Role of the cAMP-Dependent Carbon Catabolite Repression in Capsular Polysaccharide Biosynthesis in <em>Klebsiella pneumoniae</em>

<div><p><em>K. pneumoniae</em> 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 <em>K. pneumoniae</em> virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for <em>K. pneumoniae</em> virulence, in response to exogenous glucose. We found that <em>K. pneumoniae</em> produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of <em>cyaA</em> or <em>crp,</em> which respectively encode adenylate cyclase and cAMP receptor protein in <em>K. pneumoniae</em>, markedly increased CPS production, while deletion of <em>cpdA</em>, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that <em>K. pneumoniae</em> 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 (<em>orf1-2</em>, <em>orf3-15</em>, and <em>orf16-17</em>), were activated by the deletion of <em>crp</em>. Sequence analysis revealed putative CRP binding sites located on P<em>_orf3-15</em> and P<em>_orf16-17</em>, 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 <em>rcsA</em>, which encodes a <em>cps</em> transcriptional activator, demonstrating a direct repression of CRP-cAMP and P<em>_rcsA</em>. The deletion of <em>rcsA</em> in mutation of <em>crp</em> partially reduced CPS biosynthesis and the transcription of <em>orf1-2</em> but not of <em>orf3-15</em> or <em>orf16-17</em>. These results suggest that RcsA participates in the CRP-cAMP regulation of <em>orf1-2</em> 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 <em>K. pneumoniae</em> increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR.</p>