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Role of the cAMP-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.

Lin CT, Chen YC, Jinn TR, Wu CC, Hong YM, Wu WH - PLoS ONE (2013)

Bottom Line: These results were then confirmed by electrophoretic mobility shift assay.In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA).Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, China Medical University, Taichung, Taiwan. Republic of China. gingting@mail.cmu.edu.tw

ABSTRACT
K. pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. Although elevated blood glucose levels cause various immune problems, its effects on K. pneumoniae virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for K. pneumoniae virulence, in response to exogenous glucose. We found that K. pneumoniae produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of cyaA or crp, which respectively encode adenylate cyclase and cAMP receptor protein in K. pneumoniae, markedly increased CPS production, while deletion of cpdA, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that K. pneumoniae CPS biosynthesis is controlled by the cAMP-dependent carbon catabolite repression (CCR). To investigate the underlying mechanism, quantitative real-time PCR and promoter-reporter assays were used to verify that the transcription of CPS biosynthesis genes, which are organized into 3 transcription units (orf1-2, orf3-15, and orf16-17), were activated by the deletion of crp. Sequence analysis revealed putative CRP binding sites located on P(orf3-15) and P(orf16-17), suggesting direct CRP-cAMP regulation on the promoters. These results were then confirmed by electrophoretic mobility shift assay. In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA). The deletion of rcsA in mutation of crp partially reduced CPS biosynthesis and the transcription of orf1-2 but not of orf3-15 or orf16-17. These results suggest that RcsA participates in the CRP-cAMP regulation of orf1-2 transcription and influences CPS biosynthesis. Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing. We here provide evidence that K. pneumoniae increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR.

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Glucose and cAMP-related proteins affect rcsA transcription.(A) Diagrammatic representation of rcsA loci. The large arrows represent the open reading frames. The relative positions of the primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the positions relative to the translational start site. Names of the DNA probes are shown on the left. The dashed boxes indicate the predicted CRP binding sites and the alignment is shown below. (B) qRT-PCR analysis of rcsA expression was measured in WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. The results shown are an average from triplicate measurements in one single experiment representative of three independent experiments. Error bars indicate standard deviations. *P<0.05 and **P<0.01 compared with WT. (C) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid prcsAZ15 (PrcsA::lacZ) were determined using log-phased cultures grown in LB medium. **P<0.01 compared with ΔlacZ. (D) CRP binds directly to PrcsA. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream region of rcsA. Following incubation at room temperature for 30 min, the mixtures were analyzed on a 5% non-denaturing polyacrylamide gel containing 200 µM cAMP. The gel was stained with SYBR Green I dye and photographed.
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pone-0054430-g006: Glucose and cAMP-related proteins affect rcsA transcription.(A) Diagrammatic representation of rcsA loci. The large arrows represent the open reading frames. The relative positions of the primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the positions relative to the translational start site. Names of the DNA probes are shown on the left. The dashed boxes indicate the predicted CRP binding sites and the alignment is shown below. (B) qRT-PCR analysis of rcsA expression was measured in WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. The results shown are an average from triplicate measurements in one single experiment representative of three independent experiments. Error bars indicate standard deviations. *P<0.05 and **P<0.01 compared with WT. (C) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid prcsAZ15 (PrcsA::lacZ) were determined using log-phased cultures grown in LB medium. **P<0.01 compared with ΔlacZ. (D) CRP binds directly to PrcsA. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream region of rcsA. Following incubation at room temperature for 30 min, the mixtures were analyzed on a 5% non-denaturing polyacrylamide gel containing 200 µM cAMP. The gel was stained with SYBR Green I dye and photographed.

Mentions: Because no CRP binding site was found in the sequence upstream of galF, the expression of galF also appeared to be controlled by cAMP-dependent CCR, implying that CRP repression of galF transcription is indirect and that other transcription factors are involved in the CRP regulon controlling cps transcription. According to previous studies, multiple regulatory proteins, which include Fur, RcsA, RcsB, RmpA, RmpA2, KvgA, and KvhR have been shown to mediate K2 cps expression [43], [44], [45], [46]. To further investigate whether these cps regulatory proteins are involved in CRP regulon, the CRP binding site was searched in the upstream sequence of fur, rcsA/B, rmpA/A2, kvgA, and kvhR. However, we found the 2 CRP binding sites (rcsA-1 and rcsA-2) are located at −192 to −177 and −40 to −25 relative to the translation start site of RcsA (Fig. 6A), but no typical CRP binding site was found in other upstream sequence of fur, rcsB, rmpA/A2, kvgA, and kvhR. Therefore, we suggest that RcsA is a CRP-regulated transcription factor and is involved in cAMP-dependent CCR control of cps expression.


Role of the cAMP-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.

Lin CT, Chen YC, Jinn TR, Wu CC, Hong YM, Wu WH - PLoS ONE (2013)

Glucose and cAMP-related proteins affect rcsA transcription.(A) Diagrammatic representation of rcsA loci. The large arrows represent the open reading frames. The relative positions of the primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the positions relative to the translational start site. Names of the DNA probes are shown on the left. The dashed boxes indicate the predicted CRP binding sites and the alignment is shown below. (B) qRT-PCR analysis of rcsA expression was measured in WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. The results shown are an average from triplicate measurements in one single experiment representative of three independent experiments. Error bars indicate standard deviations. *P<0.05 and **P<0.01 compared with WT. (C) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid prcsAZ15 (PrcsA::lacZ) were determined using log-phased cultures grown in LB medium. **P<0.01 compared with ΔlacZ. (D) CRP binds directly to PrcsA. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream region of rcsA. Following incubation at room temperature for 30 min, the mixtures were analyzed on a 5% non-denaturing polyacrylamide gel containing 200 µM cAMP. The gel was stained with SYBR Green I dye and photographed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569464&req=5

pone-0054430-g006: Glucose and cAMP-related proteins affect rcsA transcription.(A) Diagrammatic representation of rcsA loci. The large arrows represent the open reading frames. The relative positions of the primer sets used in PCR amplification of the DNA probes are indicated, and the numbers denote the positions relative to the translational start site. Names of the DNA probes are shown on the left. The dashed boxes indicate the predicted CRP binding sites and the alignment is shown below. (B) qRT-PCR analysis of rcsA expression was measured in WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. The results shown are an average from triplicate measurements in one single experiment representative of three independent experiments. Error bars indicate standard deviations. *P<0.05 and **P<0.01 compared with WT. (C) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid prcsAZ15 (PrcsA::lacZ) were determined using log-phased cultures grown in LB medium. **P<0.01 compared with ΔlacZ. (D) CRP binds directly to PrcsA. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream region of rcsA. Following incubation at room temperature for 30 min, the mixtures were analyzed on a 5% non-denaturing polyacrylamide gel containing 200 µM cAMP. The gel was stained with SYBR Green I dye and photographed.
Mentions: Because no CRP binding site was found in the sequence upstream of galF, the expression of galF also appeared to be controlled by cAMP-dependent CCR, implying that CRP repression of galF transcription is indirect and that other transcription factors are involved in the CRP regulon controlling cps transcription. According to previous studies, multiple regulatory proteins, which include Fur, RcsA, RcsB, RmpA, RmpA2, KvgA, and KvhR have been shown to mediate K2 cps expression [43], [44], [45], [46]. To further investigate whether these cps regulatory proteins are involved in CRP regulon, the CRP binding site was searched in the upstream sequence of fur, rcsA/B, rmpA/A2, kvgA, and kvhR. However, we found the 2 CRP binding sites (rcsA-1 and rcsA-2) are located at −192 to −177 and −40 to −25 relative to the translation start site of RcsA (Fig. 6A), but no typical CRP binding site was found in other upstream sequence of fur, rcsB, rmpA/A2, kvgA, and kvhR. Therefore, we suggest that RcsA is a CRP-regulated transcription factor and is involved in cAMP-dependent CCR control of cps expression.

Bottom Line: These results were then confirmed by electrophoretic mobility shift assay.In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA).Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, China Medical University, Taichung, Taiwan. Republic of China. gingting@mail.cmu.edu.tw

ABSTRACT
K. pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. Although elevated blood glucose levels cause various immune problems, its effects on K. pneumoniae virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for K. pneumoniae virulence, in response to exogenous glucose. We found that K. pneumoniae produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of cyaA or crp, which respectively encode adenylate cyclase and cAMP receptor protein in K. pneumoniae, markedly increased CPS production, while deletion of cpdA, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that K. pneumoniae CPS biosynthesis is controlled by the cAMP-dependent carbon catabolite repression (CCR). To investigate the underlying mechanism, quantitative real-time PCR and promoter-reporter assays were used to verify that the transcription of CPS biosynthesis genes, which are organized into 3 transcription units (orf1-2, orf3-15, and orf16-17), were activated by the deletion of crp. Sequence analysis revealed putative CRP binding sites located on P(orf3-15) and P(orf16-17), suggesting direct CRP-cAMP regulation on the promoters. These results were then confirmed by electrophoretic mobility shift assay. In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and P(rcsA). The deletion of rcsA in mutation of crp partially reduced CPS biosynthesis and the transcription of orf1-2 but not of orf3-15 or orf16-17. These results suggest that RcsA participates in the CRP-cAMP regulation of orf1-2 transcription and influences CPS biosynthesis. Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing. We here provide evidence that K. pneumoniae increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR.

Show MeSH
Related in: MedlinePlus