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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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RcsA is involved in CRP regulation of CPS expression.(A) CPS levels of WT, ΔrcsA, Δcrp, and ΔcrpΔrcsA strains were determined. For complementation purposes, introduction of pRK415 and prcsA into ΔcrpΔrcsA strain were also determined. Bacterial strains were grown in LB broth 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 to the indicated group. (B) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strains (ΔlacZΔcrp, ΔlacZΔrcsA, and ΔlacZΔcrpΔrcsA) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phased cultures grown in 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 compared to the indicated group.
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pone-0054430-g007: RcsA is involved in CRP regulation of CPS expression.(A) CPS levels of WT, ΔrcsA, Δcrp, and ΔcrpΔrcsA strains were determined. For complementation purposes, introduction of pRK415 and prcsA into ΔcrpΔrcsA strain were also determined. Bacterial strains were grown in LB broth 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 to the indicated group. (B) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strains (ΔlacZΔcrp, ΔlacZΔrcsA, and ΔlacZΔcrpΔrcsA) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phased cultures grown in 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 compared to the indicated group.

Mentions: To understand whether RcsA participates in CRP regulation of CPS biosynthesis, the level of CPS was determined in WT, ΔrcsA, Δcrp, and ΔcrpΔrcsA strains. As shown in Fig. 7A, the deletion of rcsA resulted in a slight reduction in CPS level as compared to WT strain. However, the deletion of rcsA partially restored CPS production in the Δcrp strain. In addition, introducing the complementary plasmid prcsA into the ΔcrpΔrcsA strain increased CPS levels as compared to the strain carrying the empty vector control (pRK415). These results indicate that RcsA participates in CRP regulation of CPS biosynthesis.


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)

RcsA is involved in CRP regulation of CPS expression.(A) CPS levels of WT, ΔrcsA, Δcrp, and ΔcrpΔrcsA strains were determined. For complementation purposes, introduction of pRK415 and prcsA into ΔcrpΔrcsA strain were also determined. Bacterial strains were grown in LB broth 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 to the indicated group. (B) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strains (ΔlacZΔcrp, ΔlacZΔrcsA, and ΔlacZΔcrpΔrcsA) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phased cultures grown in 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 compared to the indicated group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569464&req=5

pone-0054430-g007: RcsA is involved in CRP regulation of CPS expression.(A) CPS levels of WT, ΔrcsA, Δcrp, and ΔcrpΔrcsA strains were determined. For complementation purposes, introduction of pRK415 and prcsA into ΔcrpΔrcsA strain were also determined. Bacterial strains were grown in LB broth 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 to the indicated group. (B) The β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strains (ΔlacZΔcrp, ΔlacZΔrcsA, and ΔlacZΔcrpΔrcsA) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phased cultures grown in 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 compared to the indicated group.
Mentions: To understand whether RcsA participates in CRP regulation of CPS biosynthesis, the level of CPS was determined in WT, ΔrcsA, Δcrp, and ΔcrpΔrcsA strains. As shown in Fig. 7A, the deletion of rcsA resulted in a slight reduction in CPS level as compared to WT strain. However, the deletion of rcsA partially restored CPS production in the Δcrp strain. In addition, introducing the complementary plasmid prcsA into the ΔcrpΔrcsA strain increased CPS levels as compared to the strain carrying the empty vector control (pRK415). These results indicate that RcsA participates in CRP regulation of CPS biosynthesis.

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