Role of FruR transcriptional regulator in virulence of Listeria monocytogenes and identification of its regulon

Abstract

Listeria monocytogenes is a ubiquitous opportunistic foodborne pathogen capable of survival in various adverse environmental conditions. Pathogenesis of L. monocytogenes is tightly controlled by a complex regulatory network of transcriptional regulators that are necessary for survival and adaptations to harsh environmental conditions both inside and outside host cells. Among these regulatory pathways are members of the DeoR-family transcriptional regulators that are known to play a regulatory role in sugar metabolism. In this study, we deciphered the role of FruR, a DeoR family protein, which is a fructose operon transcriptional repressor protein, in L. monocytogenes pathogenesis and growth. Following intravenous (IV) inoculation in mice, a mutant strain with deletion of fruR exhibited a significant reduction in bacterial burden in liver and spleen tissues compared to the parent strain. Further, the ΔfruR strain had a defect in cell-to-cell spread in L2 fibroblast monolayers. Constitutive activation of PrfA, a pleiotropic activator of L. monocytogenes virulence factors, did not restore virulence to the ΔfruR strain, suggesting that the attenuation was not a result of impaired PrfA activation. Transcriptome analysis revealed that FruR functions as a positive regulator for genes encoding enzymes involved in the pentose phosphate pathway (PPP) and as a repressor for genes encoding enzymes in the glycolysis pathway. These results suggested that FruR may function to facilitate NADPH regeneration, which is necessary for full protection from oxidative stress. Interestingly, deletion of fruR increased sensitivity of L. monocytogenes to H2O2, confirming a role for FruR in survival of L. monocytogenes during oxidative stress. Using anti-mouse neutrophil/monocyte monoclonal antibody RB6-8C5 (RB6) in an in vivo infection model, we found that FruR has a specific function in protecting L. monocytogenes from neutrophil/monocyte-mediated killing. Overall, this work clarifies the role of FruR in controlling L. monocytogenes carbon flow between glycolysis and PPP for NADPH homeostasis, which provides a new mechanism allowing metabolic adaptation of L. monocytogenes to oxidative stress.

Fig 1. The ΔfruR strain is attenuated in mice at 72 hours post-infection and PrfA activation does not rescue attenuation of the ΔfruR strain.

Each animal group (5 mice per cage) was IV infected with indicated strain. Livers (A and C) and spleens (B and D) were harvested at 72 hours post-infection and bacterial concentrations were determined. Data were analyzed using a nonparametric Mann-Whitney test. Median numbers for each strain are indicated by horizontal lines. Asterisks indicate significant differences (P < 0.05) compared to the wildtype.

Fig 2. The ΔfruR strain has decreased plaque size and numbers in L2 fibroblast monolayers compared to wildtype and complemented ΔfruR (ΔfruR_com).

(A) Relative plaque size is based on the percent plaque size compared to wildtype. (B) Relative plaque number is based on percent plaque number compared to wildtype. Monolayers of L2 fibroblasts were infected with the indicated strains, and plaque number and size were determined at 96 hours post infection. The experiment was repeated 3 independent times. At least 10 plaques were measured each time. Error bars represent the standard errors of the means and asterisks (*) indicate statistical significance (P < 0.05) by two-tailed Student’s t test compared to wildtype.

Fig 3. Intracellular replication of wildtype L. monocytogenes and ΔfruR.

J774A.1 macrophage cells were infected with ΔfruR, wildtype (WT), and complemented ΔfruR (ΔfruR_com). After infection, monolayers were washed, and medium containing gentamicin was added. At 2-, 4-, and 6-hours post-infection, macrophages were lysed, and released intracellular bacteria were quantified by determining CFU/ml in the lysate on BHI agar plates. All infections were performed in five replicates, and two independent assays were performed. The statistical significance between ΔfruR and wildtype at each indicated time point was determined using a student t test. Asterisks indicate statistical significance, with P < 0.05.

Fig 4. Growth curves of wildtype L. monocytogenes F2365 and ΔfruR in minimal medium with 50 mM glucose.

Growth assays were performed in a 24-well plate, and data are averages of three independent experiments with four replicates each.

Fig 5. FruR is required for L. monocytogenes response to oxidative stress.

(A) The ΔfruR strain has increased lag phase compared to wildtype during growth in BHI broth with 10 mM H₂O₂. Growth assays were performed in a 24-well plate and were repeated three independent times with five replicates. Error bars represent SEM. Asterisks mark time points at which OD₆₀₀ were significantly different (P < 0.05). (B) Survival of ΔfruR, wildtype (F2365), and complemented (ΔfruR_com) strains after exposure to H₂O₂. L. monocytogenes strains were grown to mid-log phase, resuspended in BHI, and treated with H₂O₂. After 1 hour, CFU were enumerated by colony counts on BHI agar plates. Percent survival was calculated by dividing bacterial concentration (CFU/ml) after treatment with H₂O₂ by the initial CFU/ml for the same strain prior to treatment (time point 0). Data are the mean ± SEM of three experiments with five replicates. At each concentration, Student t-test was used to compare the survival percent. Asterisks indicate significant differences (P < 0.05) compared to the wildtype.

Fig 6. The contribution of FruR to neutrophil/monocytes resistance.

The ΔfruR strain has similar virulence as wildtype strain in mice depleted of neutrophils/monocytes. Mice (5 mice per cage) were injected IV with monoclonal antibody RB6 at 24 and 6 hours before experimental infection. Livers and spleens were harvested at 72 hours post-infection. There was no significant difference in CFU observed between ΔfruR strain and wildtype.