007; Belenky et al, 2015)
While most previous studies analyzed the effects of ROS in ‘pulse-chase’ experiments with short-term ROS exposure (Wang et al, 2009; Allen and Griffiths, 2012; Drazic et al, 2015; Fu et al, 2017), we elucidated the consequences of long-term oxidative stress on serovar Typhimurium (STM) metabolism
By deletion of the cytosolic superoxide dismutases SodA and SodB, we abolish the ability of STM to neutralize cytosolic superoxide anions (SOA) and expect a higher exposure to oxidative stress compared to the WT
Summary
Pathogenic bacteria are continuously exposed to reactive oxygen species (ROS), which arise from three major sources. (i) During aerobic respiration, intrinsic ROS formation occurs by the electron transport chain (Messner and Imlay, 1999). (ii) ROS production is one of the antimicrobial mechanisms of bacterial clearance by professional phagocytes (Aussel et al, 2011). (iii) recent studies demonstrated increased concentration of intrinsic ROS induced by antibiotic treatment (Kohanski et al, 2007; Belenky et al, 2015). In contrast to HPO, SOA are negatively charged, unable to cross membranes. This gives rise to an ongoing debate of the primary targets in bacterial cells leading to clearance by immune cells (De Groote et al, 1997; Mumbengegwi et al, 2008; Craig and Slauch, 2009; Slauch, 2011). During early steps of phagosome maturation, NADPH oxidase is recruited to the phagosome membrane and increases SOA concentration within the compartment (Kamen et al, 2008; Craig and Slauch, 2009). Being much less restricted in targets, HR damage various macromolecules within the bacterial cell, leading to reduced growth or even cell death (Kobayashi et al, 2014)
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