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Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21,LipL32 and OmpL1 in Epidemic Leptospires

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 7+650-656页

作者： Xiang-Yan ZHANG~(1#) Yang YU~(2,3#) Ping HE~1 Yi-Xuan ZHANG~(2,3) Bao-Yu HU~1 Yang YANG~1 Yi-Xin NIE~4 Xiu-Gao JIANG~4 Guo-Ping ZHAO~(2,3) Xiao-Kui GUO~(1*) 1 Department of Medical Microbiology and Parasitology,shanghai Jiaotong University,shanghai 200025,china 2 Research center of Biotechnology,shanghai Institutes for Biological Sciences,chinese Academy of Sciences,shanghai 200031,china 3 chinese national human genome center at shanghai,zhangjiang high tech park,shanghai 201203,china 4 Institute for Infectious Disease Control and Prevention,chinese center for Disease Control and Prevention,Beijing 102206,china Department of Medical Microbiology and Parasitology Shanghai Jiaotong UniversityShanghai 200025 China Research center of Biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China chinese national human genome center at shanghai Zhangjiang High Tech ParkShanghai 201203 China Institute for Infectious Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206 China

Leptospiral outer membrane proteins (OMPs) are highly conserved in different species,andplay an essential role in the development of new immunoprotection and serodiagnosis *** genesencoding LipL21,LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospirainterrogans serovar lai strain Lai and expressed in *** comparison analysis revealed that the threegenes were highly conserved among distinct epidemic leptospires,including three major epidemic speciesLeptospira interrogans,Leptospira borgpetersenii and Leptospira weilii,in *** analysis wasfurther performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the *** immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that thesethree OMPs were conservatively expressed in pathogenic *** strains and other pathogenic ***,the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA)for serodiagnosis of leptospirosis was *** recombinant LipL32 and OmpL1 proteins showed ahigh degree of ELISA reactivity with sera from patients infected with *** strain Lai and otherpathogenic *** results may contribute to the identification of candidates for broad-rangevaccines and immunodiagnostic antigens in further research.

关键词： outer membrane protein expression comparative analysis epidemic leptospire

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Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai

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Acta Pharmacologica Sinica 2005年第4期26卷 453-461页

作者： Yi-xuan ZHANG~(2,3,4,5) Yan GENG(2,3,4,5) Bo BI~(2,4) Jian-yong HE~4 Chun-fu WU~4 Xiao-kui GUO~3 Guo-ping ZHAO~(2,4,6,7) 2 Research center of Biotechnology,shanghai Institutes for Biological Sciences,chinese Academy of Sciences,shanghai 200233,china 3 Department of Microbiology and Parasitology,shanghai Second Medical University,shanghai 200025,china 4 The School of Pharmaceutical Engineering,Shenyang Pharmaceutical University,Shenyang 110016,china 6 chinese national human genome center at shanghai,zhangjiang high tech park,shanghai 201203,china Research center of Biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Department of Microbiology and Parasitology Shanghai Second Medical University Shanghai China The School of Pharmaceutical Engineering Shenyang Pharmaceutical University Shenyang China chinese national human genome center at shanghai ZhangJiang High Tech Park Shanghai China

Aim:To identify and classify all potential hemolysin candidates of Leptospirainterrogans serogroup Icterohaemorrhagiae serovar ***:All of thepotential hemolysin encoding genes were characterized in *** geneswere cloned and expressed in Escherichia *** hemolytic activities of theexpressed proteins were assayed observing the hemolysis on sheep blood *** activities of the hemolysin candidates were measuredby thin-layer chromatography(TLC)and HPLC for *** and secretion of the hemolysins in L interrogans were studied byreverse transcription polymerase chain reaction,Western blot,and enzyme-linkedimmunosorbent *** and Conclusion:The hemolytic activities ofhemolysin candidates(LA0327,LA0378,LA1027,LA1029,LAI650,LA3050,LA3937,LA4004)from L interrogans strain Lai were *** were furtherdivided into two groups,sphingomyelinase hemolysins and non-sphingomyelinasehemolysins,based on their ability to hydrolyze *** of thesehemolysins were actually expressed in living L interrogans and some of themwere secreted into the *** study establishes an essential findcomplete basis for further studying the contribution of hemolysins to the patho-genesis of L interrogans.

关键词： hemolysin Leptospira interrogans sphingo myelinase

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Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 832-839页

作者： Ping HE, Xiang-Yan ZHANG, Xiao-Kui GUO*, Bao-Yu HU, Xiao-Tian HUANG, Yang YANG, and Guo-Ping ZHAO1,2* Department of Microbiology and Parasitology, shanghai Second Medical University, shanghai 200025, china 1 Research center of Biotechnology, Institutes for Biological Sciences, chinese Academy of Sciences, shanghai 200031, china 2chinese national human genome center at shanghai, zhangjiang high tech park, shanghai 201203, china Department of Microbiology and Parasitology Shanghai Second Medical UniversityShanghai 200025 China Research center of Biotechnology Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China chinese national human genome center at shanghaizhangjiang high tech park Shanghai 201203 China

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridiza- tion is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.

关键词： Leptospira interrogans serovar lai suppression subtractive hybridization (SSH) specific genes

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Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

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Cell Research 2004年第3期14卷 208-216页

作者： Yi Xuan ZHANG, Xiao Kui GUO, Chuan WU, Bo BI, Shuang Xi REN, Chun Fu WU, Guo Ping ZHAO Pharmaceutical Department, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, china. Research center of Biotechnology, shanghai Institutes for Biological Sciences, chinese Academy of Sciences, 500 Caobao Road, shanghai 200233, china. Department of Microbiology and Parasitology, shanghai Second Medical University, 280 Chongqingnan Road, shanghai 200025, china. chinese national human genome center at shanghai (CHGCS), 250 Bibo Road, zhangjiang high tech park, shanghai, 201203, china. Pharmaceutical Department Shenyang Pharmaceutical University 103 Wenhua Road Shenhe District Shenyang China Research center of Biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Department of Microbiology and Parasitology Shanghai Second Medical University Shanghai China chinese national human genome center at shanghai (CHGCS) 250 Bibo Road ZhangJiang High Tech Park Shanghai China

Comparative genomic analysis of the coding sequences （CDSs） of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequences and operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth, whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other characterized toxin-antitoxin systems of bacteria, i.e., mazEF[1], relBE[2] and chpIK[3]. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that although toxins were evolved from divergent sources and may or may not follow their speciation paths （as indicated by their 16s RNA sequences）, co-evolution with their antitoxins was obvious.

关键词： Leptospria toxin-antitoxin system VapBC co-evolution speciation.

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In silico and microarray based genomic approach for identification of potential vaccine candidates against Leptospira interrogans

In silico and microarray based genomic approach for identifi...

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首届全国微生物基因组学学术研讨会

作者： Hong-Liang YANG, Yong-Zhang ZHU, Jin-Hong QIN, Ping HE, Guo-Ping ZHAO and Xiao-Kui GUO Department of Microbiology and Parasitology, shanghai Jiao Tong University School of medicine. 200025 shanghai, PR china Institute of Medical Biology, PeKing Union Medical College & chinese Academy of Medical Sciences. 650118 Kunming, PR china School of Chemical Engineering and School of Life Science, Sichuan University, 610041 Chengdu, PR china chinese national human genome center at shanghai, zhangjiang high tech park, 201203 shanghai, china

Background Currently available vaccines against leptospirosis are low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, in addition, provide no cross-protection against the different serovars of pathogenic leptospira. There is a need to screening vaccine candidate genes encoding surface-exposed proteins in the genome of Leptospira interrogans. The reverse vaccinology, which has been successful in the identification of vaccines candidates, can be used to identifying the potential vaccine candidates against leptospirosis. Although several papers tried to identify the surface-exposed proteins of leptospire, but vaccine candidate genes encoding the surface-exposed proteins that can not only provide cross-protection against the different serovars of pathogenic leptospires but also have relatively high transcription level are still awaited. Results Bioinformatics, comparative genome analysis as well as transcriptional analysis were used for the identification of novel vaccine candidates in the genome of Leptospira interrogans strain #56601. There were 686 genes (14.5%, 686/4727) were predicted encoding surface-exposed proteins by P-CLASSIFIER, currently the most accurate predicting system for subcellular localization in Gram-negative bacteria analysis combined with signal peptides prediction, a-helix transmembrane topology prediction, integral β-barrel outer membrane protein and putative lipoproteins prediction. The DNA microarray of L. interrogans strain #56601 has been constructed for comparative genome hybridization (CGH) and transcriptome analysis at 37癈 in vitro. 307 differential genes were identified in ten pathogenic serogroups by CGH. The numbers of high transcriptional level (cy3 signal ≥342 and cy5 signal ≥363.5 respectively) are 1427 genes. The 686 genes subtract interaction set of genes encoding surface-exposed proteins and 307 differential genes, the number of genes were 625. After that, the interaction set of 625genes an

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Comparative genomic hybridization (CGH) microarray reveals genetic diversity among the main epidemic leptospira interrogans in china

Comparative genomic hybridization (CGH) microarray reveals g...

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首届全国微生物基因组学学术研讨会

作者： Ping He, Yue-Ying Sheng, Yao-Zhou Shi, Xiu-Gao Jiang, Jin-Hong Qin, Zhi-Ming Zhang, Guo-Ping Zhao, Xiao-Kui Guo Department of microbiology and parasitology. shanghai Jiaotong university, shanghai 20025, china national engineering center for biochip at shanghai, zhangjiang high tech park, 201203, china.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Comparative genomic hybridization (CGH) microarray analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with the gene contents of other L. interrogans strains. Missing genes in a tested strain were detected on a microarray slide spotted with 3,528 open reading frames (ORFs) of L. interrogans serovar Lai strain Lai. For 11 strains subjected to the CGH microarray analyses 280 ORFs were found to be absent in at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,948 ORFs. The strain-specific genes include genes encoding cell surface structures, metabolism genes and Cellular processes. Genes involved in O-antigen synthesis were highly divergent in strains belonging to the different serogroups, which revealed that antigenic differences between the serogroups are due to genes in the rfb locus. Examination of the genomic context of the strain-specific genes suggests that many are horizontally acquired. The data demonstrated that the CGH microarray technique is useful for genomic comparisons.

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Potent and Novel Nonpeptide Small Molecule Inhibtors of Caspase-3

Potent and Novel Nonpeptide Small Molecule Inhibtors of Casp...

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中国化学会第四届有机化学学术会议

作者： Huajie Zhang, Yihua Chen, Yahui Zhang, Min Gu, Jingya Li, Fang Wu, Jia Li, Fajun Nan chinese national center for Drug Screening, shanghai Institute of Materia Medica, shanghai Institutes for Biological Sciences, 189 Guoshoujing Road, zhangjiang Hi-tech park, shanghai, 201203. P. R. china fjnan＠***

＜正＞The Caspase family comprises a family of highly homologous cysteine proteases that play key roles in inflammation and apoptosis. Among several different groups of Caspase enzymes, Caspases-3 play a key role in apoptosis Therefore, Caspase-3 is a potential therapeutic target for the treatment of diseases involving dysregulated apoptosis,such as neurological disorders. Up to now several inhibitors were reported for Caspase-3, but most of them have been designed based on substrate of tetrapeptide, their utility was limited due to its intrinsic property such as poor selectivity,

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genome-Wide Transcriptional Analysis of temperature shift of L. interrogans serovar lai Strain 56601

Genome-Wide Transcriptional Analysis of temperature shift of...

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首届全国微生物基因组学学术研讨会

作者： Jin-Hong QIN, Yue-Ying SHENG, Zhi-Ming ZHANG, Ping HE, Bao-Yu HU, Yang YANG, Shi-Gui LIU, Guo-Ping Zhao, Xiao-Kui GUO School of Life Science /School of Chemical Engineering, Sichuan University, Chengdu 610041, PR china Department of Microbiology and Parasitology, shanghai Jiao Tong University School of Medicine, shanghai 200025, PR china national Engineering center for Biochip at shanghai, 151 libing Road, zhangjiang Hi-tech park, Pudong, shanghai 201203, china.

Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host Since many pathogenic ’ bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. We used the L. interrogans serovar lai Strain 56601 genome to construct the microarray. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in strain 56601 grown to mid-exponential phase at 28癈 and 37癈. A total of 112 genes expression varied significantly in expression at 37癈 relative to 28癈. The differentially expressed genes included ten kinds of genes which included membrane proteins, hemolysins encoded genes, heat shock proteins, lipoproteins, two-component system, chemotaxis and flagellar proteins, transcriptional regulators, transport and binding protein, metabolic related protein and proteins with no known homologue. Real-time reverse transcription-PCR assays confirmed the microarray data. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen results of pathogenesis investigation.

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A Novel Class of Caspase-3 Inhibitors:Tetra- and Pentasubstituted Dihydropyrroles Prepared by Multicomponent Reactions

A Novel Class of Caspase-3 Inhibitors:Tetra- and Pentasubsti...

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2011年全国药物化学学术会议——药物的源头创新

作者： Qiuhua Zhu~(,),Lixin Gao~§,Huanfeng Jiang~(*,),Shuwen Liu~(*,),Jia Li~(*,§),Zhipeng Chen~,Ronghuan Mai~ and Jinghao Li~ School of Chemistry and Chemical Engineering,South china University of technology,381 Wushan Road, Guangzhou 510640,china School of Pharmaceutical Sciences,Southern Medical University,Guangzhou,510515,china§chinese national center for Drug Screening,189 Guoshoujing Road,zhangjiang Hi-tech park,shanghai, 201203,china

＜正＞Apoptosis is an essential physiological process of tissue development and *** pathogenesis of many diseases,such as cancer and Alzheimer’s disease,is connected with aberrantly regulated *** evidences indicate that caspase-3 is a key executioner caspase and that inhibition of its activity could significantly prevented apoptosis in vitro and in ***,

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Transcription Character of LA0183-LA0219 fragment of Leptospira interrogans is similar to a prophage

Transcription Character of LA0183-LA0219 fragment of Leptosp...

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首届全国微生物基因组学学术研讨会

作者： Jin-Hong Qin, Zhi-Ming Zhang, Yang Yang, Bao-Yu Hu, Mathieu Picardeau, Shi-Gui Liu Guo-Ping Zhao, Xiao-Kui Guo School of Life Science /Chemical Engineering, Sichuan University, Chengdu 610041, PR china Department of Microbiology and Parasitology, shanghai Jiao Tong University School of Medicine, shanghai 200025,china national Engineering center for Biochip at shanghai, 151 libing Road, zhangjiang Hi-tech park, Pudong, shanghai 201203, china. Laboratoire des Spirochetes, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France.

The complete genome sequences of Leptospira interrogans, the agent of leptospirosis, have been obtained. Many sequenced bacterial genomes harbored phage-like elements or cryptic prophages. No phage-like element which has been implicated in pathogenesis, serotype conversion and phage immunity had been studied in L interrogans. BLAST searched using known phage-associated sequences with L interrogans whole genome sequence and phage-like fragment was found around LA0183-LA0219 which included integrate-like gene, repressor-like gene and other functional phage genes. Operon analysis showed that this phage-like fragment was composed of four transcripts. The function of repressor-like gene (LA0195) was tested using EMSA based on promoter sequence of promoter LA0195 and promoter LA0194 which overlapped in intergenic sequence of LA0194 and LA0195. The results showed that LA0195 protein could bind to the promoter sequence. It is not known whether this fragment still function to its host or only .is a record of the prophage relics. There is currently a lively discussion about the relative contribution of vertical and horizontal elements of evolution in bacteria. Further study of this fragment can shed light on the evolution of both prophages of *** and its hosts.

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