1.Detection of Yersinia Enterocolitica Bacteriophage PhiYe-F10 Lysis Spectrum and Analysis of the Relationship between Lysis Ability and Virulence Gene of Yersinia Enterocolitica.
Tao ZHA ; Junrong LIANG ; Yuchun XIAO ; Huaiqi JING
Chinese Journal of Virology 2016;32(2):185-189
To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.
Bacterial Proteins
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genetics
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metabolism
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Bacteriophages
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genetics
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isolation & purification
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physiology
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Host Specificity
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Virulence Factors
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genetics
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metabolism
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Yersinia enterocolitica
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genetics
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metabolism
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virology
2.Study on pathogenicity of putative virulence gene of Enterococcus faecium.
Lixian WU ; Wenxiang HUANG ; Guofu WANG ; Xiaoping SUN
Journal of Biomedical Engineering 2009;26(3):601-605
The presence of hyl gene in 364 PFGE clones of Enterococcus faecium was detected by colony hybridization under conditions of high stringency. The isogenic hyl-deficient mutant (* hyl) was constructed with suicide pTX4577 and screened by allelic replacement. Moreover, an in vitro study was made on the effect of hyl gene detection on the growth ability of hylgene detection on the mutant, and an in vivo study was made on the decrease of virulence in the mouse peritonitis model. The results showed, in the clinical isolates, the positive percentage of hyl gene was 32.8%, which was significantly higher than that (5.3%) in the non-clinical isolates. The * hyl was selected by kanamycin and identified by PCR, pulsed field gel electrophoresis (PFGE) and Southern blot. The experimental evidence indicated that the growth ability of * hyl was remarkably reduced in comparison with that of the wild-type strain. The percentage survival of mice in TX2466 groups was 0, while that of * hyl groups was 50% at the same inoculum in mouse peritonitis. The differences were significant. These data suggest that hyl gene in specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Being important in the pathogenesis, hyl gene is probably a major virulence factor of Enterococcus faecium.
Animals
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Enterococcus faecium
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genetics
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pathogenicity
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Genes, Bacterial
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Gram-Positive Bacterial Infections
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microbiology
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Hyaluronoglucosaminidase
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genetics
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Male
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Mice
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Mice, Inbred ICR
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Mutation
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Peritonitis
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microbiology
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Virulence
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Virulence Factors
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genetics
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metabolism
3.Cloning the lvgA gene of Legionella pneumophila and detecting its expression in Escherichia coli.
Mingjie LIU ; Jianping CHEN ; Tao LIAO ; Dianxiang LU ; Xian CHEN
Journal of Biomedical Engineering 2006;23(3):605-608
In order to clone lvgA gene (Legionella virulence gene) of Legionella pneumophila and detect its expression in prokaryotic cell, we amplified the lvgA gene from the total cell DNA of Legionella pneumophila with PCR,and then inserted it into the coloning vector pUC18. The recombinant plasmid pUlvgA was obtained. After the recombinant plasmid pUlvgA was identified with restriction enzyme analysis, polymerase chain reaction and nucleotide sequencing analysis, the lvgA gene was subcloned into the prokaryotic expression vector pGEX-4T-1. The prokaryotic expression recombinant plasmid pGlvgA was constructed. After IPTG induction, the E. coli JM109 containing the recombinant plasmid pGlvgA expressed fusion protein. The expression of lvgA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The results indicated that the lvgA gene of 627 bp long was amplified, the recombinant plasmids pUlvgA and pGlvgA were constructed successfully, and the GST-LvgA fusion protein of approximately 36 KDa in size was expressed in prokaryotic cell efficiently as expected.
Bacterial Proteins
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genetics
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metabolism
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Cloning, Molecular
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Cytoplasm
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metabolism
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Escherichia coli
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metabolism
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Legionella pneumophila
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genetics
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Prokaryotic Cells
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metabolism
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Recombinant Proteins
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biosynthesis
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genetics
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Virulence Factors
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biosynthesis
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genetics
4.Study on the distribution of Yersinia enterocolitica in Nantong, Jinagsu Province.
Ling GU ; Hua WANG ; Feng-cai ZHU ; Zhi-yang SHI ; Xiao-yan ZENG ; Zhao-ying TAN ; Yu-chun XIAO ; Hai-yan QIU ; Xin WANG ; Dong JIN ; Zhi-gang CUI ; Bing WANG ; Biao KAN ; Xin-sheng WANG ; Huai-qi JING ; Jian-guo XU
Chinese Journal of Epidemiology 2005;26(10):786-789
OBJECTIVETo study the distribution of Yersinia enterocolitica and its virulence factors in Nantong, Jiangsu.
METHODSYersinia strains were isolated from livestock and poultry. Conventional PCR was used to detect the virulence factors of all strains and strain 0:8 was analyzed by pulsed-field gel electrophoresis(PFGE).
RESULTSThe combined isolation rate of Yersinia enterocolitica from livestock and poultry was 31.06% and the gene distribution characters were: 39.57% of them were ail-, ystA- , ystB-, yadA- , virF-; 60.43% were ail- , ystA- , ystB + , yadA- , virF- respectively. The two reference strains from America and Denmark showed similar electrophoresis patterns but were significantly different with O:8 strains isolated from China while the serotypes of Yersinia enterocolitica O:3 and O:9 which were the main epidemic strains in China, were not found in this area.
CONCLUSIONThe pathogenic Yersinia enterocolitis O:3 and O:9 were not found in Nantong,Jiangsu province.
Animals ; Animals, Domestic ; microbiology ; China ; Electrophoresis ; Poultry ; microbiology ; Virulence Factors ; genetics ; metabolism ; Yersinia enterocolitica ; genetics ; isolation & purification ; pathogenicity
5.Expression and characterization of the dermonecrotic toxin gene of Bordetella bronchiseptica.
Yun XUE ; Zhanqin ZHAO ; Jie PEI ; Chen WANG ; Ke DING ; Xiangchao CHENG
Chinese Journal of Biotechnology 2011;27(12):1722-1728
Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.
Animals
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Animals, Newborn
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Bordetella bronchiseptica
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metabolism
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Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Mice
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Neutralization Tests
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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immunology
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Transglutaminases
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biosynthesis
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genetics
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Virulence Factors, Bordetella
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biosynthesis
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genetics
6.Protective Effects of cis-2-Dodecenoic Acid in an Experimental Mouse Model of Vaginal Candidiasis.
Dong Liang YANG ; Yu Qian ZHANG ; Yan Ling HU ; Li Xing WENG ; Gui Sheng ZENG ; Lian Hui WANG
Biomedical and Environmental Sciences 2018;31(11):816-828
OBJECTIVE:
To evaluate the efficacy of cis-2-dodecenoic acid (BDSF) in the treatment and prevention of vaginal candidiasis in vivo.
METHODS:
The activities of different concentrations of BDSF against the virulence factors of Candida albicans (C. albicans) were determined in vitro. An experimental mouse model of Candida vaginitis was treated with 250 μmol/L BDSF. Treatment efficiency was evaluated in accordance with vaginal fungal burden and inflammation symptoms.
RESULTS:
In vitro experiments indicated that BDSF attenuated the adhesion and damage of C. albicans to epithelial cells by decreasing phospholipase secretion and blocking filament formation. Treatment with 30 μmol/L BDSF reduced the adhesion and damage of C. albicans to epithelial cells by 36.9% and 42.3%, respectively. Treatment with 200 μmol/L BDSF completely inhibited phospholipase activity. In vivo mouse experiments demonstrated that BDSF could effectively eliminate vaginal infection and relieve inflammatory symptoms. Four days of treatment with 250 μmol/L BDSF reduced vaginal fungal loads by 6-fold and depressed inflammation. Moreover, BDSF treatment decreased the expression levels of the inflammatory chemokine-associated genes MCP-1 and IGFBP3 by 2.5- and 2-fold, respectively.
CONCLUSION
BDSF is a novel alternative drug that can efficiently control vaginal candidiasis by inhibiting the virulence factors of C. albicans.
Animals
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Candida albicans
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drug effects
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metabolism
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pathogenicity
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physiology
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Candidiasis, Vulvovaginal
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drug therapy
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genetics
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immunology
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microbiology
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Chemokine CCL2
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genetics
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immunology
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Disease Models, Animal
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Fatty Acids, Monounsaturated
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administration & dosage
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Female
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Fungal Proteins
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genetics
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metabolism
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Humans
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Insulin-Like Growth Factor Binding Protein 3
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genetics
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immunology
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Mice
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Virulence
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drug effects
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Virulence Factors
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genetics
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metabolism
7.Molecular recognition code between pathogenic bacterial TAL-effectors and host target genes: a review.
Yanqiang LI ; Chunlian WANG ; Kaijun ZHAO
Chinese Journal of Biotechnology 2011;27(8):1132-1141
As the pathogenic bacterial virulence and avirulence factors, transcription activator like (TAL) effectors of Xanthomonas can resulted in the host diseases or resistance responses. TAL effectors can specifically bind the target DNA of host plant with a novel protein-DNA binding pattern in which two amino acids recognize one nucleotide. The complexities of TAL-DNA binding have the feasibility in use of gene therapy through homologous recombination and site-specific mutation. By using the molecular recognition code between TAL-effectors and host target genes, we can exploit both the susceptible and resistance genes; broad spectrum resistance induced by multiple TAL effectors could also be manipulated. Deeper insight in the area of protein-DNA binding mechanism will benefit the application in the biomedical engineering and agricultural engineering. This article reviews the findings and functions of TAL effectors, the binding specificity and recognition code between TAL-effectors and host target genes. The possible applications and future prospects of the molecular recognition code have been discussed.
Base Sequence
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DNA, Plant
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metabolism
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Genes, Plant
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Genetic Code
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genetics
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Host-Pathogen Interactions
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Molecular Sequence Data
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Plant Diseases
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genetics
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prevention & control
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Transcriptional Activation
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Virulence Factors
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genetics
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metabolism
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Xanthomonas
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genetics
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pathogenicity
8.Inhibitory effects of butyl alcohol extract of Baitouweng decoction on virulence factors of Candida tropicalis.
Gui-ming YAN ; Meng-xiang ZHANG ; Dan XIA ; Ke-qiao LU ; Jing SHAO ; Tian-ming WANG ; Chang-zhong WANG
China Journal of Chinese Materia Medica 2015;40(12):2396-2402
OBJECTIVETo investigate the effects of butyl alcohol extract of baitouweng decoction (BAEB) on the fungal cell surface hydrophobicity (CSH), filamentation and biofilm formation of Candida tropicalis.
METHODGradual dilution method was used to determine the MIC. XTT assay was applied to determine the SMIC80. Time-Kill assay was employed to draw the Time-Kill curve. The water-hydrocarbon two-phase assay was used to measure the cell surface hydrophobicity. Scanning electron microscopy (SEM) was applied to observe the morphological changes of the biofilm. Confocal laser scanning microscopy (CLSM) was applied to determine the thickness of the biofilm. The quantification real-time PCR (qRT-PCR) was used to detect expression changes of releated genes (UME6, ALST3 and NRG1). result: The MICs of BAEB against C. tropicalis strains are determined as 64-128 mg x L(-1). The SMIC80 s of BAEB against the biofilm of Candida tropicalis strains are determined as 256-512 mg x L(-1). Time-Kill curve results indicate that BAEB has a promise fungicidal effect at 256 and 512 mg x L(-1). SEM results shows that 512 mg x L(-1) BAEB can inhibit the formation of C. tropicalis biofilm on Silicone catheter, and the morphology of biofilm is also affected by BAEB. The thickness of C. tropicalis biofilm is reduced by BAEB according to CLSM results. Furthermore, qRT-PCR results indicate that expression of UME6 and ALST3 are significantly down-regulated by BAEB 256,512 mg x L(-1), and NRG1 is not affected by BAEB.
CONCLUSIONBAEB inhibits effectively the CSH, filamentation and biofilm formation of VVC strains of C. tropicalis.
Antifungal Agents ; chemistry ; pharmacology ; Biofilms ; drug effects ; Candida tropicalis ; drug effects ; genetics ; physiology ; Candidiasis ; microbiology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Gene Expression Regulation, Fungal ; drug effects ; Humans ; Virulence Factors ; genetics ; metabolism
9.Studies of the expression, purification, renaturation and biologic activity of an anti-CEA immunotoxin.
Hui YANG ; Dan HE ; Kai CHAO ; Qing LIN ; Song YOU ; Hua-Liang HUANG
Chinese Journal of Biotechnology 2004;20(3):348-351
A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases
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biosynthesis
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genetics
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pharmacology
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Antibodies
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genetics
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metabolism
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pharmacology
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Antineoplastic Agents
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metabolism
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pharmacology
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Bacterial Toxins
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biosynthesis
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genetics
;
pharmacology
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Carcinoembryonic Antigen
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immunology
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Cloning, Molecular
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Escherichia coli
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genetics
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metabolism
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Exotoxins
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biosynthesis
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genetics
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pharmacology
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Humans
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Immunoglobulin Fragments
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biosynthesis
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genetics
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Immunotoxins
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genetics
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isolation & purification
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metabolism
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pharmacology
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Protein Renaturation
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Recombinant Fusion Proteins
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biosynthesis
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genetics
;
pharmacology
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Virulence Factors
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biosynthesis
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genetics
;
pharmacology
10.Pro-apoptotic effect on osteosarcoma SOSP-9607 cells by human recombinant caspase-6 fusion protein.
Ben-gen ZHOU ; Xiu-chun QIU ; Yan-ming XU ; Qing-yu FAN
Chinese Journal of Oncology 2010;32(7):497-500
OBJECTIVETo investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.
METHODSRecombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.
RESULTSThe tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.
CONCLUSIONImmunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.
ADP Ribose Transferases ; genetics ; Animals ; Apoptosis ; Bacterial Toxins ; genetics ; Bone Neoplasms ; metabolism ; pathology ; Caspase 6 ; genetics ; metabolism ; Cell Line, Tumor ; Exotoxins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Random Allocation ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; Virulence Factors ; genetics