Dataset Information

EtcABC, a Putative EII Complex, Regulates Type 3 Fimbriae via CRP-cAMP Signaling in Klebsiella pneumoniae.

ABSTRACT: Biofilm formation by Klebsiella pneumoniae on indwelling medical devices increases the risk of infection. Both type 1 and type 3 fimbriae are important factors in biofilm formation by K. pneumoniae. We found that a putative enzyme II (EII) complex of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), etcA (EIIA)-etcB (EIIB)-etcC (EIIC), regulated biofilm and type 3 fimbriae formation by K. pneumoniae STU1. In this study, the regulatory mechanism of etcABC in K. pneumoniae type 3 fimbriae formation was investigated. We found via quantitative RT-PCR that overexpression of etcABC enhanced the transcription level of the mrk operon, which is involved in type 3 fimbriae synthesis, and reduced the transcription level of the fim operon, which is involved in type 1 fimbriae synthesis. To gain further insight into the role of etcABC in type 3 fimbriae synthesis, we analyzed the region upstream of the mrk operon and found the potential cyclic 3'5'-adenosine monophosphate (cAMP) receptor protein (CRP) binding site. After crp was deleted in K. pneumoniae STU1 and two clinical isolates, these three crp mutant strains could not express MrkA, the major subunit of the fimbrial shaft, indicating that CRP positively regulated type 3 fimbriae synthesis. Moreover, a crp mutant overexpressing etcABC could not express MrkA, indicating that the regulation of type 3 fimbriae by etcABC was dependent on CRP. In addition, deletion of cyaA, which encodes the adenylyl cyclase that synthesizes cAMP, and deletion of crr, which encodes the glucose-specific EIIA, led to a reduction in lac operon regulation and therefore bacterial lactose uptake in K. pneumoniae. Exogenous cAMP but not etcABC overexpression compensated for the role of cyaA in bacterial lactose uptake. However, either etcABC overexpression or exogenous cAMP compensated for the role of crr in bacterial lac operon regulation that would eventually restore lactose uptake. We also found via ELISA and the luxCDABE reporter system that overexpression of etcABC increased intracellular cAMP levels and the transcription level of crp, respectively, in K. pneumoniae. In conclusion, overexpression of etcABC positively regulated cAMP production and cAMP-CRP activity to activate the mrk operon, resulting in increased type 3 fimbriae synthesis in K. pneumoniae.

SUBMITTER: Panjaitan NSD

PROVIDER: S-EPMC6629953 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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EtcABC, a Putative EII Complex, Regulates Type 3 Fimbriae via CRP-cAMP Signaling in Klebsiella pneumoniae.

Panjaitan Novaria Sari Dewi NSD Horng Yu-Tze YT Cheng Shih-Wen SW Chung Wen-Ting WT Soo Po-Chi PC

Frontiers in microbiology 20190709

Biofilm formation by Klebsiella pneumoniae on indwelling medical devices increases the risk of infection. Both type 1 and type 3 fimbriae are important factors in biofilm formation by K. pneumoniae. We found that a putative enzyme II (EII) complex of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), etcA (EIIA)-etcB (EIIB)-etcC (EIIC), regulated biofilm and type 3 fimbriae formation by K. pneumoniae STU1. In this study, the regu ...[more]

PMID: 31354661

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Project description: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.

Project description:Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

Project description:Klebsiella pneumoniae is a Gram-negative pathogen that has become a worldwide concern due to the emergence of multidrug-resistant isolates responsible for various invasive infectious diseases. Biofilm formation constitutes a major virulence factor for K. pneumoniae and relies on the expression of fimbrial adhesins and aggregation of bacterial cells on biotic or abiotic surfaces in a coordinated manner. During biofilm aggregation, bacterial cells communicate with each other through inter- or intra-species interactions mediated by signallng molecules, called autoinducers, in a mechanism known as quorum sensing (QS). In most Gram-negative bacteria, intra-species communication typically involves the LuxI/LuxR system: LuxI synthase produces N-acyl homoserine lactones (AHLs) as autoinducers and the LuxR transcription factor is their cognate receptor. However, K. pneumoniae does not produce AHL but encodes SdiA, an orphan LuxR-type receptor that responds to exogenous AHL molecules produced by other bacterial species. While SdiA regulates several cellular processes and the expression of virulence factors in many pathogens, the role of this regulator in K. pneumoniae remains unknown. In this study, we describe the characterization of sdiA mutant strain of K. pneumoniae. The sdiA mutant strain has increased biofilm formation, which correlates with the increased expression of type 1 fimbriae, thus revealing a repressive role of SdiA in fimbriae expression and bacterial cell adherence and aggregation. On the other hand, SdiA acts as a transcriptional activator of cell division machinery assembly in the septum, since cells lacking SdiA regulator exhibited a filamentary shape rather than the typical rod shape. We also show that K. pneumoniae cells lacking SdiA regulator present constant production of QS autoinducers at maximum levels, suggesting a putative role for SdiA in the regulation of AI-2 production. Taken together, our results demonstrate that SdiA regulates cell division and the expression of virulence factors such as fimbriae expression, biofilm formation, and production of QS autoinducers in K. pneumoniae.

Dataset Information

EtcABC, a Putative EII Complex, Regulates Type 3 Fimbriae via CRP-cAMP Signaling in Klebsiella pneumoniae.

Publications

EtcABC, a Putative EII Complex, Regulates Type 3 Fimbriae via CRP-cAMP Signaling in <i>Klebsiella pneumoniae</i>.

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