Abstract
Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP) signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP)-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP and c-di-GMP signaling pathways in K. pneumoniae.
Highlights
Cyclic AMP is a well-known second messenger that has a fundamental role in global gene regulation [1]
We found that the biosynthesis of capsular polysaccharides (CPSs) in Klebsiella pneumoniae increases in response to exogenous glucose, a process that is regulated by cAMP receptor protein (CRP)-Cyclic AMP (cAMP) [13]
When the bacteria were grown in LB broth supplemented with 0.5% glucose, the deleting effect of crp or cyaA on the MrkA production was diminished (Fig 1C), which may due to the inactivated state of CRP-cAMP signaling pathway in glucose-rich conditions
Summary
Cyclic AMP (cAMP) is a well-known second messenger that has a fundamental role in global gene regulation [1]. Its production has been demonstrated to be related to the exogenous glucose level, where bacteria grown in glucose-rich medium showed inhibited cAMP production, whereas bacteria grown in less-preferred carbon sources produced elevated levels of cAMP [1,2,3]. In Escherichia coli, almost 200 operons expression is under CRP-cAMP regulation, which containing genes coding for carbon metabolism and various virulence factors [10,11,12]. We found that the biosynthesis of capsular polysaccharides (CPSs) in Klebsiella pneumoniae increases in response to exogenous glucose, a process that is regulated by CRP-cAMP [13]. In K. pneumoniae pathogenesis, the targets regulated by CRP-cAMP remain largely unknown
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