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Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

Sudhakar P, Reck M, Wang W, He FQ, Wagner-Döbler I, Dobler IW, Zeng AP - BMC Genomics (2014)

Bottom Line: To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed.These sub-networks were significantly enriched with genes sharing common functions.Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 21073 Hamburg, Germany. iwd@helmholtz-hzi.de.

ABSTRACT

Background: Carolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed. A systems biological approach integrating time-resolved transcriptomic data, reverse engineering, transcription factor binding sites, and experimental validation was carried out.

Results: The co-expression response network constructed from transcriptomic data using the reverse engineering algorithm called the Trend Correlation method consisted of 8284 gene pairs. The regulatory response network inferred by superimposing transcription factor binding site information into the co-expression network comprised 329 putative transcriptional regulatory interactions and could be classified into 27 sub-networks each co-regulated by a transcription factor. These sub-networks were significantly enriched with genes sharing common functions. The regulatory response network displayed global hierarchy and network motifs as observed in model organisms. The sub-networks modulated by the pyrimidine biosynthesis regulator PyrR, the glutamine synthetase repressor GlnR, the cysteine metabolism regulator CysR, global regulators CcpA and CodY and the two component system response regulators VicR and MbrC among others could putatively be related to the physiological effect of carolacton. The predicted interactions from the regulatory network between MbrC, known to be involved in cell envelope stress response, and the murMN-SMU_718c genes encoding peptidoglycan biosynthetic enzymes were experimentally confirmed using Electro Mobility Shift Assays. Furthermore, gene deletion mutants of five predicted key regulators from the response networks were constructed and their sensitivities towards carolacton were investigated. Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

Conclusion: The network approach used in this study revealed important regulators and interactions as part of the response mechanisms of S. mutans biofilm cells to carolacton. It also opens a door for further studies into novel drug targets against streptococci.

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Normalized expression profiles of genes modulated by selected transcription factors in response to carolacton treatment. (A) The normalized expression profiles of the genes co-regulated by the pyrimidine biosynthesis regulatory protein in the carolacton treatment context TRRN of S. mutans. Two operons containing genes encoding enzymes involved in the biosynthesis of pyrimidine ribonucleotides were upregulated sharply by about 1.8 to 2 log2-fold 5 min post carolacton treatment. (B) The expression dynamics of the genes co-regulated by the glutamine repressor GlnR in response to carolacton treatment. (C) The normalized expression profiles of the genes commonly regulated by the downregulated essential TCS response regulator vicR in response to carolacton. VicR modulated genes include those encoding virulence attributing products such as glucosyltransferases B and D, cell wall protein WapE among others. (D) Temporal behavior of the 26 genes found within the co-regulated group/subnetwork commonly modulated by SMU.852 encoding the CysR cysteine metabolism regulatory protein. Co-regulated gene groups were constructed by overlaying predicted regulator-binding site maps onto the co-expression network as shown here (E) specifically for the network confined to cysR. The node corresponding to the lone regulator (SMU.1509 encoding a putative Rgg family transcription factor) in the gene group co-regulated by CysR is marked in grey.
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Fig5: Normalized expression profiles of genes modulated by selected transcription factors in response to carolacton treatment. (A) The normalized expression profiles of the genes co-regulated by the pyrimidine biosynthesis regulatory protein in the carolacton treatment context TRRN of S. mutans. Two operons containing genes encoding enzymes involved in the biosynthesis of pyrimidine ribonucleotides were upregulated sharply by about 1.8 to 2 log2-fold 5 min post carolacton treatment. (B) The expression dynamics of the genes co-regulated by the glutamine repressor GlnR in response to carolacton treatment. (C) The normalized expression profiles of the genes commonly regulated by the downregulated essential TCS response regulator vicR in response to carolacton. VicR modulated genes include those encoding virulence attributing products such as glucosyltransferases B and D, cell wall protein WapE among others. (D) Temporal behavior of the 26 genes found within the co-regulated group/subnetwork commonly modulated by SMU.852 encoding the CysR cysteine metabolism regulatory protein. Co-regulated gene groups were constructed by overlaying predicted regulator-binding site maps onto the co-expression network as shown here (E) specifically for the network confined to cysR. The node corresponding to the lone regulator (SMU.1509 encoding a putative Rgg family transcription factor) in the gene group co-regulated by CysR is marked in grey.

Mentions: Peptidoglycan is an important constituent of the gram positive cell-wall. It is expected that cell membrane damage and biofilm inhibition would have a substantial effect on pathways and genes related to cell wall synthesis and metabolism. UDP-N-acetylglucosamine, a key intermediate in the biosynthetic process of the cell wall component peptidoglycan, is produced by glycolysis, sugar metabolism as well as the pyrimidine metabolic pathway [61]. Expression data indicate the absence of immediate modulation and at later time points the downregulation of the glycolytic pathway as well as of the pathways related to the metabolism of various sugars such as fructose, mannose and galactose. On the contrary, two pyrimidine biosynthesis gene clusters (namely the pyrEFDZ and pyrRPBA-carB operons) belonging to the PyrR sub-network and coding for the enzymes of the pyrimidine metabolism pathway (Figure 4) were upregulated by about 1 to 1.8 log2-fold at 5 min post treatment (Figure 5A). It is of note that most of the genes in the pyrimidine metabolism pathway were not transcriptionally altered with the exception of the two strongly upregulated pyrEFDZ and pyrRPBA-carB operons. These operons encode enzymes catalyzing the biochemical steps leading to the production of UMP and UDP (see Figure 4) suggesting that this part of the pathway is specifically activated. An upregulation of the pyrimidine metabolism pathway would produce pools of UDP-N-acetlyglucosamine (UDP-N-AG) for peptidoglycan synthesis compensating carolacton-induced membrane and cell wall damage. The upregulation of pyrimidine biosynthetic steps was also observed in an S.aureus strain harboring a mutation of a two component system essential for cell wall metabolism [62].Figure 4


Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

Sudhakar P, Reck M, Wang W, He FQ, Wagner-Döbler I, Dobler IW, Zeng AP - BMC Genomics (2014)

Normalized expression profiles of genes modulated by selected transcription factors in response to carolacton treatment. (A) The normalized expression profiles of the genes co-regulated by the pyrimidine biosynthesis regulatory protein in the carolacton treatment context TRRN of S. mutans. Two operons containing genes encoding enzymes involved in the biosynthesis of pyrimidine ribonucleotides were upregulated sharply by about 1.8 to 2 log2-fold 5 min post carolacton treatment. (B) The expression dynamics of the genes co-regulated by the glutamine repressor GlnR in response to carolacton treatment. (C) The normalized expression profiles of the genes commonly regulated by the downregulated essential TCS response regulator vicR in response to carolacton. VicR modulated genes include those encoding virulence attributing products such as glucosyltransferases B and D, cell wall protein WapE among others. (D) Temporal behavior of the 26 genes found within the co-regulated group/subnetwork commonly modulated by SMU.852 encoding the CysR cysteine metabolism regulatory protein. Co-regulated gene groups were constructed by overlaying predicted regulator-binding site maps onto the co-expression network as shown here (E) specifically for the network confined to cysR. The node corresponding to the lone regulator (SMU.1509 encoding a putative Rgg family transcription factor) in the gene group co-regulated by CysR is marked in grey.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Normalized expression profiles of genes modulated by selected transcription factors in response to carolacton treatment. (A) The normalized expression profiles of the genes co-regulated by the pyrimidine biosynthesis regulatory protein in the carolacton treatment context TRRN of S. mutans. Two operons containing genes encoding enzymes involved in the biosynthesis of pyrimidine ribonucleotides were upregulated sharply by about 1.8 to 2 log2-fold 5 min post carolacton treatment. (B) The expression dynamics of the genes co-regulated by the glutamine repressor GlnR in response to carolacton treatment. (C) The normalized expression profiles of the genes commonly regulated by the downregulated essential TCS response regulator vicR in response to carolacton. VicR modulated genes include those encoding virulence attributing products such as glucosyltransferases B and D, cell wall protein WapE among others. (D) Temporal behavior of the 26 genes found within the co-regulated group/subnetwork commonly modulated by SMU.852 encoding the CysR cysteine metabolism regulatory protein. Co-regulated gene groups were constructed by overlaying predicted regulator-binding site maps onto the co-expression network as shown here (E) specifically for the network confined to cysR. The node corresponding to the lone regulator (SMU.1509 encoding a putative Rgg family transcription factor) in the gene group co-regulated by CysR is marked in grey.
Mentions: Peptidoglycan is an important constituent of the gram positive cell-wall. It is expected that cell membrane damage and biofilm inhibition would have a substantial effect on pathways and genes related to cell wall synthesis and metabolism. UDP-N-acetylglucosamine, a key intermediate in the biosynthetic process of the cell wall component peptidoglycan, is produced by glycolysis, sugar metabolism as well as the pyrimidine metabolic pathway [61]. Expression data indicate the absence of immediate modulation and at later time points the downregulation of the glycolytic pathway as well as of the pathways related to the metabolism of various sugars such as fructose, mannose and galactose. On the contrary, two pyrimidine biosynthesis gene clusters (namely the pyrEFDZ and pyrRPBA-carB operons) belonging to the PyrR sub-network and coding for the enzymes of the pyrimidine metabolism pathway (Figure 4) were upregulated by about 1 to 1.8 log2-fold at 5 min post treatment (Figure 5A). It is of note that most of the genes in the pyrimidine metabolism pathway were not transcriptionally altered with the exception of the two strongly upregulated pyrEFDZ and pyrRPBA-carB operons. These operons encode enzymes catalyzing the biochemical steps leading to the production of UMP and UDP (see Figure 4) suggesting that this part of the pathway is specifically activated. An upregulation of the pyrimidine metabolism pathway would produce pools of UDP-N-acetlyglucosamine (UDP-N-AG) for peptidoglycan synthesis compensating carolacton-induced membrane and cell wall damage. The upregulation of pyrimidine biosynthetic steps was also observed in an S.aureus strain harboring a mutation of a two component system essential for cell wall metabolism [62].Figure 4

Bottom Line: To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed.These sub-networks were significantly enriched with genes sharing common functions.Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 21073 Hamburg, Germany. iwd@helmholtz-hzi.de.

ABSTRACT

Background: Carolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed. A systems biological approach integrating time-resolved transcriptomic data, reverse engineering, transcription factor binding sites, and experimental validation was carried out.

Results: The co-expression response network constructed from transcriptomic data using the reverse engineering algorithm called the Trend Correlation method consisted of 8284 gene pairs. The regulatory response network inferred by superimposing transcription factor binding site information into the co-expression network comprised 329 putative transcriptional regulatory interactions and could be classified into 27 sub-networks each co-regulated by a transcription factor. These sub-networks were significantly enriched with genes sharing common functions. The regulatory response network displayed global hierarchy and network motifs as observed in model organisms. The sub-networks modulated by the pyrimidine biosynthesis regulator PyrR, the glutamine synthetase repressor GlnR, the cysteine metabolism regulator CysR, global regulators CcpA and CodY and the two component system response regulators VicR and MbrC among others could putatively be related to the physiological effect of carolacton. The predicted interactions from the regulatory network between MbrC, known to be involved in cell envelope stress response, and the murMN-SMU_718c genes encoding peptidoglycan biosynthetic enzymes were experimentally confirmed using Electro Mobility Shift Assays. Furthermore, gene deletion mutants of five predicted key regulators from the response networks were constructed and their sensitivities towards carolacton were investigated. Deletion of cysR, the node having the highest connectivity among the regulators chosen from the regulatory network, resulted in a mutant which was insensitive to carolacton thus demonstrating not only the essentiality of cysR for the response of S. mutans biofilms to carolacton but also the relevance of the predicted network.

Conclusion: The network approach used in this study revealed important regulators and interactions as part of the response mechanisms of S. mutans biofilm cells to carolacton. It also opens a door for further studies into novel drug targets against streptococci.

Show MeSH
Related in: MedlinePlus