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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 CCR proteins affect cps transcription.(A) qRT-PCR analyses of the expression of the K2 cps genes (orf1, orf3, and orf16) for WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. (B) β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phase 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 and **P<0.01 compared to the indicated group.
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pone-0054430-g003: Glucose and CCR proteins affect cps transcription.(A) qRT-PCR analyses of the expression of the K2 cps genes (orf1, orf3, and orf16) for WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. (B) β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phase 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 and **P<0.01 compared to the indicated group.

Mentions: The K2 cps gene cluster of K. pneumoniae Chedid contains 19 open reading frames (ORFs) organised into 3 transcription units, namely, orf1–2, orf3–15, and orf16–17[42]. To investigate the effect of glucose and cAMP-related proteins on the expression of the 3 cps gene clusters, the mRNA level of orf1 (named galF), orf3 (named wzi), and orf16 (named manC) were measured by qRT-PCR in WT grown in LB medium containing 0.5% glucose with or without 1 mM cAMP. In addition, the effect of cyaA, cpdA, andcrp mutation strains on the mRNA levels of galF, wzi, and manC were also determined. As shown in Fig. 3A, we found that the mRNA levels of galF, wzi and manC was increased in glucose-rich medium (LB+0.5% glucose), whereas addition of 1 mM cAMP to the glucose-rich medium could restore the galF, wzi and manC expression, similar to the trends observed in the WT strain. Furthermore, the mRNA levels of galF, wzi, and manC revealed an apparent increase in the ΔcyaA and Δcrp strains as compared to that observed in the WT strain. In contrast, a slight reduction in the mRNA level of cps genes was found in the ΔcpdA strain. This result indicates that cps mRNA expression is regulated by cAMP-dependent CCR, and CRP may acts a repressor 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 CCR proteins affect cps transcription.(A) qRT-PCR analyses of the expression of the K2 cps genes (orf1, orf3, and orf16) for WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. (B) β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phase 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 and **P<0.01 compared to the indicated group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0054430-g003: Glucose and CCR proteins affect cps transcription.(A) qRT-PCR analyses of the expression of the K2 cps genes (orf1, orf3, and orf16) for WT, ΔcyaA, ΔcpdA, and Δcrp strains in LB or indicated LB medium. (B) β-galactosidase activities of K. pneumoniae CG43S3ΔlacZ and the isogenic strain (ΔlacZΔcrp) carrying the reporter plasmid pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), or pOrf1617 (Porf16-17::lacZ) were determined using log-phase 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 and **P<0.01 compared to the indicated group.
Mentions: The K2 cps gene cluster of K. pneumoniae Chedid contains 19 open reading frames (ORFs) organised into 3 transcription units, namely, orf1–2, orf3–15, and orf16–17[42]. To investigate the effect of glucose and cAMP-related proteins on the expression of the 3 cps gene clusters, the mRNA level of orf1 (named galF), orf3 (named wzi), and orf16 (named manC) were measured by qRT-PCR in WT grown in LB medium containing 0.5% glucose with or without 1 mM cAMP. In addition, the effect of cyaA, cpdA, andcrp mutation strains on the mRNA levels of galF, wzi, and manC were also determined. As shown in Fig. 3A, we found that the mRNA levels of galF, wzi and manC was increased in glucose-rich medium (LB+0.5% glucose), whereas addition of 1 mM cAMP to the glucose-rich medium could restore the galF, wzi and manC expression, similar to the trends observed in the WT strain. Furthermore, the mRNA levels of galF, wzi, and manC revealed an apparent increase in the ΔcyaA and Δcrp strains as compared to that observed in the WT strain. In contrast, a slight reduction in the mRNA level of cps genes was found in the ΔcpdA strain. This result indicates that cps mRNA expression is regulated by cAMP-dependent CCR, and CRP may acts a repressor 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