Fig. 5

The knockout strains of CRISPR-Cas components are ROS-susceptible owing to elevated H₂O₂influx, and reduced antioxidant genes expression. (A)The peritoneal macrophages were infected with S. Typhimurium strain 14028s wildtype (WT), CRISPR (ΔcrisprI, ΔcrisprII, and ΔΔcrisprI crisprII) and cas operon (Δcas op) knockout strains along with their respective complements (ΔcrisprI + pcrisprI and ΔcrisprII + pcrisprII). Intracellular fold proliferation was calculated by normalizing the CFU count of intracellular bacteria at 16 h to 2 h. (B) The bacterial strains were cultivated in MgM-MES media, with and without H₂O₂ for 8 h. RNA was isolated, followed by qRT-PCR analysis of ompW. Relative expression of the gene was calculated using the 2 ^-ΔΔCt method and normalized to reference gene rpoD. (C) The bacterial strains were cultivated in MgM-MES media until they reached an OD_600nm ∼ 0.5. They were then incubated in the dark for 5 min with 1 mM H₂O₂. The H₂O₂ levels in both extracellular and intracellular fractions were measured using H₂DCFDA. The H₂O₂ untreated sample was used as a control. (D) The bacterial strains were cultivated in SPI-2 inducing conditions (MgM-MES media), and qRT-PCR was performed from isolated RNA to evaluate the expression of ROS detoxifying enzymes, superoxide dismutases (sodCI and sodA), catalase (katG), and peroxidase (ahpC). Relative expression of the gene was calculated using the 2 ^–ΔΔCt method and normalized to the reference gene, rpoD. One-way ANOVA (Dunnett’s multiple comparison test) was used to determine significant differences between the WT and knockout strains, in at least three independent experiments, with at least 3 replicates in each. Error bars indicate SD. Statistical significance is shown as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p < 0.0001; and ns, not significant