Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto

Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.     

Analysis of Leptospira Antigens by Crossed Immunoelectrophoresis

Serovars icterohaemorrhagiae, canicola , and arboreae of pathogenic Leptospira interrogans and serovar patoc of saprophytic L. biflexa were examined by crossed immunoelectrophoresis. A close antigenic relationship was found between the interrogans serovars. particularly between icterohaemorrhagiae and canicola . To a much lesser extent cross-reactions were found between interrogans serovars and patoc . Comparison of three different antigenic preparations of icterohaemorrhagiae by tandem crossed immunoelectrophoresis and by absorption experi ments with the patoc reference system showed the presence of several common broadly reactive antigens. Antigen I of the reference system, a genus-specific heat-stable protein antigen, was found in all antigenic preparations. It is a diagnostically important antigen.     

Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda)

DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.     

问号钩端螺旋体毒力相关蛋白InvA转录和表达特征的研究

目的 确定我国不同基因种问号钩端螺旋体(简称钩体)参考标准株携带毒力相关基因invA的情况,了解问号钩体赖株感染细胞前后invA基因转录和表达水平的变化.方法 采用PCR检测4个不同基因种问号钩体株及双曲钩体Patoc Ⅰ株invA基因.克隆问号钩体全长invA基因并测序,构建问号钩体黄疸出血群赖型赖株invA基因原核表达系统.Ni-NTA亲和层析法提纯目的重组蛋白rlnvA后免疫家兔获得抗血清,免疫双扩散法检测其效价.建立问号钩体赖株感染人胚肾上皮细胞HEK293模型,采用荧光定量RT-PCR和Western blot分别检测问号钩体赖株感染HEK293细胞前后invA基因的转录和表达水平的变化.结果 4个不同基因种的问号钩体株均含有invA基因,双曲钩体Patoc Ⅰ株则否.4株不同基因种的问号钩体invA基因核苷酸和氨基酸序列相似性分别为99.33%～100%和98.66%～100%.所构建的原核表达系统能有效地表达rInvA.rInvA兔抗血清免疫双扩效价为1:16.问号钩体赖株感染HEK293细胞30 min以后可大量黏附于细胞表面.问号钩体赖株感染HEK293细胞30 min时,invA基因mRNA水平明显上调,45 min时达到峰值,然后逐渐下降.问号钩体赖株感染HEK293细胞后45 min和60 min时可检出InvA蛋白,感染前及感染90 min以后检测结果均为阴性.结论 invA基因是致病性问号钩体所特有的基因.invA基因具有宿主细胞接触式表达及瞬时表达的特点,与问号钩体侵入宿主细胞密切相关.     

问号钩端螺旋体鞘磷脂酶类溶血素基因功能分析及其感染细胞后转录水平变化

目的 了解问号钩端螺旋体(简称钩体)鞘磷脂酶类溶血素基因sph1～sph4产物溶血活性及其感染细胞后转录水平的变化.方法 以致病性问号钩体黄疸出血群赖型赖株、波摩那群波摩那型罗株和非致病性双曲钩体三宝垄群Patoc型Patoc Ⅰ株基因组DNA为模板,采用PCR扩增全长sph1～spl4基因片段,扩增产物T-A克隆后测序.构建sph1～sph4基因原核表达系统,采用SDS-PAGE检测目的 重组蛋白rSph1～rSph4的表达情况,Ni-NTA亲和层析柱提纯rSph1～rSpM.采用绵羊血平板对rSph1～rSph4溶血活性进行鉴定,实时荧光定量RT-PCR检测问号钩体赖株感染J774A.1细胞前后sph1～sph4基因转录水平的变化.结果 问号钩体赖株和罗株基因组DNA中均能扩增sph1～sph4基因,双曲钩体Patoc Ⅰ株则否.与报道的相应基因序列比较,所克隆的sph1～sph4基因核苷酸序列相似性均为100%.所构建的原核表达系统能分别表达目的蕈组蛋白rSph1～rSph4.rSph1～rSph4均有溶血活性,其中以rSph2溶血活性最强.问号钩体赖株感染J774A.1细胞后,sph1～sph4基因转录水平均上调,其中sph2和sph4基因mRNA水平上调更为明显.结论 sph1～sph4基因仅存在于致病性问号钩体中,其表达产物有溶血活性.问号钩体赖株感染细胞后sph1～sph4基因转录水平的上调,提示此类鞘磷脂酶类溶血素可能在问号钩体感染宿主过程中有重要作用.     

Pulsed-field gel electrophoretic analysis of leptospiral DNA.

The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infrequently cutting restriction enzymes showed that the genome of Leptospira interrogans serovar canicola is approximately 3.1 Mb, while that of the saprophytic L. biflexa serovar patoc I is 3.5 Mb. DNA forms of approximately 2,000 and 350 kb which were present in samples from L. interrogans serovars were not readily detected in nonpathogenic serovars. Three distinct populations, designated type alpha, beta, and gamma, of L. interrogans DNA molecules were further analyzed with two-dimensional gel electrophoresis. Evidence suggested that two of these DNA forms, type alpha and gamma, were linear structures. Pulsed-field gel electrophoresis has proven to be a valuable tool with which to size bacterial genomes and to take the first steps toward characterization of a form of leptospiral DNA which behaves as a linear molecule and which may be related to the virulence of L. interrogans.     

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.     

Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA.

Primers for PCR were selected from a sequenced fragment of clone pL590, which contains a repetitive element present in the genome of Leptospira interrogans serovar hardjo type hardjoprajitno (M. L. Pacciarini, M. L. Savio, S. Tagliabue, and C. Rossi, J. Clin. Microbiol. 30:1243-1249, 1992). A specific DNA fragment was amplified from the genomic DNAs of serovar hardjo type hardjoprajitno and nine serovars also belonging to L. interrogans as a consequence of the spread of the same or a closely related repetitive element within this species (Pacciarini et al., J. Clin. Microbiol. 30:1243-1249, 1992). In addition, specific amplification was obtained from two Leptospira borgpetersenii serovars (tarassovi and hardjo type hardjobovis). Negative PCR results were observed with all of the other Leptospira serovars tested, including nonpathogenic ones (serovars patoc and andamana), another spirochete (Borrelia burgdorferi), bacteria commonly found in biological samples, and swine and bovine cell lines. Direct PCR on biological samples such as kidney samples demonstrated that preliminary isolation and culture of Leptospira cells are not required for efficient detection. Furthermore, digestion of the amplified DNA with the enzymes HinfI and DdeI yielded specific polymorphic patterns, allowing discrimination among the majority of the serovars. These methods were applied to 25 field isolates of serovar pomona, leading to the conclusion that they were suitable for the simple and rapid detection of L. interrogans and for serovar identification.     

Chemotaxis of leptospires to hemoglobin in relation to virulence.

A guinea pig-lethal line of Leptospira interrogans serovar copenhageni strain Shibaura, but not an avirulent line of the same strain, moved in larger numbers toward hemoglobin than toward distilled water (control) in a U-shaped polypropylene tube. L. interrogans serovar lai strains 017 and KH-1, which were also guinea pig lethal, showed a similar move to hemoglobin. No such move toward hemoglobin was shown by 14 avirulent strains of L. interrogans (with one exception) or any of the 8 strains of L. biflexa tested.     

Analysis of Leptospira spp., Leptonema illini, and Rickettsia rickettsii for the 39-kilodalton antigen (P39) of Borrelia burgdorferi.

Five serovars of Leptospira interrogans, Leptospira biflexa, Leptonema illini, and Rickettsia rickettsii were examined and found not to contain the 39-kDa antigen (P39) of Borrelia burgdorferi, the Lyme disease spirochete. The specificity of this antigen and its reactivity with human Lyme disease sera should exclude the possibility of false-positive serum samples from patients having had either leptospirosis or Rocky Mountain spotted fever, as well as tick-borne relapsing fever and syphilis, as reported previously (W.J. Simpson, M. E. Schrumpf, and T. G. Schwan, J. Clin. Microbiol. 28:1329-1337, 1990).     

Seroepidemiologic study of three zoonoses (leptospirosis, Q fever, and tularemia) among trappers in Québec, Canada.

This study was undertaken to evaluate the prevalence of antibodies against Francisella tularensis, Coxiella burnetii, and certain serovars of Leptospira interrogans among trappers in Québec, Canada. Muskrat trapping was identified as a risk factor for F. tularensis infection, whereas having a cat at home apparently protected trappers against infection by L. interrogans. High percentages of control sera were positive for antibodies against C. burnetii (15%) and L. interrogans (5%), most frequently serovar bratislava. This is the first report of human infection by serovar bratislava in North America.     

Identification of leptospiral flagellar antigens by gel electrophoresis and immunoblotting

Flagella extracted from five serovars, representative of the pathogenic and saprophytic species of the Leptospiraceae, were morphologically similar. Analysis of Leptospira interrogans flagellar preparations by polyacrylamide gel electrophoresis revealed three common major bands in the (30-40) x 10(3)-mol. wt region, and serovar-specific bands in the lower region of the gels. Although some differences were observed, flagella extracted from L. biflexa serovar patoc and Leptonema illini revealed similar electrophoretic profiles to those seen in L. interrogans flagella. Immunoblot analysis showed that while flagellar components in the (20-30) x 10(3)-mol. wt region were recognised only by homologous rabbit antisera, a major protein doublet of (33-34) X 10(3)-mol. wt, depending on the species, was also demonstrated by heterologous antisera. The serovar-specific bands in the (20-30) x 10(3)-mol. wt region were composed of lipopolysaccharide (LPS). These results show that leptospiral flagella are immunogenic and contain antigens which are conserved among the different genera of the family Leptospiraceae.     

Distribution of superoxide dismutase, catalase, and peroxidase activities among Treponema pallidum and other spirochetes.

Representative members of Spirochaetales were surveyed for their content of superoxide dismutase (SOD), catalase, and peroxidase activities. Only Leptospira exhibited peroxidase activity. Obligately anaerobic cultivable Treponema and Spirochaeta possessed no SOD or peroxidative capabilities. Upon polyacrylamide gel electrophoresis, Spirochaeta aurantia, Borrelia hermsi, and five Leptospira biflexa serovars showed SOD activity associated with one electrophoretic band which was inhibited by H2O2, suggesting that they were iron-containing dismutases. These spirochetes could be distinguished by differences in relative mobilities of their SODs. SOD activity, but not catalase activity, was induced aerobically in S. aurantia. All Leptospira interrogans serovars and two L. biflexa serovars lacked significant SOD activity. These SOD-deficient strains of Leptospira, with one exception, possessed high levels of catalase activity. The Nichols strain of virulent Treponema pallidum possessed SOD and catalase activities, but lacked peroxidase activity. The SOD in T. pallidum exhibited two electrophoretic bands containing copper and zinc, and its relative mobility was identical to that of purified rabbit SOD. Immunization of sheep with purified rabbit SOD resulted in antiserum which inhibited both rabbit SOD and T. pallidum SOD assayed by spectrophotometric analysis or activity staining following polyacrylamide gel electrophoresis. In agarose gel diffusion, precipitin lines of identity were observed between purified rabbit SOD and cell extracts of T. pallidum. These data indicated that the SOD activity detected in T. pallidum was host derived.     

LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species

Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.     

Comparative analysis of lipopolysaccharide and lipid antigens of Leptospira interrogans serovars.

Lipopolysaccharide (LPS) or glycolipid antigens of Leptospira interrogans have been candidates as serogroup or serotype specific antigen. In this study, therefore, we prepared the LPS and lipid antigens from L. interrogans serovars lai, icterohaemorrhagiae, copenhageni, canicola, pomona, grippotyphosa, and a Korean isolate 30R. The LPS antigens were analyzed by a polyacrylamide gel electrophoresis and lipid antigens by thin-layer chromatography, respectively. The seroreactivity of the antigens were also examined with homologous or heterologous antisera using an enzyme-linked immunosorbent assay. The LPS antigens from serovar lai and the strain 30R were closely related but different from serovar icterohaemorrhagiae. Particularly, the LPS antigens from serovars icterohaemorrhagiae and grippotyphosa were reactive only with the homologous antisera, thus indicating serovar specificity. However, the LPS antigens of the other serovars were reactive to the heterologous antisera. The lipid antigen of serovar icterohaemorrhagiae reacted only with the homologous antisera. In contrast, lipids of other serovars reacted broadly with heterologous antisera, particularly among serovars lai, copenhageni, canicola, pomona, and the strain 30R. The results thus indicated that the LPS and lipid antigens of L. interrogans may contain serovar-specific as well as cross-reactive epitopes.     

Host-inducible immunogenic sphingomyelinase-like protein, Lk73.5, of Leptospira interrogans

Leptospira interrogans causes a variety of clinical syndromes in animals and humans. Although much information has accumulated on the importance of leptospiral lipopolysaccharide in protective antibody responses, relatively little is known about proteins that participate in immune responses. Identification of those proteins induced only in the host is particularly difficult. Using a novel double-antibody screen designed to identify clones in a gene library of L. interrogans serovar Pomona expressing host-inducible proteins, we have characterized a gene (lk75.3) encoding a sphingomyelinase-like preprotein of 648 amino acids with cytotoxic activity for equine pulmonary endothelial cells and weak hemolytic activity for equine and rabbit erythrocytes. lk73.5 was found as a single gene copy in all serovars of L. interrogans but not in other Leptospira spp. except L. inadai. The open reading frame (ORF) for Lk73.5 is followed by another partially homologous sequence containing an ORF (sph-like 2) for a 28.7-kDa peptide. Lk73.5 and Sph-like 2 share 95.1 and 97.7% amino acid identity with putative sphingomyelinases Sph2 and Sph1 (N terminus) from L. interrogans serovar Lai (S.-X. Ren, G. Fu, X.-G. Jiangk, R. Zeng, Y.-G. Miao, H. Xu, Y.-X. Zhang, H. Xiong, G. Lu, L.-F. Lu, H.-Q. Jiang, J. Jia, Y.-F. Tu, J.-X. Jiang, W.-Y. Gu, Y.-Q. Zhang, Z. Cai, H.-H. Sheng, H.-F. Yin, Y. Zhang, G.-F. Zhu, M. Wank, H.-L. Huangk, Z. Qian, S.-Y. Wang, Wei Ma, Z.-J. Yao, Y. Shen, B.-Q. Qiang, Q.-C. Xia, X.-K. Guo, A. Danchinq, I. S. Girons, R. L. Somerville, Y.-M. Wen, M.-H. Shik, Z. Chen, J.-G. Xuk, and G.-P. Zhao, Nature 422:88-893, 2003). Substantial homologies to sphingomyelinases from other leptospiras and other bacteria are also present. Lk73.5 was not detected in leptospiras cultured at 30 or 37 degrees C. The recombinant protein reacted strongly with sera from recently infected mares but not with sera from horses vaccinated with commercial pentavalent bacterin. The host-inducible immunogenic Lk73.5 should have value in distinguishing vaccine from infection immune response.     

Serological interrelationship of Leptospira serovar and genus-specific antigens by enzyme-linked immunosorbent assay.

The serological interrelationship of a sonicated antigen (POM-S) and an alkali-extracted "fraction 4" antigen (POM-F4) from Leptospira interrogans serovar pomona, and an ethanol-precipitated (PAT-E) and a formolized, sonicated antigen (PAT-F) from Leptospira biflexa serovar patoc were investigated. The serological responses of rabbits immunized with these antigens were examined by the enzyme-linked immunosorbent assay (ELISA), the microscopic agglutination test, and the 2-mercaptoethanol microscopic agglutination test. Antisera from these rabbits absorbed by the homologous or heterologous antigens were examined by the ELISA. The PAT-E and PAT-F antigens were genus specific and were serologically closely related but not identical. Similarly, the POM-F4 and POM-S antigens showed some serological relatedness. By the use of absorbed antisera, the serovar pomona-derived antigens were shown to be serologically related to the PAT-F but not to the PAT-E antigen in the ELISA. It is suggested that the use of several such antigens in the ELISA may reveal differences in the kinetics of the antibody response in animals infected by different leptospiral serovars.     

Effect of heat or formalin treatment of leptospires on antibody response detected by immunoblotting.

Leptospira interrogans serovar icterohaemorrhagiae RGA (RGA), live or heated at 56 degrees C for 15 min or treated with Formalin, was injected into rabbits to prepare hyperimmune serum. The pathogens L. interrogans serovars icterohaemorrhagiae RGA, icterohaemorrhagiae 1, canicola Moulton, grippotyphosa Andaman, hardjo Hardjoprajitno, and pomona Pomona and the nonpathogen Leptospina biflexa serovar patoc Patoc 1 were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and after electrophoresis they were then transferred to nitrocellulose paper. Antiserum against RGA (live, heat killed, or Formalin killed) was used on one of each of the three blots. Formalin appeared to completely eliminate antibody response to antigens with the molecular weight of 14,000 to 20,000 (14K to 20K) but did expose an antigen at approximately 23K in the pathogens only. This same band had only slight reactivity when antiserum against heat-killed RGA was used. Heating also eliminated cross-reactivity in the 19K to 30K range and partially degraded bands in the 14K to 20K region so that one broad band rather than several discrete bands appeared. The three antiserum specimens cross-reacted with all of the serovars tested, but fewer antigens of grippotyphosa and hardjo reacted with the antisera. Against patoc, reactivity was limited primarily to the flagellar region. The most cross-reactivity was with the antiserum prepared by using live leptospires.     

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。     

Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays.

Genetic variability among Leptospira interrogans (sensu stricto) serovars was assessed by Southern blot hybridization and PCR analyses. The experiments used probes directed to sequences in a recently described insertion element, IS1500. Hybridization analysis showed that IS1500 was present on polymorphic fragments and that differences in these patterns could be used to identify serovars. Hybridization analysis was also useful in discriminating between serovar pomona type kennewicki isolates, making possible the identification of 15 previously unrecognized genetic groups. A PCR assay was developed in which the primers are positioned near the terminal inverted repeats of the element and directed outward. This assay yielded characteristic amplification patterns from isolates, allowing them to be identified. We applied these assays to several new animal isolates of L. interrogans from Nicaragua, which recently had an outbreak of human leptospirosis. Three groups of isolates were identified: one strain of serovar pomona type kennewicki and two genetically distinct groups of isolates which may be genetic intermediates between serovars canicola and portlandvere. The IS-based typing assays described should be useful for epidemiological analysis of leptospirosis.