The role of oxyR and soxRS in oxidative stress survival in Shigella flexneri

Abstract

Shigella flexneri, a facultative intracellular pathogen, is exposed to a variety of environments inside and outside of the human host. Some of these environments may contain significant oxidative stress. S. flexneri mutants were generated with deletions in the major oxidative stress regulators oxyR and/or soxRS to test their importance in Shigella biology. Strains that contained a deletion of oxyR had reduced growth and survival during aerobic growth, but not microaerobic growth. The mutants were also defective in surviving exposure to oxidative stress: oxyR mutants were sensitive to hydrogen peroxide, while soxRS mutants were sensitive to superoxide. Although the ΔsoxRS, ΔoxyR, and ΔoxyR/ΔsoxRS mutant Shigellae survived similarly to the parental strains within macrophages, the mutants formed plaques on Henle cell monolayers that were slightly smaller than the plaques formed by the wildtype strain.

Figure 1. The S. flexnerioxyR mutants show reduced growth in LB under aerobic growth conditions

Overnight cultures of the each strain [SM100, ◆; UR021, ■; UR23,Δ; and UR025, ●] were diluted to an OD₆₅₀ of 0.015 in LB and grown at 37°C under aerobic conditions. Growth was measured via optical density at 650 nm (OD₆₅₀). The data presented are the means of three experiments, and the standard deviations of the means are indicated.

Figure 2. The S. flexnerioxyR mutants show reduced viability in LB under aerobic growth conditions

Overnight cultures of the each strain were diluted to an OD₆₅₀ of 0.015 in LB and grown at 37°C under aerobic conditions for 8 hours. Serial dilutions of each culture were spotted on Congo Red plates containing 40 units of catalase per plate to determine viable bacterial colony forming units (CFU) per ml. The data presented are the means of three experiments, and the standard deviations of the means are indicated. The difference between SM100 and UR021 and between SM100 and UR025 was significant (p<0.05).

Figure 3. S. flexneri oxyR mutants show reduced colony size on agar plates

Subcultures of each strain were started from 17 hr overnight cultures (1:100 for wildtype and 1:50 for mutants) and grown under aerobic growth conditions at 37°C for 24 hr. Serial dilutions of each culture were then spread on Congo Red plates. Colony diameter was measured for each strain 22 hr after plating. The percentage of total colonies falling in each size category is reported as indicated in the accompanying legend. The data presented are the means of at least three replicate experiments. pCW1 carries the S. flexneri oxyR gene on the vector pWKS30.

Figure 4. Growth in microaerobic conditions suppresses the growth and survival defects of oxyR mutants

Overnight cultures of the each strain were subcultured into L broth and grown at 37°C under microaerobic conditions in a BD GasPak EZ Campy Pouch for 24 hours. (A) Growth was measured via optical density at 650 nm (OD₆₅₀). (B) Serial dilutions of each culture were spotted on Congo Red plates containing 40 units of catalase to determine viable bacterial colony forming units (CFU) per ml. The data presented are the means of three experiments, and the standard deviations of the means are indicated.

Figure 5. Diffusion Assays with Oxidative Stressors

Approximately 1–6 x10⁶ cells were spread on 20 mL LB + carbenicillin plates. Next, either 10 μl of 1M H₂O₂ (Panel A) or 50 mM PMS (Panel B) was added to a 6 mm BBL disk in the center of the plate. Following overnight growth, the diameters of the areas of clearing surrounding the discs were measured. The data presented are the means of three experiments, and the standard deviations of the means are indicated. pWKS30 is the vector control; pCW1 carries oxyR on pWKS30, and pRJ3 carries soxRS on pWKS30

Figure 6. Survival of the S. flexnerioxyR/soxRS mutant in activated macrophage cells

RAW264.1 macrophages, activated by mouse interferon-γ, were infected with Shigella (SM100, ◆ or UR025, ●) at an MOI of 10. Gentamicin was added to kill all extracellular bacteria, and the infected monolayers were allowed to incubate for up to 6 hours. At each time point, macrophages were lysed with Triton X-100, and lysates were diluted and plated on LB agar containing 40 U catalase. The data presented are the means of at least three experiments, and the standard deviations of the means are indicated.

Figure 7. Plaque assay with S. flexnerioxyR and soxRS mutants

Confluent Henle cell monolayers were infected with 10⁴ bacteria per 35-mm diameter plate and the diameter of the plaques were measured after 3 days. The data presented are the means of at least three experiments, and the standard deviations of the means are indicated. The difference between SM100 and UR025 was significant (p<0.05).