Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai

Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37 degrees C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30 degrees C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30 degrees C, but not at 30 degrees C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.     

问号状赖型钩端螺旋体LAg42膜蛋白基因的克隆及真核表达研究

目的:构建赖型钩端螺旋体lag42基因真核表达载体并转染哺乳动物细胞,为进一步研究奠定基础。方法:分别以问号状赖型钩体017株,56601株及双曲钩体PatocI株基因组为模板PCR扩增目的基因。构建lag42基因与质粒pcDNA3.1A 的重组真核表达质粒,克隆筛选并测序;通过脂质体介导将重组质粒转染入COS7细胞,用RT-PCR检测转染结果。结果:不同毒力赖型钩体均能扩增出约1100 bp的片段,而PatocI株则未能扩增出目的片段;PCR、双酶切及测序证实pcDNA3.1A -lag42构建成功;经RT-PCR检测证实重组质粒转染成功。结论:赖型钩体具有编码LAg42膜蛋白的基因,构建完成真核表达载体pcD-NA3.1A -lag42,并成功转染COS7细胞。     

赖型钩端螺旋体溶血素HlyX对血管内皮细胞通透性的影响

目的: 克隆表达赖型钩端螺旋体溶血素基因hlyX,观察其表达产物对人脐静脉内皮细胞(HUVECs)通透性的影响,并初步探讨其机制。方法: 将hlyX基因插入原核表达载体pET32a(+),构建重组质粒pET-hlyX,转化大肠杆菌BL21(DH3)以高效表达携带组氨酸标签的Trx-HlyX融合蛋白,并作亲和层析纯化。采用生物素标记白蛋白的酶联免疫吸附法测定目的蛋白对HUVECs单细胞层通透性的影响,并以流式细胞术及Hoechst 33258荧光染色方法检测其作用于HUVECs后的细胞凋亡率。结果: 成功构建了重组质粒pET-hlyX并高效表达出Trx-HlyX融合蛋白;与对照组相比较,蛋白Trx-HlyX作用于HUVECs后,明显增加其通透性(P< 0.05),细胞凋亡率显著增加(P<0.05)。结论: 重组质粒pET-hlyX高效表达出Trx-HlyX融合蛋白,纯化后的融合蛋白对HUVECs通透性有影响,且对HUVECs具有细胞毒性作用。     

A genomic island of the pathogen Leptospira interrogans serovar Lai can excise from its chromosome

An examination of the two Leptospira interrogans genomes sequenced so far reveals few genetic differences, including an extra DNA region, 54 kb in length, in L. interrogans serovar Lai. This locus contains 103 predicted coding sequences that are absent from the genome of L. interrogans serovar Copenhageni, of which only 20% had significant BLASTP hits in GenBank. By analyzing the L. interrogans serovar Lai genome by pulsed-field gel electrophoresis, we also found that this 54-kb DNA fragment exists as a circular plasmid. This was confirmed by amplification of a DNA fragment corresponding to that of the predicted fragment if this region excised from the chromosome and its left and right ends joined together. In addition, cloning of the putative rep gene of this DNA region was responsible for autonomous replication in Leptospira spp., therefore generating a new Escherichia coli-Leptospira sp. shuttle vector. Taken together, our results show that this genomic island can excise from the chromosome and form a replicative plasmid. Analysis of the distribution of this genomic island revealed that highly related sequences exist in other L. interrogans virulent strains. This genomic island, containing a high proportion of novel genes, may have an important role in spreading genes, including virulence factors, among bacterial populations.     

Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo.

We radiolabeled Leptospira proteins with [35S]methionine. Solubilized extracts of radiolabeled L. interrogans serovar hardjo strain hardjoprajitno were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. We compared the protein profile obtained in this manner to the protein profiles of various [35S]methionine-labeled Leptospira spp. The profiles of the pathogenic L. interrogans strains were very similar but not identical and exhibited no obvious relationship to those of the two nonpathogenic species. We used solubilized, radiolabeled hardjoprajitno extracts and a sensitive radioimmunoprecipitation procedure to identify protein antigens recognized by immunoglobulin G antibodies present in various rabbit anti-hardjo sera. Homologous hyperimmune rabbit serum efficiently precipitated a large subset of proteins, the majority of which were between 30,000 and 66,500 daltons. Radioimmunoprecipitations with sera prepared against each of four recent hardjo isolates cultured from infected cattle produced similar results. Immunoprecipitations done with various radiolabeled Leptospira extracts and anti-hardjoprajitno serum demonstrated that the pathogenic leptospires possessed a number of cross-reactive major and minor protein antigens. By cell fractionation procedures, we found that most of the major protein antigens were present in the outer envelope. These proteins were exposed on the leptospiral cell surface because intact radiolabeled leptospires bound antibodies directed against them.     

Protection of guinea pigs against Leptospira interrogans serovar Lai by LipL21 DNA vaccine

In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immunized with pcDNA3.1（＋）-lipL21, pcDNA3.1（＋） or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1（＋）-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1（＋）-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1（＋）-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.     

赖型钩端螺旋体及澳洲型钩端螺旋体的ompL1和flaB2抗原基因序列分析

目的：构建棘型钩端螺旋体017及澳洲型钩端螺旋体607株外膜蛋白抗原基因ompL1和内鞭毛抗原基因flaB2的重组质粒，并分别对ompL1及flaB2基因进行序列分析。方法：通过聚合酶链反应扩增ompL1及flaB2，并将其分别克隆到pcDNA3.1/Myc-His(＋）载体T7启动子下游，构建抗原基因表达质粒，进行序列测定分析。结果：序列分析显示赖型钩体017株与澳洲型钩体607株的ompL1相同碱基949个（98．85％），碱基变异11个（1．15％）；flaB2的相同碱基823个（96．94％），碱基变异26个（3．06％），呈很高的保守性。结论：赖型钩体017株与澳洲型钩体607株的ompL1及flaB2分别具有高度同源性。     

Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans

A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30 degrees C or when cultures were shifted to 37 degrees C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.     

Cloning of a hemolysin gene from Leptospira interrogans serovar hardjo.

A DNA fragment encoding both hemolysin and sphingomyelinase C activity was cloned from the pathogenic bacterium Leptospira interrogans serovar hardjo. Initial clones were obtained by screening a genomic library in EMBL3 for hemolytic activity. Both hemolytic and sphingomyelinase C activities were coded for by a 3.9-kilobase BamHI fragment. The hemolysin was expressed from its own promoter in Escherichia coli K-12. Similar DNA sequences were also present in the serovars tarassovi and ballum.     

Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection

We previously reported the cloning and characterization of leptospiral immunoglobulin-like proteins LigA and LigB of Leptospira interrogans. LigA and LigB are conserved at the amino-terminal region but are variable at the carboxyl-terminal region. Here, we evaluate the potential of recombinant LigA (rLigA) as a vaccine candidate against infection by L. interrogans serovar Pomona in a hamster model. rLigA was truncated into conserved (rLigAcon) and variable (rLigAvar) regions and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (rLigA). Golden Syrian hamsters were immunized at 3 and 6 weeks of age with rLigA (rLigAcon and rLigAvar) with aluminum hydroxide as an adjuvant. Hamsters given recombinant glutathione-S-transferase (rGST)-adjuvant and phosphate-buffered saline-adjuvant served as nonvaccinated controls. Three weeks after the last vaccination, all animals were challenged intraperitoneally with 10(8) L. interrogans serovar Pomona bacteria (NVSL 1427-35-093002). All hamsters immunized with recombinant LigA survived after challenge and had no significant histopathological changes. In contrast, nonimmunized and rGST-immunized hamsters were subjected to lethal doses, and the hamsters that survived showed severe tubulointerstitial nephritis. All vaccinated animals showed a rise in antibody titers against rLigA. Results from this study indicate that rLigA is a potential vaccine candidate against L. interrogans serovar Pomona infection.     

赖型钩端螺旋体溶血素基因HlyX的真核表达及基因免疫

目的 在哺乳动物细胞中表达含有赖型钩端螺旋体溶血素基因HlyX的重组质粒,并检测其作为DNA疫苗诱导小鼠体液免疫应答的效果.方法 以赖型钩端螺旋体全基因组为模板,PCR扩增出目的基因HlyX,以质粒pcDNA3.1为载体,双酶切构建重组质粒,转化大肠杆菌DH5α,双酶切、PCR及测序鉴定重组质粒.将构建成功的重组质粒转染COS-7细胞,通过RT-PCR、Western blot鉴定目的基因的表达.将重组质粒免疫BALB/c小鼠,每两周1次,共3次,ELISA法检测小鼠的体液免疫应答水平.结果 扩增出全长约1100 bp的HlyX基因,重组质粒经双酶切、PCR及测序鉴定表明重组质粒构建成功.RT-PCR检测显示重组质粒转染组能扩增出约1100 bp的目的基因片段,Western blot分析可见在相对分子质量(Mr)40×103左右出现特异性的目的条带.DNA免疫后诱导小鼠产生了较高的抗体水平(1∶6561～1∶19 683).结论 赖型钩端螺旋体溶血素基因HlyX真核表达质粒能够诱导小鼠产生高滴度的特异性抗体,为其作为钩体DNA疫苗的研究和开发奠定了基础.     

Characterization of an antigenic oligosaccharide from Leptospira interrogans serovar pomona and its role in immunity.

An antigenic oligosaccharide fraction derived from the lipopolysaccharide of Leptospira interrogans serovar pomona was isolated by endo-glycosidase H digestion and column chromatography. The oligosaccharide contained rhamnose, ribose, glucose, and glucosamine and inhibited the binding of opsonic, protective monoclonal antibodies directed against the lipopolysaccharide. When conjugated to diphtheria toxoid, the oligosaccharide elicited the production of agglutinating, opsonic antibodies.     

Bim, a new serovar of Leptospira interrogans isolated from a dog in Barbados.

A new serovar of Leptospira interrogans was isolated from a dog in Barbados. The proposed name of the new serovar is bim, and the designated type strain is 1051. The serogroup of the new serovar is the Autumnalis serogroup. The new serovar was subsequently isolated from six patients with leptospirosis in Barbados.     

Copurification of Leptospira interrogans serovar pomona hemolysin and sphingomyelinase C.

The hemolytic and sphingomyelinase C activities of supernatants of cultures of Leptospira interrogans serovar pomona tended to copurify when isoelectric fractionation was carried out. Both activities focused primarily at pH 8.1. Considered in conjunction with other circumstantial evidence, the results led to the conclusion that sphingomyelinase C is responsible for hemolysis.     

Repetitive sequences cloned from Leptospira interrogans serovar hardjo genotype hardjoprajitno and their application to serovar identification.

We selected, from a genomic library of Leptospira interrogans serovar hardjo genotype hardjoprajitno, two probes containing repetitive sequences (pL1 and pL590). The hybridization patterns of these probes to DNA isolated from a variety of Leptospira serovars were examined and their ability to detect subtle differences at the genomic organization level was established. We identified the DNA fragments within pL1 and pL590 which are sufficient to yield polymorphic hybridization patterns; these results define the upper size limit of two novel repetitive elements in the Leptospira genome. The pattern and degree of hybridization observed for the serovars tested in this work were used to divide Leptospira spp. into groups which share genetic relatedness; our conclusions are consistent with previous classifications by other authors.     

Plate Assay for Detection of Leptospira interrogans serovar pomona Hemolysin

A plate assay which utilizes the addition of a blood agar overlay for detection of hemolysin by colonies of Leptospira interrogans serovar pomona is described.     

Molecular analysis of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo.

A thermolabile hemolysin from Leptospira interrogans serovar hardjo, strain Sponselee, was shown to specifically degrade sphingomyelin. Nucleotide sequence determination revealed that sphingomyelinase activity was encoded by an open reading frame of 1,668 nucleotides. Although a putative signal sequence could be identified, no evidence for protein export in either L. interrogans or Escherichia coli was obtained. The apparent molecular mass of the expression product in E. coli minicells was 41.2 kilodaltons, whereas open reading frame 1 encoded a protein of 63,268 daltons. The observed difference may be explained by processing at the carboxy-terminal part of the hemolysin in E. coli. A high degree of similarity on the DNA and protein levels with Staphylococcus aureus beta-hemolysin and sphingomyelinase C from three Bacillus cereus strains was observed. The presence of various sphingomyelinase genes within the L. interrogans species is demonstrated.     

赖型钩端螺旋体毒力相关蛋白InvA的表达及其促细胞增殖效应

目的　在大肠杆菌中表达赖型钩端螺旋体毒力相关蛋白InvA ,并观察该蛋白对ANA 1细胞的增殖作用。方法　将invA基因克隆于pET32a( )载体 ,在大肠杆菌BL2 1(DE3)中表达带组氨酸标签的Trx InvA融合蛋白 ,经亲和层析获得纯化Trx InvA蛋白。以不同浓度的Trx InvA融合蛋白作用于ANA 1细胞 ,以四氮唑复合物 [2 ,3 bis(2 methoxy 4 nitro sulfophenyl) 5 〔(Phenylamino)carbonyl〕 2H tetrazoiumhydrixide,XTT]法检测细胞增殖的变化 ,分析InvA蛋白的细胞功能。结果　成功构建pETIN VA原核表达载体 ,在大肠杆菌BL2 1(DE3)中表达了相对分子质量 (Mr)约为 35× 10 3的Trx InvA融合蛋白 ,该蛋白能促进ANA 1细胞的增殖。结论　表达并纯化的Trx InvA融合蛋白具有促进细胞增殖的作用