Browsing by Author "Huang, Ching-Jou"

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FimK regulation on the expression of type 1 fimbriae in Klebsiella pneumoniae CG43S3
(2013-07-01)
Wang, Zhe-Chong
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Huang, Ching-Jou
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Huang, Ying-Jung
;
Wu, Chien-Chen
;
Peng, Hwei-Ling
Klebsiella pneumoniae CG43, a heavy encapsulated liver abscess isolate, mainly expresses type 3 fimbriae. Type 1 fimbriae expression was only apparent in CG43S3 Delta mrkA (the type 3 fimbriae-deficient strain). The expression of type 1 fimbriae in CG43S3 Delta mrkA was reduced by deleting the fimK gene, but was unaffected by removing the 3' end of fimK encoding the C-terminal EIL domain (EILfimK). Quantitative RT-PCR and promoter activity analysis showed that the putative DNA-binding region at the N terminus, but not the C-terminal EIL domain, of FimK positively affects transcription of the type 1 fimbrial major subunit, fimA. An electrophoretic mobility shift assay demonstrated that the recombinant FimK could specifically bind to fimS, which is located upstream of fimA and contains a vegetative promoter for the fim operon, also reflecting possible transcriptional regulation. EILfimK was shown to encode a functional phosphodiesterase (PDE) via enhancing motility in Escherichia coil JM109 and in vitro using PDE activity assays. Moreover, EILfimK exhibited higher PDE activity than FimK, implying that the N-terminal DNA-binding domain may negatively affect the PDE activity of FimK. FimA expression was detected in CG43S3 expressing EILfimK or AIL(fimK), suggesting that FimA expression is not directly influenced by the c-di-GMP level. In summary, FimK influences type 1 fimbriation by binding to fimS at the N-terminal domain, and thereafter, the altered protein structure may activate C-terminal PDE activity to reduce the intracellular c-di-GMP level.
Fur-dependent MrkHI regulation of type 3 fimbriae in Klebsiella pneumoniae CG43
(2012-04-01)
Wu, Chien-Chen
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Lin, Ching-Ting
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Cheng, Wei-Yun
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Huang, Ching-Jou
;
Wang, Zhe-Chong
Type 3 fimbriae play a crucial role in Klebsiella pneumoniae biofilm formation, but the mechanism of the regulation of the type 3 fimbrial operon is largely unknown. In K. pneumoniae CG43, three regulatory genes, mrkH, mrkI and mrkJ, are located downstream of the type 3 fimbrial genes mrkABCDF. The production of the major pilin MrkA is abolished by the deletion of mrkH or mrkI but slightly increased by the deletion of mrkJ. Additionally, quantitative RT-PCR and a promoter reporter assay of mrkHI verified that the transcription of mrkHI was activated by MrkI, suggesting autoactivation of mrkHI transcription. In addition, sequence analysis of the mrkH promoter region revealed a putative ferric uptake regulator (Fur) box. Deletion of fur decreased the transcription of mrkH, mrkI and mrkA. The expression of type 3 fimbriae and bacterial biofilm formation were also reduced by the deletion of fur. Moreover, a recombinant Fur was found to be able to bind both promoters, with higher affinity for P-mrkH than P-mrkA, implying that Fur controls type 3 fimbriae expression via MrkHI. We also proved that iron availability can influence type 3 fimbriae activity.
YjcC, a c-di-GMP Phosphodiesterase Protein, Regulates the Oxidative Stress Response and Virulence of Klebsiella pneumoniae CG43
(2013-07-23)
Huang, Ching-Jou
;
Wang, Zhe-Chong
;
Huang, Hsi-Yuan
;
Huang, Hsien-Da
;
Peng, Hwei-Ling
This study shows that the expression of yjcC, an in vivo expression (IVE) gene, and the stress response regulatory genes soxR, soxS, and rpoS are paraquat inducible in Klebsiella pneumoniae CG43. The deletion of rpoS or soxRS decreased yjcC expression, implying an RpoS- or SoxRS-dependent control. After paraquat or H2O2 treatment, the deletion of yjcC reduced bacterial survival. These effects could be complemented by introducing the DyjcC mutant with the YjcC-expression plasmid pJR1. The recombinant protein containing only the YjcC-EAL domain exhibited phosphodiesterase (PDE) activity; overexpression of yjcC has lower levels of cyclic di-GMP. The yjcC deletion mutant also exhibited increased reactive oxygen species (ROS) formation, oxidation damage, and oxidative stress scavenging activity. In addition, the yjcC deletion reduced capsular polysaccharide production in the bacteria, but increased the LD50 in mice, biofilm formation, and type 3 fimbriae major pilin MrkA production. Finally, a comparative transcriptome analysis showed 34 upregulated and 29 downregulated genes with the increased production of YjcC. The activated gene products include glutaredoxin I, thioredoxin, heat shock proteins, chaperone, and MrkHI, and proteins for energy metabolism (transporters, cell surface structure, and transcriptional regulation). In conclusion, the results of this study suggest that YjcC positively regulates the oxidative stress response and mouse virulence but negatively affects the biofilm formation and type 3 fimbriae expression by altering the c-di-GMP levels after receiving oxidative stress signaling inputs.