Chia-Fong Wei - Academia.edu (original) (raw)

Papers by Chia-Fong Wei

Research paper thumbnail of Characterization of hrpG gene from Pseudomonas syringae pv. syringae

菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin... more 菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin過敏原即利用此基因組產物所組成的第三型分泌系統被分泌到胞外。本研究主要在探討hrpC operon中的HrpG蛋白(15.4 kDa)在與植物的交互作用中所扮演的角色。利用不具有terminator的nptⅡ基因構築非極性突變的方法，證實hrpG突變株會減弱菸草的過敏性反應，利用西方點墨法得知此突變株仍可分泌Harpin蛋白，但Harpin的量減少很多；進一步將hrpG接上一個能夠以商業化的單株抗體(M2)偵測的FLAG序列，構築於廣宿主載體上，送入hrpG的突變株中作為互補株，此互補株能恢復和野生株相同的表現型，再利用細胞成份分離法以及西方點墨法可以得知HrpG蛋白質主要位於細胞質內。另外，經由北方點墨法及西方點墨法分析，結果顯示hrpG突變株的hrpZ、hrcJ、hrcQb表現量都明顯地比野生株及互補株低；然而因為構築非極性突變時所插入之nptII基因內含有較強的nptII 基因的promoter，使得下游的hrpV的表現量在突變株及互補株中又比野生株高，而HrpV具有負調控的功能，使得在hrpG的突變株中降低了其它基因的表現量，但是在互補株中這些所測試的基因又可恢復與野生株相同的表現量，因此我們推測HrpG可能是經由調控HrpV，或是其下游的相關調控蛋白，藉此對hrp/hrc基因組的表現作微調控；另外，HrpG也可能是以作為HrpV蛋白之chaperone形式來影響HrpV在胞內的量，藉此來調節HrpV之負調控功能。Pseudomonas syringae pv. syringae 61 possesses a 25 kb hrp/hrc cluster, which encodes 28 proteins responsible for eliciting the hypersensitive response (HR) in its non-host plants and causing diseases on the host plants. The products of the hrp genes are required for the Harpinp...

Research paper thumbnail of The RssAB-FlhDC-ShlBA as a Dominant Pathogenesis Pathway in Serratia marcescens

The RssAB-FlhDC-ShlBA as a Dominant Pathogenesis Pathway in Serratia marcescens Chuan-Sheng Lin, ... more The RssAB-FlhDC-ShlBA as a Dominant Pathogenesis Pathway in Serratia marcescens Chuan-Sheng Lin, Jim-Tong Horng, Chun-Hung Yang, Yu-Huan Tsai, Lin-Hui Su, Chia-Fong Wei, Chang-Chieh Chen, Shang-Chen Hsieh, Chia-Chen Lu, and Hsin-Chih Lai 1 Department of Medical Biotechnology and Laboratory Science, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. 2 Department of Biochemistry and Molecular Biology, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. 3 Department of Clinical Pathology, Chang Gung Memorial Hospital, 5 Fuxing Street, Kweishan, Taoyuan, 333 Taiwan, Republic of China. 4 Research Center for Pathogenic Bacteria, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. * Corresponding author † Chuan-Sheng Lin and Jim-Tong Horng contributed equally to this work. CORRESPONDENCE Hsin-Chih Lai: Department of Medical Biotechnology and Laboratory Science, ...

Research paper thumbnail of Cross-talk between bacterial two-component systems drives stepwise regulation of flagellar biosynthesis in swarming development

Biochemical and biophysical research communications, Jul 15, 2017

Swarming motility is a mode of bacterial movement over a solid surface driven by rotating flagell... more Swarming motility is a mode of bacterial movement over a solid surface driven by rotating flagella in a coordinated manner. Bacteria can use two-component system (TCS), which typically comprises a sensor kinase and a specific cognate response regulator, to properly react to environmental changes. We previously showed that the TCS RssAB suppresses flagellar biosynthesis master regulator flhDC specifically in swarming lag phase to control surface migration timing without affecting expansion rate in Serratia marcescens swarming development. Here we demonstrate that the TCS QseBC, which has been found in several human pathogens involved in flagellar and virulence regulation, has cross-talk with RssAB. We demonstrate that the phosphorylated QseB repressed flhDC expression, reducing swarming migration rate with modest effect on migration initiation. Unexpectedly, the QseC can dephosphorylate non-cognate response regulator RssB. Deletion of qseC prolonged RssAB signaling, reduced flhDC exp...

Research paper thumbnail of Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida

Frontiers in microbiology, 2016

Synergistic effects between the same class of antibiotics are rarely reported. Our previous study... more Synergistic effects between the same class of antibiotics are rarely reported. Our previous study found synergistic-like interaction between florfenicol (FFC) and thiamphenicol (TAP) against Staphylococcus aureus. Here, the enhanced antimicrobial activity was evaluated in 97 clinical isolates of both Gram-negative and Gram-positive bacteria. Susceptible strains were initially identified by checkerboard microdilution assay (fractional inhibitory concentration index [FICI] ≤ 0.625), followed by confirmation of synergism using the time-kill methodology (≥2 log10 CFU/ml reduction). In all, 43% of Pasteurella multocida tested were susceptible to the enhanced bactericidal effect. In chicken fowl cholera models, FFC and TAP combination at much lower dosage that is correspondent to their MIC deduction provided maximum protection in vivo. Furthermore, synergistic combination of FFC with oxytetracycline (OTC) against Pseudomonas aeruginosa in vitro was also demonstrated. Based on the enhanced...

Research paper thumbnail of The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate T3SS function and t3ss expression in Pseudomonas syringae pv. averrhoi

Molecular plant pathology, Jan 6, 2015

To ensure the optimal infectivity upon contact with host cells, pathogenic Pseudomonas syringae h... more To ensure the optimal infectivity upon contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream to hrpG, encodes a T3SS-dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on eliciting disease symptom in its host and hypersensitive response in the nonhost plant, and secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild type results in delayed HR and reduced t3ss expression. The results of protein-protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial ...

Research paper thumbnail of Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis

Journal of the Science of Food and Agriculture, 2015

The potential presence of undeclared animal by-products in pet foods is not subject to routine ex... more The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols. © 2015 Society of Chemical Industry.

Research paper thumbnail of 菜豆細菌性斑點病菌致病基因hrpG之特性分析

菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin... more 菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin過敏原即利用此基因組產物所組成的第三型分泌系統被分泌到胞外。本研究主要在探討hrpC operon中的HrpG蛋白(15.4 kDa)在與植物的交互作用中所扮演的角色。利用不具有terminator的nptⅡ基因構築非極性突變的方法，證實hrpG突變株會減弱菸草的過敏性反應，利用西方點墨法得知此突變株仍可分泌Harpin蛋白，但Harpin的量減少很多；進一步將hrpG接上一個能夠以商業化的單株抗體(M2)偵測的FLAG序列，構築於廣宿主載體上，送入hrpG的突變株中作為互補株，此互補株能恢復和野生株相同的表現型，再利用細胞成份分離法以及西方點墨法可以得知HrpG蛋白質主要位於細胞質內。另外，經由北方點墨法及西方點墨法分析，結果顯示hrpG突變株的hrpZ、hrcJ、hrcQb表現量都明顯地比野生株及互補株低；然而因為構築非極性突變時所插入之nptII基因內含有較強的nptII 基因的promoter，使得下游的hrpV的表現量在突變株及互補株中又比野生株高，而HrpV具有負調控的功能，使得在hrpG的突變株中降低了其它基因的表現量，但是在互補株中這些所測試的基因又可恢復與野生株相同的表現量，因此我們推測HrpG可能是經由調控HrpV，或是其下游的相關調控蛋白，藉此對hrp/hrc基因組的表現作微調控；另外，HrpG也可能是以作為HrpV蛋白之chaperone形式來影響HrpV在胞內的量，藉此來調節HrpV之負調控功能。 Pseudomonas syringae pv. syringae 61 possesses a 25 kb hrp/hrc cluster, which encodes 28 proteins responsible for eliciting the hypersensitive response (HR) in its non-host plants and causing diseases on the host plants. The products of the hrp genes are required for the Harpin...

Research paper thumbnail of 植物病原菌Pseudomonas syringae之HrpG、HrpV及鞭毛蛋白於致病性研究

Pseudomonas syringae為革蘭氏陰性菌，具單極生鞭毛，是重要的植物病原細菌，已發現在許多經濟作物上造成嚴重病害，依據危害寄主及病原性的不同，可被分成至少有50多種病原型 (pat... more Pseudomonas syringae為革蘭氏陰性菌，具單極生鞭毛，是重要的植物病原細菌，已發現在許多經濟作物上造成嚴重病害，依據危害寄主及病原性的不同，可被分成至少有50多種病原型 (pathovar)。而其致病機制主要受控於病原島嶼(Hrp Pai)，此病原島嶼的核心區域(core region)為組成第三型分泌系統(TTSS)的hrp/hrc基因組。在菜豆細菌性斑點病菌(P. syringae pv. syringae 61; Pss61)中含有一段約25 kb之hrp/hrc基因組可轉錄28個蛋白質。位在hrpC operon中的第二個基因hrpG可轉譯出15.4 kDa的HrpG蛋白，本研究利用含promoter的nptII基因構築hrpG非極性突變株Pss61-N674，以及不含promoter的nptII基因構築另一hrpG突變株Pss61-N826；突變株Pss61-N674的nptII promoter造成下游的負調控蛋白HrpV的大量表現，抑制了其它hrp/hrc基因表現，且在菸草上引起較慢的過敏性反應，相反地突變株Pss61-N826卻與野生株相似。以載體表現HrpG蛋白於大量表現HrpV的菌體中，則可恢復hrp/hrc基因表現及正常的過敏性反應，又從北方轉漬法中，顯示HrpG對HrpV的作用在蛋白質層次而非影響其轉錄。進一步，利用yeast two-hybrid、pull down試驗、遠西方轉漬法更發現到HrpG與HrpV無論在in vitro或是in vivo下都有交互作用存在，且經由3D-pssm分析HrpG的3D結構及根據其蛋白質特性，推測HrpG為第三型分泌系統之chaperone蛋白，然而，經由分泌試驗證實HrpV為胞內蛋白。因此，我們推測chaperone-like蛋白HrpG是HrpV的anti-repressor，除了幫助效力蛋白(effector)分泌外，亦參與調控hrp/hrc基因的表現，使病原菌能在適當的環境表現TTSS。 另外，在楊桃細菌性斑點病菌(P. syringae pv. averrhoi; Pav)中發現部分菌株沒有游動性，仍能引起過敏性反應，而部分菌株具有游動性卻引起較弱的過敏性反應。針對鞭毛於致病性的研究，我們利用高度保留性區域設計引子對，從Pav選殖出鞭毛合成相關基因組及醣基化島嶼(glyco...

Research paper thumbnail of A chaperone-like HrpG protein acts as a suppressor of HrpV in regulation of the Pseudomonas syringae pv. syringae type III secretion system

Molecular Microbiology, 2005

The cloned hrp / hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins... more The cloned hrp / hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins, and many of those are assembled into a type III secretion system (TTSS) that is responsible for eliciting the hypersensitive response (HR) in non-host plants and causing diseases on host plants (Huang et al ., 1995). hrpG , the second gene in the hrpC operon, encodes a 15.4 kDa cytoplasmic protein whose predicted structure is similar to SicP (E-value: 0.19), a TTSS chaperone of Salmonella typhimurium. Two non-polar hrpG mutants, Pss61-N826 and Pss61-N674, were produced to investigate the biological function of hrpG gene. Pss61-N826, generated by replacing the coding sequence of hrpG with an nptII gene lacking both the promoter and the terminator, was found to be capable of eliciting the wild-type HR; whereas Pss61-N674 generated by replacement of a terminatorless nptII gene in the hrpG coding sequence showed the delayed HR phenotype. Northern and Western blotting analyses showed that the expression of hrpZ , hrcJ and hrcQb genes residing on two different operons in Pss61-N674 was reduced due to the nptII promoterdriven constitutive expression of hrpV that codes for a negative regulator. Interestingly, a plasmid-borne hrpG can derepress the hrp expression in Pss61-N674 and in Pss61 overexpressing HrpV without decreasing the hrpV transcript. Moreover, results of yeast two-hybrid assay, pull-down assay and far Western analysis show that HrpG and HrpV interact with each other in vivo and in vitro. Additionally, HrpV interacts with a positive regulator HrpS according to analysis of a yeast two-hybrid system. Based on the results presented in this study, we propose that HrpG acts as a suppressor of the negative regulator HrpV mediated via protein-protein interaction, leading to modulation of hrp / hrc expression subsequently freeing HrpS to promote the activation of other downstream hrp / hrc genes.

Research paper thumbnail of Plant innate immunity induced by flagellin suppresses the hypersensitive response in non-host plants elicited by Pseudomonas syringae pv. averrhoi

PloS one, 2012

A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on c... more A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type ...

Research paper thumbnail of Exploring the Functions of Proteins Secreted by the Hrp Type III Secretion System of Pseudomonas syringae

P. syringae pathogenesis is dependent on the Hrp type III secretion system (T3SS), which injects ... more P. syringae pathogenesis is dependent on the Hrp type III secretion system (T3SS), which injects effector (Avr/Hop) proteins into plant cells. To better understand how the T3SS functions in pathogenesis, we are exploring the delivery and activity of these proteins. The model strain P. syringae pv. tomato DC3000 appears to strongly express and inject approximately 30 effectors into plant cells.

Research paper thumbnail of Effects of galU Mutation on Pseudomonas syringae –Plant Interactions

Molecular Plant-Microbe Interactions, 2010

Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role... more 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 galU mutant had reduced biofilm formation ability, was nonmotile, 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 pathogen-associated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMP-triggered immunity, which severely compromised the ability of the Psy61 galU mutant to survive in planta. Our results demonstrated that the complete LPS prot...

Research paper thumbnail of Deletions in the Repertoire of Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap among Effectors

PLoS Pathogens, 2009

The c-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III sec... more The c-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/ HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors.

Research paper thumbnail of RssAB Signaling Coordinates Early Development of Surface Multicellularity in Serratia marcescens

PLoS ONE, 2011

Bacteria can coordinate several multicellular behaviors in response to environmental changes. Amo... more Bacteria can coordinate several multicellular behaviors in response to environmental changes. Among these, swarming and biofilm formation have attracted significant attention for their correlation with bacterial pathogenicity. However, little is known about when and where the signaling occurs to trigger either swarming or biofilm formation. We have previously identified an RssAB two-component system involved in the regulation of swarming motility and biofilm formation in Serratia marcescens. Here we monitored the RssAB signaling status within single cells by tracing the location of the translational fusion protein EGFP-RssB following development of swarming or biofilm formation. RssAB signaling is specifically activated before surface migration in swarming development and during the early stage of biofilm formation. The activation results in the release of RssB from its cognate inner membrane sensor kinase, RssA, to the cytoplasm where the downstream gene promoters are located. Such dynamic localization of RssB requires phosphorylation of this regulator. By revealing the temporal activation of RssAB signaling following development of surface multicellular behavior, our findings contribute to an improved understanding of how bacteria coordinate their lifestyle on a surface.

Research paper thumbnail of Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the Prenylated Isoflavone, Osajin

PLoS ONE, 2011

Osajin is a prenylated isoflavone showing antitumor activity in different tumor cell lines. The u... more Osajin is a prenylated isoflavone showing antitumor activity in different tumor cell lines. The underlying mechanism of osajin-induced cancer cell death is not clearly understood. In the present study, the mechanisms of osajin-induced cell death of human nasopharyngeal carcinoma (NPC) cells were explored. Osajin was found to significantly induce apoptosis of NPC cells in a dose-and time-dependent manner. Multiple molecular effects were observed during osajin treatment including a significant loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, enhanced expression of Fas ligand (FasL), suppression of glucose-regulated protein 78 kDa (GRP78), and activation of caspases-9,-8,-4 and-3. In addition, up-regulation of proapoptotic Bax protein and down-regulation of antiapoptotic Bcl-2 protein were also observed. Taken together, osajin induces apoptosis in human NPC cells through multiple apoptotic pathways, including the extrinsic death receptor pathway, and intrinsic pathways relying on mitochondria and endoplasmic reticulum stress. Thus, osajin could be developed as a new effective and chemopreventive compound for human NPC.

Research paper thumbnail of Resveratrol induces apoptosis of human nasopharyngeal carcinoma cells via activation of multiple apoptotic pathways

Journal of Cellular Physiology, 2010

Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been repo... more Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been reported to trigger a variety of cancer cell types to apoptosis. Whether resveratrol shows any activity on human nasopharyngeal carcinoma (NPC) cells remained to be determined. The aim of this study was to investigate the effect and mechanism of resveratrol on human NPC cells. Treatment of resveratrol resulted in significant decrease in cell viability of NPC cell lines in a dose- and time-dependent manner. A dose-dependent apoptotic cell death was also measured by flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled resveratrol treatment resulted in a significant loss of mitochondrial transmembrane potential, release of cytochrome c, enhanced expression of Fas ligand (FasL), and suppression of glucose-regulated protein 78 kDa (GRP78). These were followed by activation of caspases-9, -8, -4, and -3, subsequently leading to DNA fragmentation and cell apoptosis. Furthermore, up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2 protein were also observed. Taken together, resveratrol induces apoptosis in human NPC cells through regulation of multiple apoptotic pathways, including death receptor, mitochondria, and endoplasmic reticulum (ER) stress. Resveratrol can be developed as an effective compound for human NPC treatment.

Research paper thumbnail of RssAB-FlhDC-ShlBA as a Major Pathogenesis Pathway in Serratia marcescens

Infection and Immunity, 2010

Serratia marcescens has long been recognized as an important opportunistic pathogen, but the unde... more Serratia marcescens has long been recognized as an important opportunistic pathogen, but the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system and its downstream elements, FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under the positive control of FlhDC, which is repressed by RssAB signaling. At 37°C, functional RssAB inhibits swarming, represses hemolysin production, and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. Hemolysin production is essential in this conte...

Research paper thumbnail of A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The Plant Journal, 2007

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Ar... more The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant antieffector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.

Research paper thumbnail of Characterization of hrpG gene from Pseudomonas syringae pv. syringae

菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin... more 菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin過敏原即利用此基因組產物所組成的第三型分泌系統被分泌到胞外。本研究主要在探討hrpC operon中的HrpG蛋白(15.4 kDa)在與植物的交互作用中所扮演的角色。利用不具有terminator的nptⅡ基因構築非極性突變的方法，證實hrpG突變株會減弱菸草的過敏性反應，利用西方點墨法得知此突變株仍可分泌Harpin蛋白，但Harpin的量減少很多；進一步將hrpG接上一個能夠以商業化的單株抗體(M2)偵測的FLAG序列，構築於廣宿主載體上，送入hrpG的突變株中作為互補株，此互補株能恢復和野生株相同的表現型，再利用細胞成份分離法以及西方點墨法可以得知HrpG蛋白質主要位於細胞質內。另外，經由北方點墨法及西方點墨法分析，結果顯示hrpG突變株的hrpZ、hrcJ、hrcQb表現量都明顯地比野生株及互補株低；然而因為構築非極性突變時所插入之nptII基因內含有較強的nptII 基因的promoter，使得下游的hrpV的表現量在突變株及互補株中又比野生株高，而HrpV具有負調控的功能，使得在hrpG的突變株中降低了其它基因的表現量，但是在互補株中這些所測試的基因又可恢復與野生株相同的表現量，因此我們推測HrpG可能是經由調控HrpV，或是其下游的相關調控蛋白，藉此對hrp/hrc基因組的表現作微調控；另外，HrpG也可能是以作為HrpV蛋白之chaperone形式來影響HrpV在胞內的量，藉此來調節HrpV之負調控功能。Pseudomonas syringae pv. syringae 61 possesses a 25 kb hrp/hrc cluster, which encodes 28 proteins responsible for eliciting the hypersensitive response (HR) in its non-host plants and causing diseases on the host plants. The products of the hrp genes are required for the Harpinp...

Research paper thumbnail of The RssAB-FlhDC-ShlBA as a Dominant Pathogenesis Pathway in Serratia marcescens

The RssAB-FlhDC-ShlBA as a Dominant Pathogenesis Pathway in Serratia marcescens Chuan-Sheng Lin, ... more The RssAB-FlhDC-ShlBA as a Dominant Pathogenesis Pathway in Serratia marcescens Chuan-Sheng Lin, Jim-Tong Horng, Chun-Hung Yang, Yu-Huan Tsai, Lin-Hui Su, Chia-Fong Wei, Chang-Chieh Chen, Shang-Chen Hsieh, Chia-Chen Lu, and Hsin-Chih Lai 1 Department of Medical Biotechnology and Laboratory Science, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. 2 Department of Biochemistry and Molecular Biology, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. 3 Department of Clinical Pathology, Chang Gung Memorial Hospital, 5 Fuxing Street, Kweishan, Taoyuan, 333 Taiwan, Republic of China. 4 Research Center for Pathogenic Bacteria, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. * Corresponding author † Chuan-Sheng Lin and Jim-Tong Horng contributed equally to this work. CORRESPONDENCE Hsin-Chih Lai: Department of Medical Biotechnology and Laboratory Science, ...

Research paper thumbnail of Cross-talk between bacterial two-component systems drives stepwise regulation of flagellar biosynthesis in swarming development

Biochemical and biophysical research communications, Jul 15, 2017

Swarming motility is a mode of bacterial movement over a solid surface driven by rotating flagell... more Swarming motility is a mode of bacterial movement over a solid surface driven by rotating flagella in a coordinated manner. Bacteria can use two-component system (TCS), which typically comprises a sensor kinase and a specific cognate response regulator, to properly react to environmental changes. We previously showed that the TCS RssAB suppresses flagellar biosynthesis master regulator flhDC specifically in swarming lag phase to control surface migration timing without affecting expansion rate in Serratia marcescens swarming development. Here we demonstrate that the TCS QseBC, which has been found in several human pathogens involved in flagellar and virulence regulation, has cross-talk with RssAB. We demonstrate that the phosphorylated QseB repressed flhDC expression, reducing swarming migration rate with modest effect on migration initiation. Unexpectedly, the QseC can dephosphorylate non-cognate response regulator RssB. Deletion of qseC prolonged RssAB signaling, reduced flhDC exp...

Research paper thumbnail of Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida

Frontiers in microbiology, 2016

Synergistic effects between the same class of antibiotics are rarely reported. Our previous study... more Synergistic effects between the same class of antibiotics are rarely reported. Our previous study found synergistic-like interaction between florfenicol (FFC) and thiamphenicol (TAP) against Staphylococcus aureus. Here, the enhanced antimicrobial activity was evaluated in 97 clinical isolates of both Gram-negative and Gram-positive bacteria. Susceptible strains were initially identified by checkerboard microdilution assay (fractional inhibitory concentration index [FICI] ≤ 0.625), followed by confirmation of synergism using the time-kill methodology (≥2 log10 CFU/ml reduction). In all, 43% of Pasteurella multocida tested were susceptible to the enhanced bactericidal effect. In chicken fowl cholera models, FFC and TAP combination at much lower dosage that is correspondent to their MIC deduction provided maximum protection in vivo. Furthermore, synergistic combination of FFC with oxytetracycline (OTC) against Pseudomonas aeruginosa in vitro was also demonstrated. Based on the enhanced...

Research paper thumbnail of The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate T3SS function and t3ss expression in Pseudomonas syringae pv. averrhoi

Molecular plant pathology, Jan 6, 2015

To ensure the optimal infectivity upon contact with host cells, pathogenic Pseudomonas syringae h... more To ensure the optimal infectivity upon contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream to hrpG, encodes a T3SS-dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on eliciting disease symptom in its host and hypersensitive response in the nonhost plant, and secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild type results in delayed HR and reduced t3ss expression. The results of protein-protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial ...

Research paper thumbnail of Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis

Journal of the Science of Food and Agriculture, 2015

The potential presence of undeclared animal by-products in pet foods is not subject to routine ex... more The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols. © 2015 Society of Chemical Industry.

Research paper thumbnail of 菜豆細菌性斑點病菌致病基因hrpG之特性分析

菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin... more 菜豆細菌性斑點病菌含有一段25 kb的hrp/hrc/hrmA基因組，可轉錄28個蛋白質，使其於非寄主植物或抗性寄主中引發過敏性反應，以及在寄主植物中造成病徵。其中由hrpZ所轉錄的Harpin過敏原即利用此基因組產物所組成的第三型分泌系統被分泌到胞外。本研究主要在探討hrpC operon中的HrpG蛋白(15.4 kDa)在與植物的交互作用中所扮演的角色。利用不具有terminator的nptⅡ基因構築非極性突變的方法，證實hrpG突變株會減弱菸草的過敏性反應，利用西方點墨法得知此突變株仍可分泌Harpin蛋白，但Harpin的量減少很多；進一步將hrpG接上一個能夠以商業化的單株抗體(M2)偵測的FLAG序列，構築於廣宿主載體上，送入hrpG的突變株中作為互補株，此互補株能恢復和野生株相同的表現型，再利用細胞成份分離法以及西方點墨法可以得知HrpG蛋白質主要位於細胞質內。另外，經由北方點墨法及西方點墨法分析，結果顯示hrpG突變株的hrpZ、hrcJ、hrcQb表現量都明顯地比野生株及互補株低；然而因為構築非極性突變時所插入之nptII基因內含有較強的nptII 基因的promoter，使得下游的hrpV的表現量在突變株及互補株中又比野生株高，而HrpV具有負調控的功能，使得在hrpG的突變株中降低了其它基因的表現量，但是在互補株中這些所測試的基因又可恢復與野生株相同的表現量，因此我們推測HrpG可能是經由調控HrpV，或是其下游的相關調控蛋白，藉此對hrp/hrc基因組的表現作微調控；另外，HrpG也可能是以作為HrpV蛋白之chaperone形式來影響HrpV在胞內的量，藉此來調節HrpV之負調控功能。 Pseudomonas syringae pv. syringae 61 possesses a 25 kb hrp/hrc cluster, which encodes 28 proteins responsible for eliciting the hypersensitive response (HR) in its non-host plants and causing diseases on the host plants. The products of the hrp genes are required for the Harpin...

Research paper thumbnail of 植物病原菌Pseudomonas syringae之HrpG、HrpV及鞭毛蛋白於致病性研究

Pseudomonas syringae為革蘭氏陰性菌，具單極生鞭毛，是重要的植物病原細菌，已發現在許多經濟作物上造成嚴重病害，依據危害寄主及病原性的不同，可被分成至少有50多種病原型 (pat... more Pseudomonas syringae為革蘭氏陰性菌，具單極生鞭毛，是重要的植物病原細菌，已發現在許多經濟作物上造成嚴重病害，依據危害寄主及病原性的不同，可被分成至少有50多種病原型 (pathovar)。而其致病機制主要受控於病原島嶼(Hrp Pai)，此病原島嶼的核心區域(core region)為組成第三型分泌系統(TTSS)的hrp/hrc基因組。在菜豆細菌性斑點病菌(P. syringae pv. syringae 61; Pss61)中含有一段約25 kb之hrp/hrc基因組可轉錄28個蛋白質。位在hrpC operon中的第二個基因hrpG可轉譯出15.4 kDa的HrpG蛋白，本研究利用含promoter的nptII基因構築hrpG非極性突變株Pss61-N674，以及不含promoter的nptII基因構築另一hrpG突變株Pss61-N826；突變株Pss61-N674的nptII promoter造成下游的負調控蛋白HrpV的大量表現，抑制了其它hrp/hrc基因表現，且在菸草上引起較慢的過敏性反應，相反地突變株Pss61-N826卻與野生株相似。以載體表現HrpG蛋白於大量表現HrpV的菌體中，則可恢復hrp/hrc基因表現及正常的過敏性反應，又從北方轉漬法中，顯示HrpG對HrpV的作用在蛋白質層次而非影響其轉錄。進一步，利用yeast two-hybrid、pull down試驗、遠西方轉漬法更發現到HrpG與HrpV無論在in vitro或是in vivo下都有交互作用存在，且經由3D-pssm分析HrpG的3D結構及根據其蛋白質特性，推測HrpG為第三型分泌系統之chaperone蛋白，然而，經由分泌試驗證實HrpV為胞內蛋白。因此，我們推測chaperone-like蛋白HrpG是HrpV的anti-repressor，除了幫助效力蛋白(effector)分泌外，亦參與調控hrp/hrc基因的表現，使病原菌能在適當的環境表現TTSS。 另外，在楊桃細菌性斑點病菌(P. syringae pv. averrhoi; Pav)中發現部分菌株沒有游動性，仍能引起過敏性反應，而部分菌株具有游動性卻引起較弱的過敏性反應。針對鞭毛於致病性的研究，我們利用高度保留性區域設計引子對，從Pav選殖出鞭毛合成相關基因組及醣基化島嶼(glyco...

Research paper thumbnail of A chaperone-like HrpG protein acts as a suppressor of HrpV in regulation of the Pseudomonas syringae pv. syringae type III secretion system

Molecular Microbiology, 2005

The cloned hrp / hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins... more The cloned hrp / hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins, and many of those are assembled into a type III secretion system (TTSS) that is responsible for eliciting the hypersensitive response (HR) in non-host plants and causing diseases on host plants (Huang et al ., 1995). hrpG , the second gene in the hrpC operon, encodes a 15.4 kDa cytoplasmic protein whose predicted structure is similar to SicP (E-value: 0.19), a TTSS chaperone of Salmonella typhimurium. Two non-polar hrpG mutants, Pss61-N826 and Pss61-N674, were produced to investigate the biological function of hrpG gene. Pss61-N826, generated by replacing the coding sequence of hrpG with an nptII gene lacking both the promoter and the terminator, was found to be capable of eliciting the wild-type HR; whereas Pss61-N674 generated by replacement of a terminatorless nptII gene in the hrpG coding sequence showed the delayed HR phenotype. Northern and Western blotting analyses showed that the expression of hrpZ , hrcJ and hrcQb genes residing on two different operons in Pss61-N674 was reduced due to the nptII promoterdriven constitutive expression of hrpV that codes for a negative regulator. Interestingly, a plasmid-borne hrpG can derepress the hrp expression in Pss61-N674 and in Pss61 overexpressing HrpV without decreasing the hrpV transcript. Moreover, results of yeast two-hybrid assay, pull-down assay and far Western analysis show that HrpG and HrpV interact with each other in vivo and in vitro. Additionally, HrpV interacts with a positive regulator HrpS according to analysis of a yeast two-hybrid system. Based on the results presented in this study, we propose that HrpG acts as a suppressor of the negative regulator HrpV mediated via protein-protein interaction, leading to modulation of hrp / hrc expression subsequently freeing HrpS to promote the activation of other downstream hrp / hrc genes.

Research paper thumbnail of Plant innate immunity induced by flagellin suppresses the hypersensitive response in non-host plants elicited by Pseudomonas syringae pv. averrhoi

PloS one, 2012

A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on c... more A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type ...

Research paper thumbnail of Exploring the Functions of Proteins Secreted by the Hrp Type III Secretion System of Pseudomonas syringae

P. syringae pathogenesis is dependent on the Hrp type III secretion system (T3SS), which injects ... more P. syringae pathogenesis is dependent on the Hrp type III secretion system (T3SS), which injects effector (Avr/Hop) proteins into plant cells. To better understand how the T3SS functions in pathogenesis, we are exploring the delivery and activity of these proteins. The model strain P. syringae pv. tomato DC3000 appears to strongly express and inject approximately 30 effectors into plant cells.

Research paper thumbnail of Effects of galU Mutation on Pseudomonas syringae –Plant Interactions

Molecular Plant-Microbe Interactions, 2010

Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role... more 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 galU mutant had reduced biofilm formation ability, was nonmotile, 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 pathogen-associated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMP-triggered immunity, which severely compromised the ability of the Psy61 galU mutant to survive in planta. Our results demonstrated that the complete LPS prot...

Research paper thumbnail of Deletions in the Repertoire of Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap among Effectors

PLoS Pathogens, 2009

The c-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III sec... more The c-proteobacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the type III secretion system to inject ca. 28 Avr/Hop effector proteins into plants, which enables the bacterium to grow from low inoculum levels to produce bacterial speck symptoms in tomato, Arabidopsis thaliana, and (when lacking hopQ1-1) Nicotiana benthamiana. The effectors are collectively essential but individually dispensable for the ability of the bacteria to defeat defenses, grow, and produce symptoms in plants. Eighteen of the effector genes are clustered in six genomic islands/islets. Combinatorial deletions involving these clusters and two of the remaining effector genes revealed a redundancy-based structure in the effector repertoire, such that some deletions diminished growth in N. benthamiana only in combination with other deletions. Much of the ability of DC3000 to grow in N. benthamiana was found to be due to five effectors in two redundant-effector groups (REGs), which appear to separately target two high-level processes in plant defense: perception of external pathogen signals (AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE, HopM1, HopR1). Further support for the membership of HopR1 in the same REG as AvrE was gained through bioinformatic analysis, revealing the existence of an AvrE/DspA/E/ HopR effector superfamily, which has representatives in virtually all groups of proteobacterial plant pathogens that deploy type III effectors.

Research paper thumbnail of RssAB Signaling Coordinates Early Development of Surface Multicellularity in Serratia marcescens

PLoS ONE, 2011

Bacteria can coordinate several multicellular behaviors in response to environmental changes. Amo... more Bacteria can coordinate several multicellular behaviors in response to environmental changes. Among these, swarming and biofilm formation have attracted significant attention for their correlation with bacterial pathogenicity. However, little is known about when and where the signaling occurs to trigger either swarming or biofilm formation. We have previously identified an RssAB two-component system involved in the regulation of swarming motility and biofilm formation in Serratia marcescens. Here we monitored the RssAB signaling status within single cells by tracing the location of the translational fusion protein EGFP-RssB following development of swarming or biofilm formation. RssAB signaling is specifically activated before surface migration in swarming development and during the early stage of biofilm formation. The activation results in the release of RssB from its cognate inner membrane sensor kinase, RssA, to the cytoplasm where the downstream gene promoters are located. Such dynamic localization of RssB requires phosphorylation of this regulator. By revealing the temporal activation of RssAB signaling following development of surface multicellular behavior, our findings contribute to an improved understanding of how bacteria coordinate their lifestyle on a surface.

Research paper thumbnail of Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the Prenylated Isoflavone, Osajin

PLoS ONE, 2011

Osajin is a prenylated isoflavone showing antitumor activity in different tumor cell lines. The u... more Osajin is a prenylated isoflavone showing antitumor activity in different tumor cell lines. The underlying mechanism of osajin-induced cancer cell death is not clearly understood. In the present study, the mechanisms of osajin-induced cell death of human nasopharyngeal carcinoma (NPC) cells were explored. Osajin was found to significantly induce apoptosis of NPC cells in a dose-and time-dependent manner. Multiple molecular effects were observed during osajin treatment including a significant loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, enhanced expression of Fas ligand (FasL), suppression of glucose-regulated protein 78 kDa (GRP78), and activation of caspases-9,-8,-4 and-3. In addition, up-regulation of proapoptotic Bax protein and down-regulation of antiapoptotic Bcl-2 protein were also observed. Taken together, osajin induces apoptosis in human NPC cells through multiple apoptotic pathways, including the extrinsic death receptor pathway, and intrinsic pathways relying on mitochondria and endoplasmic reticulum stress. Thus, osajin could be developed as a new effective and chemopreventive compound for human NPC.

Research paper thumbnail of Resveratrol induces apoptosis of human nasopharyngeal carcinoma cells via activation of multiple apoptotic pathways

Journal of Cellular Physiology, 2010

Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been repo... more Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been reported to trigger a variety of cancer cell types to apoptosis. Whether resveratrol shows any activity on human nasopharyngeal carcinoma (NPC) cells remained to be determined. The aim of this study was to investigate the effect and mechanism of resveratrol on human NPC cells. Treatment of resveratrol resulted in significant decrease in cell viability of NPC cell lines in a dose- and time-dependent manner. A dose-dependent apoptotic cell death was also measured by flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled resveratrol treatment resulted in a significant loss of mitochondrial transmembrane potential, release of cytochrome c, enhanced expression of Fas ligand (FasL), and suppression of glucose-regulated protein 78 kDa (GRP78). These were followed by activation of caspases-9, -8, -4, and -3, subsequently leading to DNA fragmentation and cell apoptosis. Furthermore, up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2 protein were also observed. Taken together, resveratrol induces apoptosis in human NPC cells through regulation of multiple apoptotic pathways, including death receptor, mitochondria, and endoplasmic reticulum (ER) stress. Resveratrol can be developed as an effective compound for human NPC treatment.

Research paper thumbnail of RssAB-FlhDC-ShlBA as a Major Pathogenesis Pathway in Serratia marcescens

Infection and Immunity, 2010

Serratia marcescens has long been recognized as an important opportunistic pathogen, but the unde... more Serratia marcescens has long been recognized as an important opportunistic pathogen, but the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system and its downstream elements, FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under the positive control of FlhDC, which is repressed by RssAB signaling. At 37°C, functional RssAB inhibits swarming, represses hemolysin production, and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. Hemolysin production is essential in this conte...

Research paper thumbnail of A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The Plant Journal, 2007

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Ar... more The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant antieffector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.