Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

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

  • Hossam Abdelhamed ,

    Roles Funding acquisition, Methodology, Writing – original draft, Writing – review & editing

    abdelhamed@cvm.msstate.edu

    Affiliation Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, United States of America

    ⨯
  • Reshma Ramachandran,

    Roles Methodology

    Affiliations Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, United States of America, Department of Poultry Science, Mississippi State University, Starkville, MS, United States of America

    ⨯
  • Lakshmi Narayanan,

    Roles Methodology

    Affiliation Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, United States of America

    ⨯
  • Shamima Islam,

    Roles Methodology

    Affiliation Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, United States of America

    ⨯
  • Ozdemir Ozan,

    Roles Methodology

    Affiliation Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, United States of America

    ⨯
  • Nancy Freitag,

    Roles Conceptualization, Investigation, Supervision, Writing – review & editing

    Affiliation Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America

    ⨯
  • Mark L. Lawrence

    Roles Conceptualization, Investigation, Supervision, Validation, Writing – review & editing

    Affiliation Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, United States of America

    ⨯

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.

Citation: Abdelhamed H, Ramachandran R, Narayanan L, Islam S, Ozan O, Freitag N, et al. (2022) Role of FruR transcriptional regulator in virulence of Listeria monocytogenes and identification of its regulon. PLoS ONE 17(9): e0274005. https://doi.org/10.1371/journal.pone.0274005

Editor: Z. Tom Wen, LSU Health Sciences Center School of Dentistry, UNITED STATES

Received: May 4, 2022; Accepted: August 19, 2022; Published: September 2, 2022

Copyright: © 2022 Abdelhamed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This study was supported (HA) by the Center for Biomedical Research Excellence in Pathogen-Host Interactions, National Institute of General Medical Sciences, National Institutes of Health (P20 GM103646-08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Listeria monocytogenes, a facultative intracellular pathogen, is the causative agent of listeriosis, a potentially fatal foodborne infection [1]. L. monocytogenes is a frequent contaminant of food processing facilities and has been responsible for some of the largest and most expensive food recalls in US history [2–5]. Although healthy individuals generally exhibit febrile gastroenteritis, immunocompromised persons, pregnant women, and elderly persons often suffer more severe invasive forms of illness [6]. L. monocytogenes invasive disease has a high mortality rate even with antibiotic treatment [6].

L. monocytogenes has a biphasic lifestyle, transitioning from a saprophyte in the environment to a facultative intracellular pathogen within host cells. This transition is mainly mediated by virulence regulator PrfA, a member of the Crp/Fnr family [7–10]. PrfA regulates the transcription of core virulence factors necessary for survival and replication of L. monocytogenes within host environment [11,12]. PrfA activity is retained at a low basal level in the outside environment, while PrfA activity becomes high upon vacuolar escape and access to host cytosol [11,13]. Published data suggest that binding of PrfA to glutathione in the cytosol of the host cell is necessary for increasing the activity of PrfA at target genes [14].

L. monocytogenes moves through various niches and responds to each environment it encounters, including extracellular, abiotic, and intracellular environments [7,15]. For this to occur, L. monocytogenes requires a well-coordinated regulatory system that can sense and respond to a unique environment through signaling pathways that lead to the expression of proteins/factors aiding in survival [16]. These transcriptional networks are highly complex and require the coordination of a large number of regulatory elements [17,18]. To fully understand the pathogenic processes that allow L. monocytogenes to survive and adapt in stress-related environmental conditions and host colonization, it is important to clarify the function of transcriptional networks and how they are regulated. Many features and components of these transcriptional factors have not been well characterized.

Transcriptional regulators of the DeoR-family are widespread in Gram-positive and Gram-negative bacteria, and most of the characterized regulators in this family are involved in sugar metabolism or nucleoside metabolism [19,20]. However, there are some examples of DeoR-family members participating in bacterial responses to environmental conditions and stress [21–24]. Proteins of the DeoR family exhibit several common features. First, their length is highly conserved, ranging from 240 to 260 amino acids. Second, DeoR-family proteins contain a highly-conserved N-terminal region with a helix-turn-helix DNA-binding motif [22,25]. Third, the C-terminal region acts as a sensor domain and is responsible for oligomerization and binding effectors, which are generally phosphorylated intermediates of the relevant metabolic pathway [25]. By analyzing the genome of L. monocytogenes strain F2365, we found seven uncharacterized members of the DeoR-family of regulators. A phylogenetic tree of the seven DeoR-family regulators revealed that they can be classified into two main clades (S1 Fig).

One of the DeoR-family regulators encoded by L. monocytogens is FruR, which is a transcriptional repressor of the fructose operon (fruRBA) [26]. FruA is a fructose phosphotransferase, and FruB is phosphofructokinase [27–29]. However, FruR influences the regulation of other genes encoding enzymes involved in different pathways such as glycolysis, gluconeogenesis, and the Krebs cycle [29,30]. In Escherichia coli and Salmonella typhimurium, a fruR null mutation altered the rates of utilization of at least 36 carbon sources [31]. Therefore, it was concluded that FruR serves as a pleiotropic transcriptional regulator that modulates the flow of carbon through different pathways [31–34]. The function of FruR in L. monocytogenes is unexplored, even though sugar metabolism is crucial for virulence [12,35,36]. This provoked us to investigate the role of FruR in L. monocytogenes virulence and to determine the FruR regulon in L. monocytogenes.

Material and methods

Bacterial strains and growth conditions

Listeria monocytogenes F2365 strain and derivative mutants were cultured in brain heart infusion (BHI) medium (Difco) at 37°C. Escherichia coli DH5α was grown in Luria–Bertani (LB) (Difco), either broth or agar. Bacterial strains and plasmids used in this study are described in Table 1. Murine fibroblast (ATCC CRL-2648) cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC, Manassas, VA) supplemented with 20% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA) and 1% glutamine. Murine macrophage cell line J774A.1 was grown in DMEM supplemented with 10% FBS. Cultures were maintained at 37°C with 5% CO2 under humidified conditions. When necessary, 10 μg/ml erythromycin, 100 μg/ml ampicillin, 50 μg/ml neomycin, and/or 10 μg/ml gentamicin were added to the growth media.

thumbnail
Download:
Table 1. Bacterial strains, plasmids, and primers used in this study.

https://doi.org/10.1371/journal.pone.0274005.t001

Construction of ΔfruR mutant and complemented strain

The fruR gene (LMOf2365_2307) encoding fructose operon transcriptional repressor was targeted for in-frame deletion using splice overlap extension and allelic exchange with pHoss1 as previously published [39]. The sequence of primers used for construction and validation of the mutant is listed in Table 1. PCR analysis with flanking primers (A and D) was done to confirm deletion of the fruR gene. The fruR deletion was further confirmed by sanger sequencing of the amplified deletion fragment. For complementation assays, a DNA fragment containing the entire fruR gene and its upstream promoter was amplified from the F2365 genome and ligated into pPL2 shuttle integration vector [40]. The fruR-complementing plasmid was electrotransformed into F2365ΔfruR to obtain the complemented strain. Successful complementation was confirmed by PCR analysis and sequencing of genomic DNA.

Growth assay

Growth of wildtype F2365, F2365ΔfruR, and the complemented strain in BHI broth and minimal medium (MM) were compared [41]. Bacterial growth curves were generated by inoculating media with a standard dose of overnight culture in a 24-well plate and incubated on a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) at 37°C under static growth conditions. The optical density at 600 nm (OD600) was monitored automatically every hour for 24 hours with moderate shaking (160 rpm) for 15 seconds before reading. The growth assays were conducted in three independent experiments, and each experiment was run with four replicates. Growth curves with different carbon sources were obtained by inoculating the strains into MM supplemented with 50 mM glucose, mannose, fructose, maltose, or sucrose.

Virulence in mice

All animal experiments were done in accordance with a protocol (18–508) approved by the Institutional Animal Care and Use Committee at Mississippi State University. The virulence of ΔfruR was compared with L. monocytogenes strain F2365 and complemented strain in BALB/c mice (Jackson Laboratory) through intravenous (IV) injection via the tail vein and through oral routes as previously described [42–44]. Twelve-week-old female BALB/c mice were housed in 5 cages (5 mice/cage) and kept under specific pathogen-free conditions. After prewarming with a heat pad, each mouse was infected with approximately 2 x 104 CFU/mouse in 200 μl volume for each bacterial strain. Control mice were inoculated with sterile saline. Animals were monitored daily for signs of disease. At 24- and 72-hours post-infection, mice were euthanized. After euthanasia, spleen and liver tissues were aseptically removed, weighed, and homogenized in sterile saline. Serial dilutions of homogenized tissues were spread on BHI agar plates to determine bacterial concentration in the tissues. For oral infection studies, approximately 5.5 × 106 CFU/ml of each bacterial strain was orally infused by gavage needle in each mouse and mice were euthanized on day 5 post-infection to determine the number of CFU per gram of tissue.

PrfA* expression in ΔfruR

The constitutively activated form of PrfA (PrfA*) was introduced into ΔfruR strain using U153 bateriophage-mediated phage transduction as described [45,46]. Briefly, U153 phage were grown in the presence of L. monocytogenes strain NF-L1775 containing the transcriptional fusion fragment (prfA* actA-neo-gus); the resulting phage lysate was incubated with the ΔfruR strain for 40 min at room temperature. After incubation, the mixture was spread on BHI plates containing 50 μg/ml neomycin and 50 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (Inalco, Milano Italy). Positive transductants that were both neomycin resistant and dark-blue in color (conferred a high level of prfA* actA-neo-gus expression and activity) were isolated and used in further analysis. The integration of prfA* actA-neo-gus fragment in the chromosome of the ΔfruR strain was then reconfirmed by PCR analysis using actA promoter primer (Table 1) and sequencing of amplified product. Virulence of the resulting ΔfruRprfA* strain was evaluated in BALB/c mice using IV injection with 200 μl of 104 CFU/ml. Spleens and livers were harvested after 72 h, and bacterial CFUs were determined.

Plaque assay in murine L2 fibroblasts

Plaque assays using murine fibroblast (ATCC CRL-2648) monolayers were performed as described [47]. Briefly, fibroblasts were grown in DMEM in six-well tissue culture plates until they reached confluence. Confluent monolayers were infected with wildtype F2365, F2365ΔfruR, and the complemented strain for 1 hour with a multiplicity of infection (MOI) of approximately 1 to 10. DMEM agar containing 10 μg/ml of gentamicin was added to each well, and plates were incubated at 37°C and 5% CO2. After 96 hours, plaques were visualized by adding an overlay consisting of DMEM, 0.5% agarose, and 0.2% neutral red. The plates were stained overnight, and plaque size was evaluated using ImageJ from at least three independent experiments.

Infection of macrophages

Macrophage cell line J774A.1 was infected with wildtype F2365, F2365ΔfruR, and the complemented mutant strain at a mean MOI of 1 to 10. After infection, extracellular bacteria were killed by adding 0.5 ml DMEM medium containing 10 μg/ml gentamicin. After incubation for 2, 4, and 6 hours, infected cells were washed and lysed, and lysate was spread on BHI agar plates to quantify intracellular bacteria.

RNA-seq analysis

L. monocytogenes wildtype F2365 and ΔfruR (four independent biological replicates) were grown in BHI at 37°C until the growth reached mid-logarithmic phase (OD600 reached ∼ 0.06). Bacterial pellets were collected by centrifugation. The resulting pellet was maintained on ice and placed into RNase-free tubes (ThermoFisher Scientific) that contained 10 volumes of RNAlater solution (Ambion, Austin, TX) to minimize RNA degradation. RNA was isolated using a FastRNA™ SPIN Kit for Microbes with a FastPrep-24™ (MP Biomedicals, Santa Ana, CA) following the manufacturer’s instructions. Genomic DNA was eliminated from total RNA using on-column DNase treatment with an RNase-Free DNase Set (QIAGEN, Hilden, Germany). The quantity and quality of total RNA were analyzed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA) by measuring the OD260 nm/OD280 nm ratio. RNA samples (obtained in triplicate) were submitted to Novogene Corporation for quality control, library preparation, and sequencing using the HiSeq platform (Illumina) as described [48]. Before analyzing data, raw reads were filtered to remove reads containing adapters or low-quality reads. Filtered reads were mapped to the F2365 genome using Bowtie2 [49]. Transcriptomic analysis was conducted using Bioconductor Edge R [50] to identify differentially expressed genes of the wild type compared to mutant strain. To minimize false-positive results, a stringent False Discovery Rate (FDR) cutoff of 1 was applied.

Verification of gene expression by quantitative real-time PCR (qRT-PCR)

To validate RNA-seq results, qRT-PCR was performed on the same RNAs used for RNA-seq. Primers and gene information are listed in Table 1. cDNA was synthesized from RNA (5 μg) using the SuperScript cDNA synthesis kit (Thermo Scientific). The product of first-strand cDNA synthesis was diluted 50 times, and qRT-PCR was performed in a 20 μl reaction containing SYBR Green Real-time PCR master mix (Roche Diagnostic GmbH, Mannheim, Germany). Amplification and detection of specific products were performed with the Mx3000P™ real-time PCR system (Stratagene) with the following cycle profile: initial denaturation at 95°C for 10 min, 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. Expression of each gene was normalized against the expression of 16S rRNA before comparative analysis. For each gene, triplicate assays were performed, and transcription levels were quantified by 2-△△CT method [51].

Hydrogen peroxide sensitivity

Growth and survival of the ΔfruR strain were compared to wildtype during growth in BHI medium at 37°C supplemented with different concentrations of H2O2. Growth and survival experiments were performed on exponentially growing bacteria (OD600 < 0.2), which were harvested by centrifugation and resuspended in BHI supplemented with different concentrations of H2O2. Hydrogen peroxide was added to BHI to a final concentration of 10, 20, 50, and 100 mM. For the growth experiments, OD600 values were automatically determined every hour for 24 hours at 37°C using a Cytation 5 Cell Imaging. In the survival experiments, CFUs were quantified for wildtype, ΔfruR, and the complemented mutant strain at zero time and after 1 hour incubation with different concentrations of H2O2. The survival rate of bacteria was determined by comparing CFUs after treatment with H2O2 with the initial number of CFUs for the same strain present before treatment (time point 0).

ΔfruR virulence in neutrophil/monocyte depleted mice

Female Balb/c mice aged 10–12 weeks were neutrophil/monocyte depleted using intraperitoneal injection with monoclonal antibody RB6-8C5 (RB6) (500 μg) at 24 and 6 hours before initiation of bacterial infection. Neutrophil/monocyte depletion was microscopically confirmed using peripheral blood of the mice. Neutrophil/monocyte depleted mice were infected IV with wildtype or ΔfruR strain. At 72 hours post-infection, mice were euthanized, and bacterial concentrations were determined in liver and spleen.

Statistical analysis

The dot plots and median values of bacterial tissue concentrations in each mouse were generated using GraphPad Prism 8. For in vivo experiments, a non-parametric Mann-Whitney test was used to compare bacterial concentrations in liver and spleen of infected mice. Fold changes were calculated for each gene using 2−ΔΔCt and used for statistical analysis to determine significant differences in gene expression between F2365ΔfruR and wildtype strain. Intracellular bacteria concentrations and plaque sizes were compared using a two-tailed Student’s t test. P value < 0.05 was considered significant.

Results

fruR contributes to L. monocytogenes virulence

In L. monocytogenes, fruR is located in the fructose operon with fruA and fruB, with fruA encoding fructose phosphotransferase and fruB encoding phosphofructokinase. The ΔfruR strain did not show growth defects in brain heart infusion (BHI) broth compared to wildtype strain F2365 (S2 Fig).

In 10–12-week-old female Balb/c mice, animals infected with ΔfruR exhibited 2.3 and 1.6 log10 decreased bacterial CFU at 72 hours post-infection in spleens and livers compared to animals infected with wildtype bacteria, respectively (Fig 1A and 1B). However, no significant difference in bacterial numbers were observed between ΔfruR and wildtype at 24-hour post-infection (S3 Fig). The difference in the ΔfruR strain clearance between early and late time points indicates that FruR is not required for early colonization or for the initial establishment of infection and suggests a possible role for FruR during the later stages of infection or in resistance to the innate immune responses. The complemented strain carrying a wildtype copy of fruR was able to restore bacterial concentrations to a level similar to the wildtype strain in the infected animals. Similar to IV infection, oral infection of BALB/c mice with ΔfruR resulted in lower bacterial concentrations in the liver and spleen at 5 days post-infection (S3 Fig).

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0274005.g001

Constitutive activation of PrfA (PrfA*) does not restore virulence to ΔfruR

PrfA regulates expression of L. monocytogenes virulence genes and is essential for virulence [12,52]. We tested the hypothesis that the virulence defect in the ΔfruR strain is due to FruR directly affecting PrfA expression and/or activity. At 72 hours post-infection, bacterial concentrations in livers and spleens of mice infected with ΔfruRprfA* were significantly lower than wildtype (Fig 1C and 1D). This result indicates that the constitutive expression of PrfA* did not restore virulence to ΔfruR strain.

ΔfruR is defective in cell-to-cell spread

The ability of L. monocytogenes to form zones of clearing or plaques in murine L2 fibroblast cells is an indication of both bacterial escape from host cell vacuoles as well as bacterial cell-to-cell spread [53]. We therefore examined the ability of ΔfruR mutants to form visible plaques in monolayers of L2 fibroblasts (Fig 2). Mutants lacking fruR exhibited fewer numbers of plaques (an average of 49.8% reduction in plaque numbers) and the plaques formed were smaller in size (28.3% reduction in plaque size) in comparison to cell infected with wildtype (Fig 2A and 2B). These findings indicate a significant contribution of FruR to cellular invasion and suggest an additional defect exists that reduces intracellular replication and/or cell-to-cell spread.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0274005.g002

Contribution of FruR to intracellular replication

J774A.1 macrophage cells were used to assess the intracellular replication of the ΔfruR strain in comparison to cells infected with wildtype [54]. We found that intracellular numbers of ΔfruR were significantly decreased (P < 0.05) compared to wildtype F2365 at 2-, 4-, and 6-hours post-infection (Fig 3). However, the pattern of intracellular growth for ΔfruR was indistinguishable from wildtype. Therefore, while the plaque assay points to a defect in intracellular replication and/or cell-to-cell spread, this assay suggests that intracellular replication occurs, and thus the plaque assay results may be due to a defect in cell-to-cell spread.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0274005.g003

ΔfruR has decreased growth rate in defined media supplemented with glucose

To determine whether FruR facilitates uptake and utilization of carbon sources, we compared growth of ΔfruR with F2365 and complemented strain in minimal medium (MM) supplemented with different carbon sources. After 16 hours, growth of ΔfruR was significantly lower than the growth of wildtype in MM with glucose (Fig 4). In contrast, ΔfruR did not have decreased growth in MM supplemented with fructose, mannose, maltose, or sucrose (S4 Fig). This result suggests that FruR plays a specific role in glucose uptake and/or metabolism during stationary growth phase.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0274005.g004

Glycolysis and pentose phosphate pathway (PPP) genes are regulated by fruR

To determine the impact of FruR on L. monocytogenes gene expression, RNA-seq was used to compare ΔfruR to wildtype grown in BHI. Results of genes with false discovery rate <0.05 are shown in Table 2. In total, 12 genes were upregulated (threshold >1.5-fold), while 16 genes were downregulated in ΔfruR. In particular, genes encoding pentose phosphate pathway (PPP) enzymes and enzymes for NADPH synthesis were decreased in the ΔfruR strain, including glucose 6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydrogenase (gnd), transketolase (tkt), and transaldolase (taldo). Moreover, ΔfruR had upregulated genes encoding glycolysis enzymes, such as phosphofructokinase (fruB) and glucokinase (glck) [55]. These findings suggest that (i) FruR controls L. monocytogenes carbon metabolism by regulating the expression of genes encoding glycolysis and PPP enzymes, and (ii) FruR induces expression of genes encoding PPP enzymes and suppresses glycolysis genes.

thumbnail
Download:
Table 2. Genes with significantly changed expression in ΔfruR strain.

https://doi.org/10.1371/journal.pone.0274005.t002

In addition, four different regulators of sigma B factor (rsbR, rsbS, rsbT, and rsbU) were downregulated in ΔfruR. These genes are in the sigB operon of L. monocytogenes and contribute to regulation of σB activity in response to environmental stresses and energy stresses [56]. RNA-seq results were confirmed using qRT-PCR and primers for fruA, fruB, glck, and rsbT genes (S5 Fig).

FruR is required for L. monocytogenes response to oxidative stress

Based on RNA-seq findings, we hypothesized that ΔfruR strain might be more sensitive to oxidative stress due to defective NADPH production via PPP. Exposure to H2O2 is widely used to study oxidative stress in bacteria [57,58]. First, we compared growth in BHI medium with 10 mM H2O2, and we found that ΔfruR had an increased lag phase compared to wildtype (Fig 5A). By comparison, ΔfruR did not show growth defects in BHI broth (without H2O2) relative to wildtype.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0274005.g005

Next, we compared survival of wildtype, ΔfruR, and complemented strain after exposure to H2O2 for 1 hour in BHI. At 20, 50, and 100 mM of H2O2, wildtype strain viability was significantly higher than viability of ΔfruR after exposure for 1 hour, indicating that FruR contributes to survival of L. monocytogenes during oxidative stress (Fig 5B). These results suggest a novel virulence mechanism whereby FruR upregulates the PPP in L. monocytogenes, which is necessary for full protection from oxidative stress.

FruR contributes to L. monocytogenes resistance to neutrophil/monocyte-mediated killing in mice

We investigated whether attenuation of ΔfruR could be caused by increased susceptibility to innate immune cells. For this experiment, we used monoclonal antibody RB6 to deplete both neutrophils and inflammatory monocytes from mice [59]. The specificity and efficacy of RB6 is well established [60]. Following neutrophil/monocyte depletion, ΔfruR had similar virulence to wildtype in mice at 72 hours post-infection (Fig 6). These results suggest that the virulence attenuation of ΔfruR is neutrophil/monocyte-dependent and supports the new hypothesis that FruR has a specific function in protecting L. monocytogenes from neutrophil/monocyte-mediated killing.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0274005.g006

Discussion

FruR is annotated as a DeoR-family transcriptional regulator. L. monocytogenes FruR comprises 250 amino acids and includes a helix-turn-helix DNA-binding motif at the N-terminal region and a sensor domain at the C-terminal region. A previous study has shown significant upregulation of a gene encoding a DeoR-family protein (LMOF6854_2396, paralogous to FruR), when comparing transcriptomic sequencing of persistent and nonpersistent L. monocytogenes EGD-e strains grown in the presence of quaternary ammonium compounds (QACs) [61]. In another study, a DeoR-family transcriptional regulator gene (paralogous to FruR) was downregulated after 4 hours of exposure of L. monocytogenes to carnocyclin A, an antimicrobial peptide used to control the growth of L. monocytogenes in meat products [62]. Thus, it is probable that DeoR-family proteins, including FruR, have complex regulatory roles in L. monocytogenes, and their functions appear to include several cellular processes. The current study was undertaken to understand the importance of FruR in L. monocytogenes pathogenesis.

Our results indicate, for the first time, that FruR significantly contributes to in vitro and in vivo pathogenesis, highlighted by attenuation in mice following IV injection and impaired cell-to-cell spread in fibroblast cells. This finding is corroborated by previous studies that have connected DeoR-family regulators with bacterial pathogenesis. For example, FruR of Streptococcus pyogenes is important for survival in whole human blood and resistance to killing by human neutrophils and primary monocytes. However, FruR did not contribute to virulence in a murine model of infection, perhaps relevant to its replication niche (S. pyogenes is often found extracellular) [45,63]. In addition, a Brucella suis deoR1 mutant was shown to be strongly attenuated in human macrophages [64].

Previous studies demonstrated an existence link between regulation of utilizable carbon sources and expression of virulence genes in L. monocytogenes [35,65]. Given the role of DeoR family of transcriptional regulators in sugar metabolism, we asked the question whether the attenuation of ΔfruR strain could be explained by reduction in the PrfA activity. The introduction of the prfA* mutation to the fruR strain did not restore the virulence of ΔfruR strain, demonstrating that FruR has no regulatory role or connection with PrfA activity and suggesting that the ΔfruR colonization defect is due to a mechanism distinct from L. monocytogenes PrfA activation.

To provide further insight into the regulatory role of FruR in L. monocytogenes, we identified the genes controlled by FruR using RNA-seq. Of particular interest is that FruR functions to induce the expression of genes encoding PPP enzymes and to suppress glycolysis. Deletion of fruR downregulated genes encoding two PPP enzymes, glucose 6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd). Zwf catalyzes conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone (6PGL), generating NADPH from NADP. Gnd converts 6PGL into ribulose-5-phosphate (Ru5P), generating a second molecule of NADPH (S6 Fig). The PPP plays a crucial role in maintaining redox homeostasis in bacteria [66]. In L. monocytogenes, NADPH is used as the reducing agent for numerous reductase mechanisms involved in detoxification and protection from reactive oxygen species [14,67–69]. Therefore, our findings may indicate that a functional FruR is important for L. monocytogenes pathogenesis to regenerate sufficient NADPH to resist oxidative stress by directing carbon flow from glycolysis to the PPP pathway.

We therefore asked whether FruR contributes to L. monocytogenes resistance to oxidative stress using hydrogen peroxide (H2O2), which is widely used to study oxidative stress [70,71]. The ΔfruR mutant had a significant growth defect and reduced viability as result of exposure to H2O2. This result correlates with our RNA-seq findings showing that FruR induces the PPP, which is needed for NADPH production. Redirection of metabolic flux from glycolysis to the PPP provides protection against oxidative stress in yeast and mammalian cells, but this has not been thoroughly examined in prokaryotes [72–76]. Future work will explore the role of FruR in the induction of this shift and appropriately adjusting the L. monocytogenes response to oxidative stress. We will also explore the role of NADPH production enzymes in oxidative stress response.

Transcriptome analysis of ΔfruR also revealed downregulation of four genes encoding regulators of the sigB operon: rsbR, rsbS, rsbT, and rsbU (Table 2). In L. monocytogenes, the sigB operon encodes seven regulators of sigma B: RsbR, RsbS, RsbT, RsbU, RsbV, RsbW, and RsbX [56,77]. Bacterial σB plays an important role in coordinating gene expression in response to various environmental stresses, including high osmolarity, low pH, cold exposure, and antimicrobial stress [78]. In addition to its stress-protective function, σB regulates many other L. monocytogenes processes, including biofilm formation, cell-wall turnover, survival within the mammalian gastrointestinal (GI) tract, and regulation of carbon and energy metabolism [16,79]. It is possible that downregulation of σB in ΔfruR contributes to its attenuation and its survival defect in an oxidative environment. Thus, FruR may contribute to induction of σB activity under stress conditions. However, further studies are necessary to determine the nature of the interaction between FruR and σB factor.

Oxidative stress damage generated by production of reactive oxygen species (ROS) (such as superoxide radicals, hydrogen peroxide, hypochlorous acid, and hydroxyl radicals) is a potent bactericidal mechanism by which professional phagocytes (neutrophils, monocytes, and macrophages) limit L. monocytogenes systemic spread [80,81]. Therefore, we tested the hypothesis that FruR protects L. monocytogenes from oxidative stress imposed by neutrophils and monocytes. To this end, neutrophils/monocytes were depleted in mice with the monoclonal antibody RB6-8C5 prior to infection with wildtype and ΔfruR strains. At 72 hours after infection, ΔfruR exhibited a similar level of virulence compared to wildtype as indicated by bacterial concentrations in the liver and spleen of neutrophil-depleted mice. This suggests that neutrophils/monocytes play a significant role in the attenuation of ΔfruR compared to wildtype. It was reported that FruR is important for Streptococcus pyogenes survival in whole human blood and resistance to killing by human neutrophils and primary monocytes [82]. However, the molecular mechanism by which FruR mediates resistance to neutrophils was not determined. Our results showed that ΔfruR strain failed to upregulate PPP, and ΔfruR is more susceptible to oxidative killing. Because oxygen radicals are an important killing mechanism used by phagocytes, it is logical that generation of NADPH via the PPP is important for listerial resistance to phagocytes. Thus, we propose a new hypothesis whereby FruR mediates resistance of L. monocytogenes to phagocytes and oxidative stress by upregulation of PPP and increased NADPH production. Further, our results provide evidence that regulation of carbon flux from glycolysis to PPP plays a significant role in the pathogenesis of L. monocytogenes.

Reduced growth of ΔfruR in defined medium with glucose as the only carbon source was another interesting finding in our study. The ΔfruR strain did not show any growth defects in BHI, a nutrient-rich media. The defined medium contains limited resources and nutrients compared to nutrient-rich BHI. This result suggests that FruR contribute to glucose uptake in a nutrient-limited environment. FruR belongs to the DeoR family of transcriptional regulators, which in most instances act as transcriptional repressors in sugar metabolism [83]. Our transcriptome comparison between wildtype and ΔfruR revealed that FruR functions as a negative regulator for PTS fructose transporter subunit IIC (FruA) and 1-phosphofructokinase (FruB). Repression of FruA and FruB by FruR has been observed in other bacterial species [20,63]. The growth defect of ΔfruR in MM with glucose may be due to the upregulation of glucokinase (glck), which encodes an enzyme that catalyzes phosphorylation of glucose to glucose-6-phosphate (G6P) [55].

In conclusion, we showed that FruR contributes to L. monocytogenes virulence, survival during oxidative stress, and resistance to neutrophils/monocytes. Moreover, we found that FruR of L. monocytogenes act as a transcriptional activator for PPP, which is an important pathway for generation of NADPH and protection from oxidative stress. Transcriptional analysis also showed that FruR induces the sigma B factor operon. Sigma B is important for stress adaptation and controls the response to several stress cues, such as osmotic stress, light, and acid stress. Overall, this work suggests a novel virulence mechanism in which FruR upregulates the PPP in L. monocytogenes, and this upregulation is necessary for full protection from oxidative stress and phagocytic killing. FruR is the first transcriptional regulator of the DeoR-type characterized to date in L. monocytogenes. Further understanding of the regulatory mechanisms of FruR in L. monocytogenes could provide important information on how the pathogen uses regulation of metabolic pathways to adapt to stressful environments, including host phagocytic killing mechanisms.

Supporting information

S1 Fig. A phylogenetic tree of the DeoR-family regulators in L. monocytogenes F2365 strain.

The seven DeoR-family regulators are classified into two clades. In the first clade, the DeoR-family regulators are characterized by the presence of both DNA-binding domain at the N-terminus and sensor domain at the C-terminus. Six DeoR-family members, including FruR, are in the first clade. The second clade has one DeoR-family protein; it has the DNA-binding domain and sugar binding domain. The phylogenetic tree was constructed using the unweighted-pair group method with arithmetic mean. Multiple sequence alignment was conducted with CLUSTALW.

https://doi.org/10.1371/journal.pone.0274005.s001

(TIF)

S2 Fig. The ΔfruR strain did not show growth defects in BHI, as enriched medium.

The growth assay was conducted with L. monocytogenes strain F2365, ΔfruR, and complement strain. The growth kinetic was monitored using optical density measurements at 600 nm. The experiment was repeated three independent times with four replicates and the figure shows a representative experiment. Error bars represent SEM. The optical density values between ΔfruR strain and wildtype were not statistically significant (P > 0.05).

https://doi.org/10.1371/journal.pone.0274005.s002

(TIF)

S3 Fig. Bacterial counts (CFU/g) of L. monocytogenes strain F2365, ΔfruR strain, and complemented strain following IV and oral infection in Balb/c mice.

(A and B) Bacterial concentrations in livers and spleens upon IV infection via the tail vein with 2 x104 CFU and dissected at 24 hours post-infection. Differences in bacterial concentrations between ΔfruR strain and wildtype were not statistically significant (P > 0.05). (C and D) Bacterial concentrations in livers and spleens upon oral infection by gavage needle with 5.5x106 CFU and dissected at 5 days post-infection. 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.

https://doi.org/10.1371/journal.pone.0274005.s003

(TIF)

S4 Fig.

ΔfruR strain exhibits normal growth patterns in MM supplemented with fructose (A), mannose (B), maltose (C), or sucrose (D) as a sole carbon source. Bacterial growth curves were determined by optical density measurements at 600 nm. All growth data are the results from three independent experiments with four replicates and the figure shows a representative experiment. Error bars represent SEM. Differences between ΔfruR strain and wildtype were not statistically significant (P > 0.05).

https://doi.org/10.1371/journal.pone.0274005.s004

(TIF)

S5 Fig. RT-PCR analyses of upregulated genes (fruA and fruB) and downregulated genes (glck and rsbT) in ΔfruR compared to wildtype.

The data represent means ± standard errors from three biological replicates.

https://doi.org/10.1371/journal.pone.0274005.s005

(TIF)

S6 Fig. Simplified diagram of glycolysis and PPP in L. monocytogenes.

https://doi.org/10.1371/journal.pone.0274005.s006

(TIF)

Acknowledgments

We thank the Laboratory Animal Resources and Care Unit at Mississippi State University for animal and veterinary care.

References

  1. 1. Freitag NE (2006) From hot dogs to host cells: how the bacterial pathogen Listeria monocytogenes regulates virulence gene expression. Future Microbiology 1: 89–101.
  2. 2. Centers for Disease C, Prevention (1998) Multistate outbreak of listeriosis—United States, 1998. MMWR Morb Mortal Wkly Rep 47: 1085–1086.
  3. 3. Centers for Disease C, Prevention (2011) Multistate outbreak of listeriosis associated with Jensen Farms cantaloupe—United States, August-September 2011. MMWR Morb Mortal Wkly Rep 60: 1357–1358.
  4. 4. Centers for Disease C, Prevention (2000) Multistate outbreak of listeriosis—United States, 2000. MMWR Morb Mortal Wkly Rep 49: 1129–1130.
  5. 5. Mead PS, Dunne EF, Graves L, Wiedmann M, Patrick M, et al. (2005) Nationwide outbreak of listeriosis due to contaminated meat. Epidemiology and Infection 134: 744–751. pmid:16318652
  6. 6. Drevets DA, Bronze MS (2008) Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion. FEMS Immunol Med Microbiol 53: 151–165.
  7. 7. Xayarath B, Freitag NE (2012) Optimizing the balance between host and environmental survival skills: lessons learned from Listeria monocytogenes. Future microbiology 7: 839–852.
  8. 8. Joseph B, Przybilla K, Stuhler C, Schauer K, Slaghuis J, et al. (2006) Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening. J Bacteriol 188: 556–568.
  9. 9. Anaya-Sanchez A, Feng Y, Berude JC, Portnoy DA (2021) Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes. PLOS Pathogens 17: e1009819.
  10. 10. Chakraborty T, Leimeister-Wachter M, Domann E, Hartl M, Goebel W, et al. (1992) Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol 174: 568–574.
  11. 11. Port GC, Freitag NE (2007) Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA. Infect Immun 75: 5886–5897.
  12. 12. Scortti M, Monzó HJ, Lacharme-Lora L, Lewis DA, Vázquez-Boland JA (2007) The PrfA virulence regulon. Microbes Infect 9: 1196–1207. pmid:17764998
  13. 13. Bruno JC Jr., Freitag NE (2010) Constitutive activation of PrfA tilts the balance of Listeria monocytogenes fitness towards life within the host versus environmental survival. PLoS One 5: e15138.
  14. 14. Reniere ML, Whiteley AT, Hamilton KL, John SM, Lauer P, et al. (2015) Glutathione activates virulence gene expression of an intracellular pathogen. Nature 517: 170–173. pmid:25567281
  15. 15. Chaturongakul S, Raengpradub S, Wiedmann M, Boor KJ (2008) Modulation of stress and virulence in Listeria monocytogenes. Trends in Microbiology 16: 388–396.
  16. 16. Tiensuu T, Guerreiro DN, Oliveira AH, O’Byrne C, Johansson J (2019) Flick of a switch: regulatory mechanisms allowing Listeria monocytogenes to transition from a saprophyte to a killer. Microbiology 165: 819–833.
  17. 17. Schauer K, Geginat G, Liang C, Goebel W, Dandekar T, et al. (2010) Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling. BMC Genomics 11: 573.
  18. 18. Toledo-Arana A, Dussurget O, Nikitas G, Sesto N, Guet-Revillet H, et al. (2009) The Listeria transcriptional landscape from saprophytism to virulence. Nature 459: 950–956.
  19. 19. Engels V, Wendisch VF (2007) The DeoR-Type Regulator SugR Represses Expression of ptsG in Corynebacterium glutamicum. Journal of Bacteriology 189: 2955–2966.
  20. 20. Voigt C, Bahl H, Fischer RJ (2014) Identification of PTS(Fru) as the major fructose uptake system of Clostridium acetobutylicum. Appl Microbiol Biotechnol 98: 7161–7172.
  21. 21. Balleza E, Lopez-Bojorquez LN, Martinez-Antonio A, Resendis-Antonio O, Lozada-Chavez I, et al. (2009) Regulation by transcription factors in bacteria: beyond description. FEMS Microbiol Rev 33: 133–151. pmid:19076632
  22. 22. Elgrably-Weiss M, Schlosser-Silverman E, Rosenshine I, Altuvia S (2006) DeoT, a DeoR-type transcriptional regulator of multiple target genes. FEMS Microbiol Lett 254: 141–148. pmid:16451192
  23. 23. Ramos-Aires J, Plesiat P, Kocjancic-Curty L, Kohler T (2004) Selection of an antibiotic-hypersusceptible mutant of Pseudomonas aeruginosa: identification of the GlmR transcriptional regulator. Antimicrob Agents Chemother 48: 843–851.
  24. 24. Beck von Bodman S, Hayman GT, Farrand SK (1992) Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor. Proc Natl Acad Sci U S A 89: 643–647. pmid:1731335
  25. 25. Mortensen L, Dandanell G, Hammer K (1989) Purification and characterization of the deoR repressor of Escherichia coli. EMBO J 8: 325–331.
  26. 26. Turner SE, Pang Y-Y, O’Malley MR, Weisberg AJ, Fraser VN, et al. (2020) A DeoR-Type Transcription Regulator Is Required for Sugar-Induced Expression of Type III Secretion-Encoding Genes in Pseudomonas syringae pv. tomato DC3000. Molecular Plant-Microbe Interactions® 33: 509–518.
  27. 27. Benthin S, Nielsen J, Villadsen J (1993) Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation. Appl Environ Microbiol 59: 3206–3211.
  28. 28. Liu Y, Yoo BB, Hwang CA, Suo Y, Sheen S, et al. (2017) LMOf2365_0442 Encoding for a Fructose Specific PTS Permease IIA May Be Required for Virulence in L. monocytogenes Strain F2365. Front Microbiol 8: 1611.
  29. 29. Ramseier TM (1996) Cra and the control of carbon flux via metabolic pathways. Res Microbiol 147: 489–493. pmid:9084760
  30. 30. Chin AM, Feucht BU, Saier MH Jr., (1987) Evidence for regulation of gluconeogenesis by the fructose phosphotransferase system in Salmonella typhimurium. Journal of bacteriology 169: 897–899.
  31. 31. Ramseier TM, Bledig S, Michotey V, Feghali R, Saier MH Jr. (1995) The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli. Mol Microbiol 16: 1157–1169.
  32. 32. Ramseier TM, Negre D, Cortay JC, Scarabel M, Cozzone AJ, et al. (1993) In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium. J Mol Biol 234: 28–44.
  33. 33. Barriere C, Veiga-da-Cunha M, Pons N, Guedon E, van Hijum SA, et al. (2005) Fructose utilization in Lactococcus lactis as a model for low-GC gram-positive bacteria: its regulator, signal, and DNA-binding site. J Bacteriol 187: 3752–3761.
  34. 34. Gaurivaud P, Laigret F, Garnier M, Bové JM (2001) Characterization of FruR as a putative activator of the fructose operon of Spiroplasma citri. FEMS Microbiology Letters 198: 73–78.
  35. 35. Ripio MT, Brehm K, Lara M, Suárez M, Vázquez-Boland JA (1997) Glucose-1-phosphate utilization by Listeria monocytogenes is PrfA dependent and coordinately expressed with virulence factors. Journal of Bacteriology 179: 7174–7180.
  36. 36. Milenbachs AA, Brown DP, Moors M, Youngman P (1997) Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Mol Microbiol 23: 1075–1085.
  37. 37. Nelson KE, Fouts DE, Mongodin EF, Ravel J, DeBoy RT, et al. (2004) Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res 32: 2386–2395.
  38. 38. Miner MD, Port GC, Freitag NE (2008) Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA. Microbiology (Reading) 154: 3579–3589.
  39. 39. Abdelhamed H, Lawrence ML, Karsi A (2015) A novel suicide plasmid for efficient gene mutation in Listeria monocytogenes. Plasmid 81: 1–8.
  40. 40. Lauer P, Chow MY, Loessner MJ, Portnoy DA, Calendar R (2002) Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J Bacteriol 184: 4177–4186.
  41. 41. Premaratne RJ, Lin WJ, Johnson EA (1991) Development of an improved chemically defined minimal medium for Listeria monocytogenes. Appl Environ Microbiol 57: 3046–3048.
  42. 42. Bou Ghanem EN, Jones GS, Myers-Morales T, Patil PD, Hidayatullah AN, et al. (2012) InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice. PLoS Pathog 8: e1003015.
  43. 43. Olier M, Pierre F, Rousseaux S, Lemaître J-P, Rousset A, et al. (2003) Expression of truncated Internalin A is involved in impaired internalization of some Listeria monocytogenes isolates carried asymptomatically by humans. Infection and immunity 71: 1217–1224.
  44. 44. Abdelhamed H, Ramachandran R, Narayanan L, Ozdemir O, Cooper A, et al. (2020) Contributions of a LysR Transcriptional Regulator to Listeria monocytogenes Virulence and Identification of Its Regulons. J Bacteriol 202.
  45. 45. Wong KKY, Freitag NE (2004) A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products. Journal of Bacteriology 186: 6265–6276.
  46. 46. Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1: 190–206. pmid:13267987
  47. 47. Jones S, Portnoy DA (1994) Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect Immun 62: 5608–5613.
  48. 48. Parkhomchuk D, Borodina T, Amstislavskiy V, Banaru M, Hallen L, et al. (2009) Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res 37: e123. pmid:19620212
  49. 49. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25. pmid:19261174
  50. 50. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139–140. pmid:19910308
  51. 51. Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H (2006) Quantitative real-time RT-PCR data analysis: current concepts and the novel "gene expression’s CT difference" formula. J Mol Med (Berl) 84: 901–910. pmid:16972087
  52. 52. Joseph B, Mertins S, Stoll R, Schar J, Umesha KR, et al. (2008) Glycerol metabolism and PrfA activity in Listeria monocytogenes. J Bacteriol 190: 5412–5430.
  53. 53. Sun AN, Camilli A, Portnoy DA (1990) Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect Immun 58: 3770–3778.
  54. 54. Abdelhamed H, Ramachandran R, Narayanan L, Ozdemir O, Cooper A, et al. (2020) Contributions of a LysR Transcriptional Regulator to Listeria monocytogenes Virulence and Identification of Its Regulons. Journal of Bacteriology 202: e00087–00020.
  55. 55. Ziveri J, Tros F, Guerrera IC, Chhuon C, Audry M, et al. (2017) The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella. Nat Commun 8: 853. pmid:29021545
  56. 56. Chaturongakul S, Boor KJ (2004) RsbT and RsbV Contribute to sigmaB-dependent Survival under Environmental, Energy, and Intracellular Stress Conditions in Listeria monocytogenes. Applied and Environmental Microbiology 70: 5349–5356.
  57. 57. Moreau PL (2004) Diversion of the metabolic flux from pyruvate dehydrogenase to pyruvate oxidase decreases oxidative stress during glucose metabolism in nongrowing Escherichia coli cells incubated under aerobic, phosphate starvation conditions. J Bacteriol 186: 7364–7368.
  58. 58. Christodoulou D, Link H, Fuhrer T, Kochanowski K, Gerosa L, et al. (2018) Reserve Flux Capacity in the Pentose Phosphate Pathway Enables Escherichia coli’s Rapid Response to Oxidative Stress. Cell Syst 6: 569–578 e567.
  59. 59. Shi C, Hohl TM, Leiner I, Equinda MJ, Fan X, et al. (2011) Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection. J Immunol 187: 5293–5298.
  60. 60. Lopez S, Marco AJ, Prats N, Czuprynski CJ (2000) Critical role of neutrophils in eliminating Listeria monocytogenes from the central nervous system during experimental murine listeriosis. Infect Immun 68: 4789–4791.
  61. 61. Fox EM, Leonard N, Jordan K (2011) Physiological and transcriptional characterization of persistent and nonpersistent Listeria monocytogenes isolates. Appl Environ Microbiol 77: 6559–6569.
  62. 62. Liu X, Basu U, Miller P, McMullen LM (2014) Stress response and adaptation of Listeria monocytogenes 08–5923 exposed to a sublethal dose of carnocyclin A. Appl Environ Microbiol 80: 3835–3841.
  63. 63. Valdes KM, Sundar GS, Vega LA, Belew AT, Islam E, et al. (2016) The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood. Infect Immun 84: 1016–1031.
  64. 64. Kohler S, Foulongne V, Ouahrani-Bettache S, Bourg G, Teyssier J, et al. (2002) The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell. Proc Natl Acad Sci U S A 99: 15711–15716.
  65. 65. Behari J, Youngman P (1998) Regulation of hly expression in Listeria monocytogenes by carbon sources and pH occurs through separate mechanisms mediated by PrfA. Infect Immun 66: 3635–3642.
  66. 66. Stincone A, Prigione A, Cramer T, Wamelink MMC, Campbell K, et al. (2015) The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biological reviews of the Cambridge Philosophical Society 90: 927–963. pmid:25243985
  67. 67. Cheng C, Dong Z, Han X, Wang H, Jiang L, et al. (2017) Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions. Frontiers in Cellular and Infection Microbiology 7.
  68. 68. Comtois SL, Gidley MD, Kelly DJ (2003) Role of the thioredoxin system and the thiol-peroxidases Tpx and Bcp in mediating resistance to oxidative and nitrosative stress in Helicobacter pylori. Microbiology (Reading) 149: 121–129.
  69. 69. Dons LE, Mosa A, Rottenberg ME, Rosenkrantz JT, Kristensson K, et al. (2014) Role of the Listeria monocytogenes 2-Cys peroxiredoxin homologue in protection against oxidative and nitrosative stress and in virulence. Pathog Dis 70: 70–74.
  70. 70. Mains DR, Eallonardo SJ, Freitag NE, Torres VJ (2021) Identification of Listeria monocytogenes Genes Contributing to Oxidative Stress Resistance under Conditions Relevant to Host Infection. Infection and Immunity 89: e00700–00720.
  71. 71. Dons LE, Mosa A, Rottenberg ME, Rosenkrantz JT, Kristensson K, et al. (2014) Role of the Listeria monocytogenes 2-Cys peroxiredoxin homologue in protection against oxidative and nitrosative stress and in virulence. Pathogens and Disease 70: 70–74.
  72. 72. Han TL, Cannon RD, Gallo SM, Villas-Boas SG (2019) A metabolomic study of the effect of Candida albicans glutamate dehydrogenase deletion on growth and morphogenesis. NPJ Biofilms Microbiomes 5: 13.
  73. 73. Leal SM Jr., Vareechon C, Cowden S, Cobb BA, Latge JP, et al. (2012) Fungal antioxidant pathways promote survival against neutrophils during infection. J Clin Invest 122: 2482–2498. pmid:22706306
  74. 74. Jiang P, Du W, Wu M (2014) Regulation of the pentose phosphate pathway in cancer. Protein Cell 5: 592–602. pmid:25015087
  75. 75. Liu X, Olszewski K, Zhang Y, Lim EW, Shi J, et al. (2020) Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer. Nat Cell Biol 22: 476–486. pmid:32231310
  76. 76. Kuehne A, Emmert H, Soehle J, Winnefeld M, Fischer F, et al. (2015) Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells. Molecular Cell 59: 359–371. pmid:26190262
  77. 77. Wiedmann M, Arvik TJ, Hurley RJ, Boor KJ (1998) General Stress Transcription Factor ςB and Its Role in Acid Tolerance and Virulence of Listeria monocytogenes. Journal of Bacteriology 180: 3650–3656.
  78. 78. O’Byrne CP, Karatzas KA (2008) The role of sigma B (sigma B) in the stress adaptations of Listeria monocytogenes: overlaps between stress adaptation and virulence. Adv Appl Microbiol 65: 115–140.
  79. 79. Garner MR, Njaa BL, Wiedmann M, Boor KJ (2006) Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model. Infect Immun 74: 876–886.
  80. 80. Singh R, Lemire J, Mailloux RJ, Appanna VD (2008) A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network. PLoS One 3: e2682. pmid:18628998
  81. 81. Dinauer MC, Deck MB, Unanue ER (1997) Mice lacking reduced nicotinamide adenine dinucleotide phosphate oxidase activity show increased susceptibility to early infection with Listeria monocytogenes. J Immunol 158: 5581–5583.
  82. 82. Valdes KM, Sundar GS, Vega LA, Belew AT, Islam E, et al. (2016) The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood. Infection and Immunity 84: 1016–1031.
  83. 83. Zeng X, Saxild HH, Switzer RL (2000) Purification and Characterization of the DeoR Repressor of Bacillus subtilis. Journal of Bacteriology 182: 1916–1922.