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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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CRP directly binds to Pwzi and PmanC.Diagrammatic representation of the wzi loci (Porf3-15) (A) and the manC loci (Porf16-17) (B). 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 consensus sequences. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream regions of wzi or manC. 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-g005: CRP directly binds to Pwzi and PmanC.Diagrammatic representation of the wzi loci (Porf3-15) (A) and the manC loci (Porf16-17) (B). 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 consensus sequences. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream regions of wzi or manC. 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: To demonstrate whether CRP binds directly to the upstream sequence of wzi and manC via the CRP binding sites, electric mobility shift assay (EMSA) was performed using the recombinant His6-CRP protein and different DNA fragments containing truncated forms of Pwzi (Pwzi-1, Pwzi-2, Pwzi-3, and Pwzi-4) and PmanC (PmanC-1 and PmanC-2), as described in the Materials and Methods. Using Pwzi fragments of different lengths, binding of His6-CRP could be observed for Pwzi-1, Pwzi-2, and Pwzi-3 but not for Pwzi-4 (Fig. 5A). As shown in Fig. 5A, DNA-protein-binding complexes were observed after the incubation of 100 nM purified His6-CRP with 10 ng Pwzi-1 or Pwzi-2. However, formation of the Pwzi-3/CRP complex required the incubation with 200 nM purified His6-CRP. This suggests that the lower binding ability of His6-CRP in Pwzi-3 is due to the location of only one CRP binding site in Pwzi-3 as compared to 2 and 3 CRP binding sites in Pwzi-1 and Pwzi-2, respectively. In addition, we found that His6-CRP was able to bind to the PmanC-1 DNA fragment, but not to the PmanC-2 fragment in which the CRP binding site had been removed (Fig. 5B). This result indicates that the recombinant His6-CRP protein can bind directly to the predicted CRP binding sites located in Pwzi and PmanC.


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)

CRP directly binds to Pwzi and PmanC.Diagrammatic representation of the wzi loci (Porf3-15) (A) and the manC loci (Porf16-17) (B). 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 consensus sequences. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream regions of wzi or manC. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569464&req=5

pone-0054430-g005: CRP directly binds to Pwzi and PmanC.Diagrammatic representation of the wzi loci (Porf3-15) (A) and the manC loci (Porf16-17) (B). 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 consensus sequences. Different concentrations of purified His6-CRP were incubated with 10 ng of various truncated DNA fragments of the upstream regions of wzi or manC. 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: To demonstrate whether CRP binds directly to the upstream sequence of wzi and manC via the CRP binding sites, electric mobility shift assay (EMSA) was performed using the recombinant His6-CRP protein and different DNA fragments containing truncated forms of Pwzi (Pwzi-1, Pwzi-2, Pwzi-3, and Pwzi-4) and PmanC (PmanC-1 and PmanC-2), as described in the Materials and Methods. Using Pwzi fragments of different lengths, binding of His6-CRP could be observed for Pwzi-1, Pwzi-2, and Pwzi-3 but not for Pwzi-4 (Fig. 5A). As shown in Fig. 5A, DNA-protein-binding complexes were observed after the incubation of 100 nM purified His6-CRP with 10 ng Pwzi-1 or Pwzi-2. However, formation of the Pwzi-3/CRP complex required the incubation with 200 nM purified His6-CRP. This suggests that the lower binding ability of His6-CRP in Pwzi-3 is due to the location of only one CRP binding site in Pwzi-3 as compared to 2 and 3 CRP binding sites in Pwzi-1 and Pwzi-2, respectively. In addition, we found that His6-CRP was able to bind to the PmanC-1 DNA fragment, but not to the PmanC-2 fragment in which the CRP binding site had been removed (Fig. 5B). This result indicates that the recombinant His6-CRP protein can bind directly to the predicted CRP binding sites located in Pwzi and PmanC.

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