Browsing by Author "Wu, Chien-Chen"

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Characterization of the type 3 fimbriae with different MrkD adhesins: Possible role of the MrkD containing an RGD motif
(2006-11-24)
Huang, Ying-Jung
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Wu, Chien-Chen
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Chen, Mei-Chen
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Fung, Chang-Phone
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Peng, Hwei-Ling
Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneuntoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E coli JM 109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha 5 beta 1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin. (c) 2006 Elsevier Inc. All rights reserved.
CRP-Cyclic AMP Regulates the Expression of Type 3 Fimbriae via Cyclic di-GMP in Klebsiella pneumoniae
(2016-09-15)
Lin, Ching-Ting
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Lin, Tien-Huang
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Wu, Chien-Chen
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Wan, Lei
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Huang, Chun-Fa
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 P-mrkA were increased in Delta crp 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 P-mrkHI and P-mrkJ were decreased in Delta lacZ Delta crp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Delta crp 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 P-mrkHI in the Delta lacZ Delta crp strain was diminished in Delta lacZ Delta crp Delta mrkHI, but increased in the Delta lacZ Delta crp Delta mrkJ 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 Delta crp 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.
Evidence of DNA transfer through F-pilus channels during Escherichia coli conjugation
(2008-07-01)
Shu, An-Chi
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Wu, Chien-Chen
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Chen, Yi-Yang
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Peng, Hwei-Ling
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Chang, Hwan-You
The mechanism of DNA transfer from Escherichia coli (E. coli) Hfr donor strain AT2453 to recipient strain AB1157 during the conjugation process has been investigated by liquid atomic force microscopy (AFM). With the success of immobilizing both E. coli strains on gelatin-treated glass under aqueous solution, the F-pilus between an E. coli mating pair could be clearly imaged and dissected by an AFM probe. Another AFM probe functionalized with an anti-single-stranded DNA (ssDNA) antibody was then applied to detect transferring ssDNA. According to the AFM force spectrum, the transferring ssDNA could be detected only in the dissected area with a binding force of 109 5 pN measured. Our results provide direct evidence indicating that the DNA was transferred through the F-pilus channel between an E. coli mating pair during their conjugation.
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
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Wu, Chien-Chen
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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 regulation of the capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43
(2011-02-01)
Lin, Ching-Ting
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Wu, Chien-Chen
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Chen, Yu-Sheng
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Lai, Yi-Chyi
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Chi, Chia
The ferric uptake regulator Fur has been reported to repress the expression of rmpA, a regulatory gene for the mucoid phenotype, leading to decreased capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae CG43. Here, quantitative real-time FOR (qRT-PCR) analyses and electrophoretic mobility shift assays showed that Fur also repressed the expression of the CPS regulatory genes rmpA2 and rcsA. Interestingly, deletion of rmpA or rcsA but not rmpA2 from the Delta fur strain was able to suppress the deletion effect of Fur. The availability of extracellular iron affected the amount of CPS, suggesting that Fur regulates CPS biosynthesis in an Fe(II)-dependent manner. Increased production of siderophores was observed in the Delta fur strain, suggesting that uptake of extracellular iron in K. pneumoniae is regulated by Fur. Fur titration assays and qRT-PCR analyses demonstrated that at least six of the eight putative iron-acquisition systems, identified by a BLAST search in the contig database of K. pneumoniae CG43, were directly repressed by Fur. We conclude that Fur has a dual role in the regulation of CPS biosynthesis and iron acquisition in K. pneumoniae.
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
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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.
MrkF is a component of type 3 fimbriae in Klebsiella pneumoniae
(2009-01-01)
Huang, Ying-Jung
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Liao, Hsin-Wei
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Wu, Chien-Chen
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Peng, Hwei-Ling
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生物科技學系
Klebsiella pneumoniae type 3 fimbriae are encoded by mrkABCDF genes which produce the major pilin subunit MrkA, chaperone MrkB, outer membrane usher MrkC, adhesin MrkD and MrkF of unknown function, respectively. RT-PCR analysis demonstrated that the mrkF gene is contained within the mrk operon. Deletion of mrkF in K. pneumoniae CG43 was found to reduce biofilm formation. A higher level of biofilm formation activity was also observed in recombinant Escherichia coli JM109[pmrkABCDF] compared to that observed for JM109[pmrkABCD]. Immunoelectron microscopy analysis of recombinant type 3 fimbriae using anti-MrkA and anti-MrkF antibody-labeled gold particles revealed that MrkF intermittently inserted into the MrkA filament. An interaction between recombinant MrkA and MrkF was demonstrated by co-immunoprecipitation analysis, further supporting the notion that MrkF is a structural component of the fimbriae. Intriguingly, the incorporation of MrkF appeared to decrease fimbrial length but increased activity of autoaggregation and biofilm formation in the bacteria JM109[pmrkABCDF]. This suggested that MrkF may play a role in assembly of the filament. (C) 2008 Elsevier Masson SAS. All rights reserved.
Novel microchip for in situ TEM imaging of living organisms and bio-reactions in aqueous conditions
(2008)
Liu, Kuo-Liang
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Wu, Chien-Chen
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Huang, Ying-Jung
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Peng, Hwei-Ling
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Chang, Hwan-You
A novel and disposable microchip (K-kit) with SiO(2) nano-membranes was developed and used as a specimen kit for in situ imaging of living organisms in an aqueous condition using transmission electron microscopy (TEM) without equipment modi. cation. This K-kit enabled the successful TEM observation of living Escherichia coli cells and the tellurite reduction process in Klebsiella pneumoniae. The K. pneumoniae and Saccharomyces cerevisiae can stay alive in K-kit after continuous TEM imaging for up to 14 s and 42 s, respectively. Besides, different tellurite reduction profiles in cells grown in aerobic and anaerobic environments can be clearly revealed. These results demonstrate that the K-kit developed in this paper can be useful for observing living organisms and monitoring biological processes in situ.
Regulation of the Klebsiella pneumoniae Kpc fimbriae by the site-specific recombinase Kpcl
(2010-07-01)
Wu, Chien-Chen
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Huang, Ying-Jung
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Fung, Chang-Phone
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Peng, Hwei-Ling
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生物科技學系
In the genome of Klebsiella pneumoniae NTUH-K2044, nine fimbrial gene clusters were identified. Besides type 1 and type 3 fimbriae, the others are novel and were named Kpa, Kpb, Kpc, Kpd, Kpe, Kpf and Kpg fimbriae. Prevalence analysis among 105 K. pneumoniae clinical isolates revealed that the kpc genes were highly associated with the K1 serotype isolates. Induced expression of the recombinant kpcABCD genes in Escherichia colt resulted in Kpc fimbriation and increased biofilm formation. A putative site-specific recombinase encoding gene kpcl and a 302 bp intergenic DNA flanked by 11 bp inverted repeats, namely kpcS, were identified in the upstream region of the kpcABCD genes. Using LacZ as the reporter, a dramatic difference in promoter activity of kpcS in two different orientations was observed and accordingly assigned as ON and OFF phase. kpcl expression was found to be able to invert kpcS in trans from phase ON to OFF and vice versa. Using the two-plasmid system, expression of kpcA, encoding the major component of the Kpc fimbriae, could be observed upon the induced expression of kpcl. These results indicate that Kpcl is involved in the regulation of Kpc fimbriation in a phase-variable manner.
Role of the cAMP-Dependent Carbon Catabolite Repression in Capsular Polysaccharide Biosynthesis in Klebsiella pneumoniae
(2013-02-11)
Lin, Ching-Ting
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Chen, Yu-Ching
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Jinn, Tzyy-Rong
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Wu, Chien-Chen
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Hong, Yi-Ming
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 Porf3-15 and Porf16-17, 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
(1970-01-01)
Huang, Su-Hua
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Wang, Chien-Kuo
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Peng, Hwei-Ling
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Wu, Chien-Chen
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Chen, Ying-Tsong
"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 Delta fur 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 Delta fur 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 Delta fur 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."
Surface Rigidity Change of Escherichia coli after Filamentous Bacteriophage Infection
(2009-04-21)
Chen, Yi-Yang
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Wu, Chien-Chen
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Hsu, Jye-Lin
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Peng, Hwei-Ling
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Chang, Hwan-You
In this study, the feasibility using atomic force microscopy (AFM) to study the interaction between bacteriophages (phages) and bacteria in situ was demonstrated here. Filamentous phage M 13 specifically infects the male Escherichia coli, which expresses F-pili. After infection, E. coli become fragile and grows at a slower rate. AFM provides a powerful tool for investigating these changes in a near-physiological environment. Using high-resolution AFM in phosphate-buffered saline, the damage to the lipopolysaccharide (LPS) layer on the outer membrane of the M13 phage-infected E. coli was observed. The membrane became smoother and more featureless compared to those that were not infected. Besides, the force-distance (f-d) curves were measured to reveal the surface rigidity change in E. coli after M13 phage infection. The effective spring constant and Young's modulus of E. coli decreased after M13 phage infection. Furthermore, the AFM tip was pressed against E. coli to stud), the response of E. coli under load before and after M 13 phage infection. The results showed that after infection E. coli became less rigid and the membrane was also damaged. However, the stiffness changes, including the spring constant and Young's modulus of E. coli, are negligible after M 13 phage infection compared with those in previous reports, which may be one of the reasons that E. coli still can maintain its viability after filamentous phage infection.