Role of the cAMP-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.

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.

Role of the small RNA RyhB in the Fur regulon in mediating the capsular polysaccharide biosynthesis and iron acquisition systems in Klebsiella pneumoniae.

BACKGROUND: The capsular polysaccharide (CPS) and iron acquisition systems are important determinants of Klebsiella pneumoniae infections, and we have previously reported that the ferric uptake repressor (Fur) can play dual role in iron acquisition and CPS biosynthesis. In many bacteria, Fur negatively controls the transcription of the small non-coding RNA RyhB to modulate cellular functions and virulence. However, in K. pneumoniae, the role played by RyhB in the Fur regulon has not been characterised. This study investigated Fur regulation of ryhB transcription and the functional role of RyhB in K. pneumoniae. RESULTS: Deletion of fur from K. pneumoniae increased the transcription of ryhB; the electric mobility shift assay and the Fur-titration assay revealed that Fur could bind to the promoter region of ryhB, suggesting that Fur directly represses ryhB transcription. Additionally, in a Δfur strain with elevated CPS production, deletion of ryhB obviously reduced CPS production. The following promoter-reporter assay and quantitative real-time PCR of cps genes verified that RyhB activated orf1 and orf16 transcription to elevate CPS production. However, deletion of ryhB did not affect the mRNA levels of rcsA, rmpA, or rmpA2. These results imply that Fur represses the transcription of ryhB to mediate the biosynthesis of CPS, which is independent of RcsA, RmpA, and RmpA2. In addition, the Δfur strain's high level of serum resistance was attenuated by the deletion of ryhB, indicating that RyhB plays a positive role in protecting the bacterium from serum killing. Finally, deletion of ryhB in Δfur reduced the expression of several genes corresponding to 3 iron acquisition systems in K. pneumoniae, and resulted in reduced siderophore production. CONCLUSIONS: The regulation and functional role of RyhB in K. pneumoniae is characterized in this study. RyhB participates in Fur regulon to modulate the bacterial CPS biosynthesis and iron acquisition systems in K. pneumoniae.