DegU-mediated suppression of carbohydrate uptake in Listeria monocytogenes increases adaptation to oxidative stress

Abstract

The foodborne bacterial pathogen Listeria monocytogenes exhibits remarkable survival capabilities under challenging conditions, severely threatening food safety and human health. The orphan regulator DegU is a pleiotropic regulator required for bacterial environmental adaptation. However, the specific mechanism of how DegU participates in oxidative stress tolerance remains unknown in L. monocytogenes. In this study, we demonstrate that DegU suppresses carbohydrate uptake under stress conditions by altering global transcriptional profiles, particularly by modulating the transcription of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS)-related genes, such as ptsH, ptsI, and hprK. Specifically, in the absence of degU, the transcripts of ptsI are significantly upregulated and those of hprK are significantly downregulated in response to copper ion-induced stress. Overexpression of ptsI significantly increases bacterial growth in vitro, while overexpression of hprK leads to a decrease in growth. We further demonstrate that DegU directly senses oxidative stress, downregulates ptsI transcription, and upregulates hprK transcription. Additionally, through an electrophoretic mobility shift assay, we demonstrate that DegU directly regulates the transcription of ptsI and hprK by binding to specific regions within their respective promoter sequences. Notably, the putative pivotal DegU binding sequence for ptsI is located from 38 to 68 base pairs upstream of the ptsH transcription start site (TSS), whereas for hprK, it is mapped from 36 to 124 base pairs upstream of the hprK TSS. In summary, we elucidate that DegU plays a significant role in suppressing carbohydrate uptake in response to oxidative stress through the direct regulation of ptsI and hprK.ImportanceUnderstanding the adaptive mechanisms employed by Listeria monocytogenes in harsh environments is of great significance. This study focuses on investigating the role of DegU in response to oxidative stress by examining global transcriptional profiles. The results highlight the noteworthy involvement of DegU in this stress response. Specifically, DegU acts as a direct sensor of oxidative stress, leading to the modulation of gene transcription. It downregulates ptsI transcription while it upregulates hprK transcription through direct binding to their promoters. Consequently, these regulatory actions impede bacterial growth, providing a defense mechanism against stress-induced damage. These findings gained from this study may have broader implications, serving as a reference for studying adaptive mechanisms in other pathogenic bacteria and aiding in the development of targeted strategies to control L. monocytogenes and ensure food safety.

Keywords: Listeria monocytogenes; PTS; hprK; orphan response regulator DegU; oxidative tolerance; ptsI.

Fig 1

Deletion of degU increased the growth of L. monocytogenes under copper ion stress conditions. Overnight cultures of wild-type L. monocytogenes strain EGD-e, degU deletion strain ΔdegU, and degU complemented strain CΔdegU_P_dlt were prepared. Subsequently, the bacterial cultures were washed with PBS, serially diluted, and spotted onto BHI plates containing varying concentrations of Cu²⁺. These plates were then incubated at 37°C for 24–48 hours. Following the incubation period, the survival of L. monocytogenes was evaluated by scanning the plates. The red boxes indicate that the growth ability of ΔdegU and EGD-e exhibits a more pronounced difference under copper ion stress conditions at the concentrations of 0.5 and 1 mmol/L. The data presented in the study are based on three replicates.

Fig 2

DegU regulates PTS transcription under oxidative stress. (A) The volcano plot illustrates the overall changes in transcript levels between the wild-type (WT) strain and the degU deletion strain ΔdegU when cultured with 1-mmol/L copper chloride. Transcripts with a log₂ fold change greater than 1.0 or less than −1.0 and a significance level (P-value) below 0.05 are highlighted. (B) KEGG annotation analysis was conducted to examine the genes regulated by DegU. (C) KEGG enrichment analysis was performed to determine the enrichment of specific gene sets regulated by DegU. (D) The transcription of genes related to the PTS was initially identified through transcriptome sequencing and subsequently validated by quantitative real-time PCR (qRT-qPCR). Gene transcription levels were normalized to the housekeeping gene rpoB. The presented results represent the means ± standard deviation (SEM) obtained from three independent experiments.

Fig 3

DegU binds to the ptsH promoter. (A) An electrophoretic mobility shift assay (EMSA) was conducted to investigate the direct binding of DegU to the full-length ptsH promoter and three different truncated forms. The degU promoter regions served as the positive control for comparison. (B) The DNA sequences of the ptsH promoter region were analyzed to determine the specific binding site of DegU. The putative pivotal DegU binding site was highlighted in blue and enclosed in a blue box. The −35, −10, + 1, and RBS (ribosome binding site) regions were underlined and shown in purple. The start codon of ptsH was indicated in red. The data presented in the study are based on three replicates.

Fig 4

Overexpression of ptsI increased the growth of L. monocytogenes under copper ion stress conditions. Overnight cultures of L. monocytogenes EGD-e and the ptsI overexpression strain CptsI_P_dlt were serially diluted and then spotted onto BHI plates containing varying concentrations of Cu²⁺. The plates were incubated at 37°C for 24–48 hours. The red boxes indicate that the growth ability of CptsI_P_dlt and EGD-e displays a more noticeable difference under copper ion stress conditions at the concentrations of 0.25 and 0.5 mmol/L. (B) The transcription of ptsI was analyzed using qRT-PCR in EGD-e. A comparison was made between the transcription levels under copper ion stress conditions and normal culture conditions. (C) The transcription of ptsI was examined using qRT-PCR in both EGD-e and CptsI_P_dlt under the influence of 1 mmol/L copper chloride. The presented results represent the means ± standard deviation (SEM) obtained from three independent experiments.

Fig 5

DegU binds to the hprK promoter. (A) An electrophoretic mobility shift assay (EMSA) was performed to investigate the direct binding of DegU to the full-length hprK promoter and seven different truncated forms. The degU promoter regions were used as positive controls for comparison. The assay determined the presence of band shifts, indicating the binding interaction between DegU and the respective hprK promoter fragments. (B) The DNA sequences of the hprK promoter region were analyzed to identify the specific binding site of DegU. The putative pivotal DegU binding site was indicated by the presence of blue nucleotides, which are enclosed in a blue box. Additionally, the −35, −10, + 1, and RBS regions were underlined and represented in purple. The start codon of ptsH was highlighted in red. The data presented in the study are based on three replicates.

Fig 6

Overexpression of hprK reduced the growth of L. monocytogenes under copper ion stress conditions, as well as the transcription of CcpA/P-Ser-HPr-dependent CCR-related genes iiB^man and lmo2004. (A) Overnight cultures of L. monocytogenes EGD-e and the hprK overexpression strain ChprK_P_dlt were serially diluted and spotted onto BHI plates containing varying concentrations of Cu²⁺. Subsequently, the plates were incubated at 37°C for 24–48 hours. The red boxes highlight that the growth ability between ChprK_P_dlt and EGD-e is more distinct under copper ion stress conditions at the concentration of 0.5 mmol/L. (B) The transcription levels of hprK were assessed using qRT-PCR in both EGD-e and ChprK_P_dlt, specifically under the influence of 1 mmol/L copper chloride. (C) The transcription levels of iiB^man, lmo2004, and hprK were examined using qRT-PCR in EGD-e and ChprK_P_dlt. The presented results represent the means ± standard deviation (SEM) obtained from three independent experiments.

Fig 7

A proposed model depicting the role of DegU in mediating the oxidative stress response. (a) Under oxidative stress conditions, L. monocytogenes activates the direct binding of DegU to the ptsH promoter, leading to the suppression of the ptsH-ptsI operon transcription. Simultaneously, DegU binds directly to the hprK promoter, resulting in the activation of hprK transcription. The upregulation of hprK leads to a decrease in the transcription of CcpA/P-Ser-HPr-dependent CCR-related genes. Ultimately, the suppression of ptsH-ptsI transcription and the activation of hprK transcription result in reduced carbohydrate transport and increased CCR, thereby slowing bacterial growth. (b) In the degU deletion strain ΔdegU, the response to oxidative stress is altered. Without DegU binding to the ptsH promoter, the transcription of the ptsH-ptsI operon is upregulated. Additionally, in the absence of DegU binding to the hprK promoter, the transcription of hprK is downregulated. This downregulation of hprK leads to an increase in the transcription of CcpA/P-Ser-HPr-dependent CCR-related genes. Consequently, the upregulation of ptsH-ptsI transcription and downregulation of hprK transcription cause increased carbohydrate transport and decreased CCR, thereby accelerating bacterial growth. The model provides insights into the regulatory role of DegU in the oxidative stress response, the control of PTS gene transcription, and the modulation of CCR-related gene transcription in L. monocytogenes.