Browsing by Author "Peng, Hwei-Ling"

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Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1
(2008-04-11)
Hsu, Jye-Lin
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Chen, Hsuan-Cheng
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Peng, Hwei-Ling
;
Chang, Hwan-You
;
生物科技學系
Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346-PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.
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.
Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1
(2007-06-15)
Hung, Ruei-Jiun
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Chien, Han-Sheng
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Lin, Ruei-Zeng
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Lin, Ching-Ting
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Vatsyayan, Jaya
UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDPglucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP- glucose as a substrate to generate UDP- glucuronic acid. The Km values of PA2022 and PA3559 for UDP-glucose are similar to 0.1 and 0.4 mM, whereas the K-m values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP- glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg2+, which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.
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
;
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.
Effects of gaIU Mutation on Pseudomonas syringae-Plant Interactions
(2010-09-01)
Deng, Wen-Ling
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Lin, Yuan-Chun
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Lin, Rong-Hwa
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Wei, Chia-Fong
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Huang, Yi-Chiao
Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role in carbohydrates biosynthesis, including synthesis of lipopolysaccharides (LPS), membrane-derived oligosaccharides, and capsular polysaccharides. In this study, we characterized the galU mutant of Pseudomonas syringae pv. syringae 61 (Psy61), a necrotizing plant pathogen whose pathogenicity depends on a functional type III secretion system (T3SS), and showed that the Psy61 gall] mutant had reduced biofilm formation ability, was nomnotile, and had an assembled T3SS structure but failed to elicit hypersensitive response in resistant plants and necrotic lesions in susceptible plants. Moreover, the defective LPS and other pathogenassociated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMPtriggered immunity, which severely compromised the ability of the Psy61 gall] mutant to survive in planta. Our results demonstrated that the complete LPS protected plant-pathogenic bacteria from host innate immunity, similar to what was found in animal pathogens, prior to the translocation of T3S effectors and bacterial multiplication.
Enhancing ATP-based bacteria and biofilm detection by enzymatic pyrophosphate regeneration
(2010-04-15)
Lee, Hui-Ju
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Ho, Ming-Rong
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Bhuwan, Manish
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Hsu, Ching-Yi
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Huang, Meng-Shun
The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PP(i) in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5'-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PPi and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudo monas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines. (C) 2009 Elsevier Inc. All rights reserved.
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.
Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria
(2011-11-01)
Lee, Hui-Ju
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Ho, Min-Rong
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Tseng, Chih-Sian
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Hsu, Ching-Yi
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Huang, Meng-Shun
Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia cob is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water. (C) 2011 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.
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.
Genetic Requirements for Klebsiella pneumoniae-Induced Liver Abscess in an Oral Infection Model
(2009-07-01)
Tu, Ya-Chun
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Lu, Min-Chi
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Chiang, Ming-Ko
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Huang, Shu-Ping
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Peng, Hwei-Ling
Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.
Gut microbiome changes in overweight male adults following bowel preparation
(2018-12-31)
Chen, Hui-Mei
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Chen, Chung-Chu
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Chen, Chien-Chi
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Wang, Shen-Chih
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Wang, Chun-Lin
BackgroundHuman gut microbiome has an essential role in human health and disease. Although the major dominant microbiota within individuals have been reported, the change of gut microbiome caused by external factors, such as antibiotic use and bowel cleansing, remains unclear. We conducted this study to investigate the change of gut microbiome in overweight male adults after bowel preparation, where none of the participants had been diagnosed with any systemic diseases.MethodsA total of 20 overweight, male Taiwanese adults were recruited, and all participants were omnivorous. The participants provided fecal samples and blood samples at three time points: prior to bowel preparation, 7days after colonoscopy, and 28days after colonoscopy. The microbiota composition in fecal samples was analyzed using 16S ribosome RNA gene amplicon sequencing.ResultsOur results demonstrated that the relative abundance of the most dominant bacteria hardly changed from prior to bowel preparation to 28days after colonoscopy. Using the ratio of Prevotella to the sum of Prevotella and Bacteroides in the fecal samples at baseline, the participants were separated into two groups. The fecal samples of the Type 1 group was Bacteroides-dominant, and that of the Type 2 group was Prevotella-dominant with a noticeable presence Bacteroides. Bulleidia appears more in the Type 1 fecal samples, while Akkermensia appears more in the Type 2 fecal samples. Of each type, the gut microbial diversity differed slightly among the three collection times. Additionally, the Type 2 fecal microbiota was temporarily susceptible to bowel cleansing. Predictive functional analysis of microbial community reveals that their activities for the mineral absorption metabolism and arachidonic acid metabolism differed significantly between the two types. Depending on their fecal type, the variance of triglycerides and C-reactive protein also differed between the two types of participants.ConclusionsDepending upon the fecal type, the microbial diversity and the predictive functional modules of microbial community differed significantly after bowel preparation. In addition, blood biochemical markers presented somewhat associated with fecal type. Therefore, our results might provide some insights as to how knowledge of the microbial community could be used to promote health through personalized clinical treatment.
Histidine-containing Phosphotransfer Protein-B (HptB) Regulates Swarming Motility through Partner-switching System in Pseudomonas aeruginosa PAO1 Strain
(2012-01-13)
Bhuwan, Manish
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Lee, Hui-Ju
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Peng, Hwei-Ling
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Chang, Hwan-You
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生物科技學系
The histidine-containing phosphotransfer protein-B (HptB; PA3345) is an intermediate protein involved in transferring a phosphoryl group from multiple sensor kinases to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. The objective of this study was to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription profiling analysis of an hptB knock-out mutant showed that the expression of a number of motility-related genes was altered consistent with the non-swarming phenotype observed for the mutant. Domain analysis indicated that the PA3346 C-terminal region (PA3346C) exhibits similar to 30% identity with the anti-sigma factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity targeting an anti-sigma antagonist, PA3347, at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-sigma(28) factor FlgM were found to interact with PA3347 individually both in vivo and in vitro. FlgM displaced PA3346C in binding of PA3347 and was then competitively displaced by sigma(28) from the PA3347-FlgM complex, forming a phosphorylation-dependent partner-switching system. The significance of PA3347 phosphorylation in linking the partner-switching system and swarming motility was established by analyzing the swarming phenotype of the PA3347 knock-out mutant and its complement strains.
Homologous response regulators KvgA, KvbA and KvhR regulate the synthesis of capsular polysaccharide in Klebsiella pneumoniae CG43 in a coordinated manner
(2006-09-01)
Lin, Ching-Ting
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Huang, Teng-Yi
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Liang, Wan-Chun
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Peng, Hwei-Ling
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生物科技學系
On the basis of phenotypic analysis, the Klebsiella pneumoniae CG43 derived mutants with deletions of the gene encoding respectively the response regulators KvgA, KvhA, and KvhR were classified into two groups. Group I bacteria carrying either kvgA(-) or kvhR(-) exhibited less mucoidy, lower level of capsular polysaccharide (CPS) synthesis and higher LD50 than the parental strain. No apparent change of the group II, including kvhA(-) and hvhA(-)kvhR(-) mutants, was observed. However, the mucoidy of kvhA(-)kvhR(-) mutant was found to be diminished after introducing into a kvhA(-) expressing plasmid. Via promoter-lacZ fusion analysis, kvhA deletion was found to reduce kvhR expression. A regulatory role of KvhA for the expression of kvhR was supported further by EMSA showing a specific binding of KvhA to the putative promoter of kvhR. The promoter activity measurement and EMSA also revealed that KvgA acted as an autoregulator and an activator for the expression of kvhAS and kvhR. In addition, deletion of kvgA suppressed slightly the promoter activity of the cps-orf16-17, and the expression of all three cps transcripts orf1-2, orf3-15, and orf16-17 were reduced in the kvhR(-) mutant. These suggest that the three homologous response regulators interact to control, in coordination, the bacterial cps expression.
Identification of amino acid residues important for the phosphomannose isomerase activity of Ps1B in Pseudomonas aeruginosa PAO1
(2008-10-15)
Lee, Hui-Ju
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Chang, Hwan-You
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Venkatesan, Nandinin
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Peng, Hwei-Ling
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生物科技學院
Phosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co2+-dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8-20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI.
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.
Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
(2011)
Huang, Chen-Ji
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Peng, Hwei-Ling
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Cheng, Chih-Yu
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生物科技學系
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Department of Biological Science and Technology
In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W(84), P(95), P(110), or V(129). The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W(84)S, P(110)S and V(129)L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 mu M(-1). Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.
Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue
(2013)
Ho, Chen-Ta
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Lin, Ruei-Zeng
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Chen, Rong-Jhe
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Chin, Chung-Kuang
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Gong, Song-En
A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.
Localization and characterization of VVA0331, a 489-kDa RTX-like protein, in Vibrio vulnificus YJ016
(2009-05-01)
Chou, Li-Fang
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Peng, Hwei-Ling
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Yang, Yu-Chung
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Kuo, Min-Chieh
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Chang, Hwan-You
Vibrio vulnificus YJ016 contains three genes encoding proteins homologous to repeats-in-toxin proteins. One of these genes, vva0331, possesses a long open reading frame of 13,971 bp in length and resides on the small chromosome between two gene clusters encoding a type I secretion system and several regulatory proteins, respectively. Bioinformatic analysis revealed that VVA0331 consist of nineteen 87-amino acid repeats, two Arg-Gly-Asp motifs, four cysteine residues, an outer membrane protein domain, a polysaccharide-binding site and several motifs related to cell adhesions. These features are distinct from those of typical repeat-in-toxins and autotransporter adhesins. Real-time quantitative PCR analysis indicates that vva0331 gene expression is activated at 30A degrees C and regulated by iron. In addition, VVA0331 is present primarily in a secreted form as determined by cell fractionation assay and Western blot analysis. No significant difference in Hep2 cell adherence, cytotoxicity, and virulence was observed between the wild type and vva0331 mutant strains. In contrast, these strains exhibited apparently different outer membrane protein profiles, and antiserum raised against C-terminal region of VVA0331 reacted with an 85-kDa outer membrane protein of V. vulnificus YJ016.