Figure 4. NOX expression restores virulence to respiration-deficient L. monocytogenes strains.

(A) Plaque formation by cell-to-cell spread of L. monocytogenes strains in monolayers of mouse L2 fibroblast cells. The mean plaque size of each strain is shown as a percentage relative to the wildtype plaque size. Error bars represent standard deviations of the mean plaque size from two independent experiments. Statistical analysis was performed using the unpaired two-tailed t test. ****, p<0.0001; ns, no significant difference (p>0.05). (B) Intracellular growth of L. monocytogenes strains in murine bone marrow-derived macrophages (BMMs). At 1-hour post-infection, infected BMMs were treated with 50 μg/mL of gentamicin to kill extracellular bacteria. Colony-forming units (CFU) were enumerated at the indicated times. Results are representative of three independent experiments. (C) Bacterial burdens in murine spleens and livers 48 hours post-intravenous infection with indicated L. monocytogenes strains. The median values of the CFUs are denoted by black bars. The dashed lines represent the limit of detection. Data were combined from two independent experiments, n = 10 mice per strain, but for the wildtype +NOX strain (n = 9 mice). Statistical significance was evaluated using one-way ANOVA and Dunnett’s post-test using the wildtype control strain to compare with the NOX-complemented strains. Significance between the parental and the NOX-complemented strains was determined using the unpaired two-tailed t test. ****, p<0.0001; **, p<0.01; ns, no significant difference (p>0.05). ΔQC, ΔqoxA/ΔcydAB; ΔQC/fmnB, ΔqoxA/ΔcydAB/fmnB::tn; Δndh1/ndh2, Δndh1/ndh2::tn; + NOX, strains complemented with Lactococcus lactis nox.