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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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CCR proteins affect bacterial CPS levels.(A) CPS levels of WT, ΔcyaA, ΔcpdA strains and complementation of cyaA and cpdA strains were determined. (B) CPS levels in mutation and complementation of crp strains were determined. Bacteria were grown in LB medium at 37°C with agitation. *P<0.05 and **P<0.01 compared to the indicated group. (C) Intracellular levels of cAMP in WT, ΔcyaA, ΔcpdA, and Δcrp strains, as determined by ELISA. 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.
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pone-0054430-g002: CCR proteins affect bacterial CPS levels.(A) CPS levels of WT, ΔcyaA, ΔcpdA strains and complementation of cyaA and cpdA strains were determined. (B) CPS levels in mutation and complementation of crp strains were determined. Bacteria were grown in LB medium at 37°C with agitation. *P<0.05 and **P<0.01 compared to the indicated group. (C) Intracellular levels of cAMP in WT, ΔcyaA, ΔcpdA, and Δcrp strains, as determined by ELISA. 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.

Mentions: To confirm that K. pneumoniae CPS biosynthesis is regulated by cAMP, individual strains with deletion of cyaA and cpdA, which respectively encodes adenylate cyclase and cAMP phosphodiesterase from CG43S3, were constructed, and the effects of the deletions on CPS production were analysed. As shown in Fig. 2A, compared to wild type (WT) strain, we found that CPS levels increased in ΔcyaA, and introduction of pcyaA, but not the empty vector control (pACYC184), into ΔcyaA could reverse the effect of cyaA mutation. In contrast, the deletion of cpdA caused a decreased in CPS levels, which could be complemented by introducing a plasmid-carried cpdA (pETQ-cpdA) into the ΔcpdA strain. These results confirmed that cAMP can act as a signalling molecule for regulation of CPS biosynthesis. In addition, since cAMP affects gene transcription through its effector protein CRP, we assessed the effect of deletion of crp on CPS levels. As shown in Fig. 2B, compared to WT, Δcrp produced higher levels of CPS. Introduction of the complement plasmid pcrp, but not the empty vector control (pACYC184), into Δcrp reversed the effect of the deletion. This result indicates that the CRP-cAMP signalling pathway is involved in the regulation of CPS biosynthesis, and that CRP acts as a negative regulator of CPS biosynthesis. In addition, since the functions of CyaA, CpdA, and CRP in controlling the cAMP level in K. pneumoniae have not yet been demonstrated, enzyme-linked immunosorbent assays were performed to determine the intracellular cAMP level upon the deletion of cyaA, cpdA, or crp. Compared to WT (9.75±0.35 nM), the intracellular cAMP level was almost undetectable in ΔcyaA (<1 nM), whereas a higher cAMP level was found in ΔcpdA (38±2.8 nM) (Fig. 2C). In addition, a slight increase in the cAMP level was found in Δcrp (14.5±0.7 nM). These result confirmed that CyaA and CpdA are responsible for cAMP biosynthesis and degradation, respectively, in K. pneumoniae.


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)

CCR proteins affect bacterial CPS levels.(A) CPS levels of WT, ΔcyaA, ΔcpdA strains and complementation of cyaA and cpdA strains were determined. (B) CPS levels in mutation and complementation of crp strains were determined. Bacteria were grown in LB medium at 37°C with agitation. *P<0.05 and **P<0.01 compared to the indicated group. (C) Intracellular levels of cAMP in WT, ΔcyaA, ΔcpdA, and Δcrp strains, as determined by ELISA. 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569464&req=5

pone-0054430-g002: CCR proteins affect bacterial CPS levels.(A) CPS levels of WT, ΔcyaA, ΔcpdA strains and complementation of cyaA and cpdA strains were determined. (B) CPS levels in mutation and complementation of crp strains were determined. Bacteria were grown in LB medium at 37°C with agitation. *P<0.05 and **P<0.01 compared to the indicated group. (C) Intracellular levels of cAMP in WT, ΔcyaA, ΔcpdA, and Δcrp strains, as determined by ELISA. 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.
Mentions: To confirm that K. pneumoniae CPS biosynthesis is regulated by cAMP, individual strains with deletion of cyaA and cpdA, which respectively encodes adenylate cyclase and cAMP phosphodiesterase from CG43S3, were constructed, and the effects of the deletions on CPS production were analysed. As shown in Fig. 2A, compared to wild type (WT) strain, we found that CPS levels increased in ΔcyaA, and introduction of pcyaA, but not the empty vector control (pACYC184), into ΔcyaA could reverse the effect of cyaA mutation. In contrast, the deletion of cpdA caused a decreased in CPS levels, which could be complemented by introducing a plasmid-carried cpdA (pETQ-cpdA) into the ΔcpdA strain. These results confirmed that cAMP can act as a signalling molecule for regulation of CPS biosynthesis. In addition, since cAMP affects gene transcription through its effector protein CRP, we assessed the effect of deletion of crp on CPS levels. As shown in Fig. 2B, compared to WT, Δcrp produced higher levels of CPS. Introduction of the complement plasmid pcrp, but not the empty vector control (pACYC184), into Δcrp reversed the effect of the deletion. This result indicates that the CRP-cAMP signalling pathway is involved in the regulation of CPS biosynthesis, and that CRP acts as a negative regulator of CPS biosynthesis. In addition, since the functions of CyaA, CpdA, and CRP in controlling the cAMP level in K. pneumoniae have not yet been demonstrated, enzyme-linked immunosorbent assays were performed to determine the intracellular cAMP level upon the deletion of cyaA, cpdA, or crp. Compared to WT (9.75±0.35 nM), the intracellular cAMP level was almost undetectable in ΔcyaA (<1 nM), whereas a higher cAMP level was found in ΔcpdA (38±2.8 nM) (Fig. 2C). In addition, a slight increase in the cAMP level was found in Δcrp (14.5±0.7 nM). These result confirmed that CyaA and CpdA are responsible for cAMP biosynthesis and degradation, respectively, in K. pneumoniae.

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