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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 affects the CPS levels of K. pneumoniae CG43S3.CPS levels of K. pneumoniae CG43S3 were activated by increasing environmental glucose. Bacterial strains were grown in LB broth supplemented with glucose and cAMP as indicated at 37°C with agitation. After 16 h of growth, the bacterial glucuronic acid content was determined. *P<0.05 and **P<0.01 compared with no addition. #P<0.05 compared to the indicated group.
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pone-0054430-g001: Glucose and cAMP affects the CPS levels of K. pneumoniae CG43S3.CPS levels of K. pneumoniae CG43S3 were activated by increasing environmental glucose. Bacterial strains were grown in LB broth supplemented with glucose and cAMP as indicated at 37°C with agitation. After 16 h of growth, the bacterial glucuronic acid content was determined. *P<0.05 and **P<0.01 compared with no addition. #P<0.05 compared to the indicated group.

Mentions: To analyse if exogenous glucose affects K. pneumoniae CPS biosynthesis, CG43S3 was grown in LB broth supplemented with increasing amount of glucose for the quantification of CPS. As shown in Fig. 1, the CPS level increased when bacteria were grown in LB broth supplemented with 0.25% and 0.5% glucose, while the addition of 0.1% glucose did not have an obvious effect. Since the presence of glucose in the growth medium has been demonstrated to inhibit the cAMP production in many bacteria [17], [21], we examined whether the elevated CPS production was regulated by cAMP. Increasing amounts of exogenous cAMP were added to LB broth supplemented with 0.5% glucose, and bacterial CPS production was determined. The result showed that the addition of exogenous cAMP repressed the effect of glucose on CPS production, suggesting that environmental glucose can activate K. pneumoniae CPS biosynthesis through a reduction of cAMP level.


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 affects the CPS levels of K. pneumoniae CG43S3.CPS levels of K. pneumoniae CG43S3 were activated by increasing environmental glucose. Bacterial strains were grown in LB broth supplemented with glucose and cAMP as indicated at 37°C with agitation. After 16 h of growth, the bacterial glucuronic acid content was determined. *P<0.05 and **P<0.01 compared with no addition. #P<0.05 compared to the indicated group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0054430-g001: Glucose and cAMP affects the CPS levels of K. pneumoniae CG43S3.CPS levels of K. pneumoniae CG43S3 were activated by increasing environmental glucose. Bacterial strains were grown in LB broth supplemented with glucose and cAMP as indicated at 37°C with agitation. After 16 h of growth, the bacterial glucuronic acid content was determined. *P<0.05 and **P<0.01 compared with no addition. #P<0.05 compared to the indicated group.
Mentions: To analyse if exogenous glucose affects K. pneumoniae CPS biosynthesis, CG43S3 was grown in LB broth supplemented with increasing amount of glucose for the quantification of CPS. As shown in Fig. 1, the CPS level increased when bacteria were grown in LB broth supplemented with 0.25% and 0.5% glucose, while the addition of 0.1% glucose did not have an obvious effect. Since the presence of glucose in the growth medium has been demonstrated to inhibit the cAMP production in many bacteria [17], [21], we examined whether the elevated CPS production was regulated by cAMP. Increasing amounts of exogenous cAMP were added to LB broth supplemented with 0.5% glucose, and bacterial CPS production was determined. The result showed that the addition of exogenous cAMP repressed the effect of glucose on CPS production, suggesting that environmental glucose can activate K. pneumoniae CPS biosynthesis through a reduction of cAMP level.

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