The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium

Abstract

Salmonella enterica Serovar Typhimurium (S. Typhimurium) is an intracellular bacterium that overcomes host immune system barriers for successful infection. The bacterium colonizes the proximal small intestine, penetrates the epithelial layer, and is engulfed by macrophages and neutrophils. Intracellularly, S. Typhimurium encounters highly toxic reactive oxygen species including hydrogen peroxide and hypochlorous acid. The molecular mechanisms of Salmonella resistance to intracellular oxidative stress is not completely understood. The ArcAB two-component system is a global regulatory system that responds to oxygen. In this work, we show that the ArcA response regulator participates in Salmonella adaptation to changing oxygen levels and is also involved in promoting intracellular survival in macrophages and neutrophils, enabling S. Typhimurium to successfully establish a systemic infection.

Fig 1. Relative CFUs harvested from mutant vs. wild type S. Typhimurium strains.

Strains from S. Typhimurium were incubated at a MOI of 100 with HEp-2, RAW 264.7 and bone-marrow derived neutrophils. Intracellular bacteria were harvested at 1 hpi (A) and 3 hpi (B) by lysing the cells with sodium deoxycholate. Values are the number of CFU from each strain relative to the number of CFU from the parental strain. Data represent mean ± SD from 5 independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Bonferroni post hoc test.

Fig 2. Total reactive oxygen species production.

S. Typhimurium strains were incubated at a MOI of 100 with each cell type and ROS were evaluated at 1 and 3 hpi. The amount of ROS was determined by quantifying DCF fluorescence in neutrophils and macrophages co-cultured with the parental S. Typhimurium 14028s, and the ΔarcA and ΔarcB mutants. Values indicate Arbitrary Fluoresce Units (AFU) normalized by the number of bacteria recovered. One-way ANOVA followed by Bonferroni post hoc test.; no significant differences found at α = 0.05. Biological replicates n = 5, three technical replicates per experiment.

Fig 3. Relative expression of SPI-1 genes sipC and hilA in S. Typhimurium ΔarcA and ΔarcB strains inside bone-marrow-derived murine neutrophils.

The effect of ΔarcA and ΔarcB on SPI-1 genes sipC and hilA was determined from bacteria isolated from infected bone-marrow-derived murine neutrophils. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with the Bonferroni correction comparing mutant strains with a wild type strain separately at 1 and 3 h postinfection. Biological replicates n = 5, three technical replicates per experiment.

Fig 4. Relative expressions of genes related to detoxification (sodA, sodB, katN, katG, katE, ahpC and ahpF) in S. Typhimurium ΔarcA and ΔarcB strains inside bone-marrow-derived murine neutrophils.

*P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with the Bonferroni correction comparing mutant strains with a wild type strain separately at 1 and 3 h postinfection. Biological replicates n = 5, three technical replicates per experiment.

Fig 5. Relative expression of porin genes (ompC, ompD, ompF and ompW) in S. Typhimurium ΔarcA and ΔarcB strains inside bone-marrow-derived murine neutrophils.

ΔarcA and ΔarcB strains inside bone-marrow-derived murine neutrophils. *P < 0 05; **P < 0 01; ***P < 0 001. One-way ANOVA with the Bonferroni correction comparing mutant strains with a wild type strain separately at 1 and 3 h postinfection. Biological replicates n = 5, three technical replicates per experiment.

Fig 6. Relative expression of metabolic genes (manZ, pgi, pmgI and fbaB) in S. Typhimurium ΔarcA and ΔarcB strains inside bone-marrow-derived murine neutrophils.

ΔarcA and ΔarcB strains inside bone-marrow-derived murine neutrophils. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with the Bonferroni correction comparing mutant strains with a wild type strain separately at 1 and 3 hours postinfection. Biological replicates n = 5, three technical replicates per experiment.

Fig 7. Recovery of Salmonella in C57BL/6 mice.

(A) Mice orally infected with 1×10⁵ bacteria/100 μl S. Typhimurium 14028s. (B) Mice intraperitoneally infected with 1×10³ bacteria/100 μl S. Typhimurium 14028s. ΔarcA and ΔarcB strains were grown until OD₆₀₀ = 0.2 in microaerophilic conditions. One-way ANOVA with the Bonferroni correction. *P < 0.05; **P < 0.01, ***P < 0.001. Biological replicates n = 5, five technical replicates per experiment.