Browsing by Author "Huang, Ying-Jung"

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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.
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.
Identification of Protein Domains on Major Pilin MrkA That Affects the Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae
(2012-05-15)
Chan, Chia-Han
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Chen, Feng-Jung
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Huang, Ying-Jung
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Chen, Shin-Yu
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Liu, Kuo-Liang
The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains that are critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution at either F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal number of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wildtype. This study has demonstrated that the C-terminal beta strands of MrkA are required for the assembly and structural stability of fimbriae.
Measurement of the adhesive force between a single Klebsiella pneumoniae type 3 fimbria and collagen IV using optical tweezers
(2006-11-10)
Chang, Bo-Jui
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Huang, Ying-Jung
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Chan, Chia-Han
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Hsu, Long
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Peng, Hwei-Ling
Type 3 fimbriae are important adhesive filaments that assist Klebsiella pneumoniae to establish an infection. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins, although the difference has not been determined physically. For this reason, the adhesive force between type 3 fimbriae and collagen IV were measured using optical tweezers. The measured force data displayed a periodic histogram thus Fourier analysis was applied to group it to extract the adhesive force of a single molecular pair. Specifically, we showed that grouping should begin with an offset at the first half of the period. Finally, we first present the adhesive force between each mrkD(V2)-, mrkD(V3)-, and mrkD(V4)-expressed fimbriae and collagen IV is 2.03, 3.79, and 2.87 pN, respectively, This result can be referred to further research on mrkD allelic effect on bacteria infection. (c) 2006 Elsevier Inc. All rights reserved.
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.
PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43
(2015-12-01)
Wang, Zhe-Chong
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Liu, Chia-Jui
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Huang, Ying-Jung
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Wang, Yu-Seng
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Peng, Hwei-Ling
In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3 Delta cpxAR, the derived mutants CG43S3 Delta cpxAR Delta pecS and CG43S3 Delta cpxAR Delta pecS Delta pecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3 Delta cpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3 Delta cpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.
PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43
(2015-12)
Wang, Zhe-Chong
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Liu, Chia-Jui
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Huang, Ying-Jung
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Wang, Yu-Seng
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Peng, Hwei-Ling
In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3 Delta cpxAR, the derived mutants CG43S3 Delta cpxAR Delta pecS and CG43S3 Delta cpxAR Delta pecS Delta pecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3 Delta cpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3 Delta cpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.
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.
Structural and Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae
(2011-04-01)
Chen, Feng-Jung
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Chan, Chia-Han
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Huang, Ying-Jung
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Liu, Kuo-Liang
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Peng, Hwei-Ling
This study investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium. Transmission electron microscopy and optical tweezers were used to understand the ability of the bacterium to survive flushes. An individual K. pneumoniae type 3 fimbria exhibited a helix-like structure with a pitch of 4.1 nm and a three-phase force-extension curve. The fimbria was first nonlinearly stretched with increasing force. Then, it started to uncoil and extended several micrometers at a fixed force of 66 +/- 4 pN (n +/- 22). Finally, the extension of the fimbria shifted to the third phase, with a characteristic force of 102 +/- 9 pN (n = 14) at the inflection point. Compared with the P fimbriae and type 1 fimbriae of uropathogenic Escherichia coli, K. pneumoniae type 3 fimbriae have a larger pitch in the helix-like structure and stronger uncoiling and characteristic forces.
Uncoiling mechanism of Klebsiella pneumoniae type 3 pili measured by using optical tweezers
(2007-01-01)
Chen, Feng-Jung
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Chan, Chia-Han
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Liu, Kuo-Liang
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Huang, Ying-Jung
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Peng, Hwei-Ling
Pili are bacterial appendages that play many important roles in bacterial behaviors, physiology and interaction with hosts. Via pili, bacteria are able to adhere to, migrate onto, and colonize on host cells, mechanically. Different from the most studied type I and P type pili, which are rigid and thick with an average of 6 similar to 7 nm in diameter, type 3 pili are relatively tiny (3-5 nm in diameter) and flexible, and their biophysical properties remains unclear. By using optical tweezers, we found that the elongation processes of type 3 pili are divided into three phases: (1) elastic elongation, (2) uncoiling-elongation, and (3) intrinsic elongation, separately. Besides, the uncoiling force of the recombinant pili displayed on the surface of E. coli [pmrkABCD(VI)F] is measured 20 pN in average stronger than that of E. coli [pmrkABCD(VI)]. This suggests that pilin MrkF is involved in determining the mechanical properties of the type 3 pili.
Uncoiling mechanism of Klebsiella pneumoniae type 3 pili measured by using optical tweezers - art. no. 66441W
(2007)
Chen, Feng-Jung
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Chan, Chia-Han
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Liu, Kuo-Liang
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Huang, Ying-Jung
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Peng, Hwei-Ling
Pili are bacterial appendages that play many important roles in bacterial behaviors, physiology and interaction with hosts. Via pili, bacteria are able to adhere to, migrate onto, and colonize on host cells, mechanically. Different from the most studied type I and P type pili, which are rigid and thick with an average of 6 similar to 7 nm in diameter, type 3 pili are relatively tiny (3-5 nm in diameter) and flexible, and their biophysical properties remains unclear. By using optical tweezers, we found that the elongation processes of type 3 pili are divided into three phases: (1) elastic elongation, (2) uncoiling-elongation, and (3) intrinsic elongation, separately. Besides, the uncoiling force of the recombinant pili displayed on the surface of E. coli [pmrkABCD(VI)F] is measured 20 pN in average stronger than that of E. coli [pmrkABCD(VI)]. This suggests that pilin MrkF is involved in determining the mechanical properties of the type 3 pili.