问号钩端螺旋体T和B细胞表位联合基因的原核表达及其产物免疫性分析

目的 构建问号钩端螺旋体(简称钩体)LipL32、OmpL1和LipL21蛋白的优势T-和B-细胞联合表位融合基因及其原核表达系统,并对表达产物的免疫原性进行鉴定.方法 人工合成多表位联合基因并构建其原核表达系统.采用SDS-PAGE检测重组蛋白;采用MAT检测重组蛋白兔抗血清与我国钩体标准参考株的凝集效价;Western blot和ELISA检测重组蛋白的免疫原性.结果 获得了多表位融合基因并构建了原核表达系统.表达产物的相对分子质量约为23×103,且主要以可溶性形式存在;重组蛋白兔抗血清免疫双扩散效价为1∶8,该抗血清能与我国15群的钩体标准参考株发生凝集反应,ELISA证明该重组蛋白能检测不同群型钩体感染患者血清中的抗钩体抗体.结论 成功构建了包含钩体LipL32、OmpL1和LipL21蛋白的优势T和B细胞联合表位基因及其原核表达系统,表达产物具有良好的抗原性和交叉免疫反应性,可作为研制通用型问号钩体基因工程疫苗及血清学检测的抗原.     

钩端螺旋体多抗原肽的构建及免疫性分析

目的 构建问号钩端螺旋体(简称钩体)主要外膜蛋白OmpL1、LipL21和LipL32优势抗原表位的串联基因及其表达系统,了解该重组蛋白的免疫活性.方法 采用噬菌体M13KE表面展示技术结合Western blot分析,鉴定了OmpLl、LipL21和LipL32的优势抗原表位,人工合成优势抗原表位串联基因并构建其原核表达系统.SDS-PAGE检测重组蛋白的表达情况;Western blot及ELISA鉴定重组蛋白的免疫活性.结果 该合成基因在原核表达系统中得到了有效表达,且表达产物主要以可溶性形式存在.Western blot和ELISA结果 显示该重组蛋白能与兔抗钩体全菌抗体及不同血清群的钩体病人血清中的抗体产生免疫反应.结论 本研究成功构建了钩体多表位串联基因及其表达系统,所表达目的 蛋白具有良好的免疫活性,且对不同血清群型抗体之间均有免疫原活性.     

问号钩端螺旋体脂蛋白LipL32膜定位及其免疫应答

目的 确定问号钩端螺旋体(简称钩体)属特异性脂蛋白抗原LipL32膜定位及其自然抗体应答情况和抗体类型.方法 IPTG诱导目的 重组蛋白rLipL32-1和rLipL32-2表达,Ni-NTA亲和层析法提纯rLipL32.采用显微镜凝集试验(MAT)检测四川地区钩体患者血清标本及rLipL32兔抗血清与我国问号钩体参考标准株的交叉凝集情况.采用胶体金免疫电镜技术对LipL32进行膜定位.建立基于rLipL32的ELISA,检测钩体患者血清中特异性抗体类型及其水平.结果 黄疸出血群是四川地区最主要的优势钩体血清群.rLipL32兔抗血清均能与我国问号钩体参考标准株发生MAT效价为1∶80～1∶320的交叉凝集反应.LipL32是位于钩体外膜表面的蛋白质分子.156例MAT阳性钩体患者血清标本中,rLipL32-1和rLipL32-2特异性IgM阳性率分别为91.0%～92.9%和90.4%～92.3%,特异性IgG阳性率分别为99.4%和97.4%～98.1%.结论 LipL32是问号钩体属特异性表面蛋白抗原.自然感染钩体时,LipL32-1和LipL32-2可诱导机体产生IgM和IgG两类血清抗体.rLipL32-1和rLipL32-2可作为研制检测试剂盒的候选抗原.     

问号钩端螺旋体血清群LipL41基因型分析及其表达产物的免疫学鉴定

目的 确定我国15群15株问号钩端螺旋体(简称钩体)参考标准株和2群2株双曲钩体国际标准株携带LipL41基因情况，构建该基因的原核表达系统，鉴定表达产物的免疫原性。方法 常规酚—氯仿法提取上述17株钩体基因组DNA，高保真PCR扩增全长LipL41基因片段，T—A克隆后测序分型。构建LipL41基因原核表达系统，SDS-PAGE检测重组目的蛋白(rLipL41)表达情况。分别用钩体属特异性TP/patoe Ⅰ抗原、rLipL41兔抗血清的Western blot鉴定其免疫反应性和抗原性。分别用显微镜凝集试验(MAT)、钩体黏附J774A．1细胞模型检测兔抗rLipL41血清的交叉凝集效价和黏附阻断作用。结果 15株问号钩体均有LipL41基因，并可分为LipL41／1和LipL41／2两种基因型，2株双曲钩体则否。11个LipL41／1基因和4个LipL41／2基因克隆之间的核苷酸和氨基酸序列相似性分别为88．61％—88．67％和93．24％—97．18％。所构建的原核表达系统rLipL41／1和rLipL41／2的表达量分别占钩体总蛋白的30％和40％。rLipL41／1和rLipL41／2均能与TP/patoe Ⅰ抗血清发生结合反应，免疫家兔能产生抗体。rLipL41／1和rLipL41／2兔抗血清对上述15株问号钩体MAT效价为1：8，1：128、1：16～1：2．S6稀释时均能有效地阻断钩体对细胞的黏附。结论 我国主要的15群问号钩体代表株均有LipL41／1或LipL41／2基因。所构建原核表达系统能高效表达rLipL41／1和rLipL41／2。rLipL41／1和rLipL41／2是具有良好抗原性和免疫反应性、广泛存在于不同血清群问号钩体表面的蛋白抗原。     

钩端螺旋体抗原表位预测、重组、表达及免疫原性分析

目的应用生物信息学方法预测两种钩端螺旋体外膜蛋白的表位，结合基因工程手段进行表位重组、表达和免疫原性分析。方法用预测程序ProPred和ANTIGENIC预测LipL32和OmpL1的表位，应用PCR技术合成重组表位基因片段，克隆PCR产物构建重组质粒，测序验证。在BL21（DE3）中诱导表达融合蛋白。纯化该融合蛋白，免疫BALB／c小鼠，显微镜凝集试验（MAT法）测定抗体效价。结果在LipL32和OmpL1中各预测到2个既具有MHC结合肽特性又具有B细胞表位特征的肽段。PCR合成的重组表位基因序列中没有出现移码和碱基置换。纯化后融合蛋白纯度〉90％。融合蛋白产生的抗体效价为75．79，融合头（运载蛋白）抗体效价为10．62。结论重组表位具有一定免疫原性。为相关蛋白的表位重组和亚单位疫苗等方面的研究打下了良好的基础。     

幽门螺杆菌UreB抗原表位预测及其有效表位噬菌体展示的筛选

目的 筛选幽门螺杆菌（Hp）尿素酶B亚单位（UreB）分子中的抗原表位，为研制多抗原肽（MAP）疫苗奠定基础。方法 采用生物信息学技术对UreB的T细胞和B细胞表位进行预测和分析。构建ureB基因原核表达系统，Ni-NTA亲和层析法提纯目的重组表达产物rUreB，常规皮内免疫法制备兔抗血清。选择UreB主要T细胞和B细胞联合表位肽并构建其噬菌体展示系统，PEG／NaCl沉淀法提纯重组噬菌体，SDS-PAGE鉴定目的重组PⅢ蛋白（rPⅢ）。分别以商品化抗跏全菌IsG和rUreB抗血清为一抗，采用Western blot对上述表位肽进行鉴定和筛选。结果 与GenBank中相关序列比较，所克隆的ureB基因核苷酸和氨基酸相似性分别为96％～99．5％和96％～100％。rUreB表达量约为细菌总蛋白的52％，提纯后仅见单一蛋白条带。预测的4个主要表位肽UreB230、UreB322、UreB479和UreB527在M13噬菌体中获得成功表达，采用不同一抗的Westernblot均显示相似的阳性结果，但UreB322和UreB527反应强度明显高于UreB230和UreB479。结论 本研究成功地构建ureB基因高效原核表达系统和UreB主要T细胞和B细胞联合表位肽噬菌体展示系统。所采用的生物信息学技术可有效地预测抗原表位。UreB322和UreB527是UreB有效抗原表位，可作为幽门螺杆菌MAP疫苗的候选表位。     

幽门螺杆菌黏附素A有效T和B联合抗原表位噬菌体展示和抗原性测定

目的 分析并确定幽门螺杆菌黏附素A(HpaA)分子中有效T细胞和B细胞联合(T/B)抗原表位.方法 重组表达HpaA(rHpaA)并免疫家兔制备抗血清.采用生物信息学技术预测和分析HpaA分子中T细胞和B细胞表位,PCB扩增T/B联合表位肽片段并构建其噬菌体展示系统.采用PEG/NaCl沉淀法提纯展示了T/B联合表位的重组噬菌体PⅢ蛋白(rPⅢ).分别以商品化幽门螺杆菌全菌IgG抗体和rHpaA抗血清为一抗,采用Western blot和ELISA对rPⅢ蛋白中展示的T/B联合表位进行筛选和鉴定.采用MTT检测rHpaA免疫小鼠脾细胞在不同重组噬菌体蛋白刺激下的增殖情况.结果 HpaA分子中共有5个T/B联合表位:HpaA10、HpaA37、HpaA79、HpaA116和HpaA143.所有T/B联合表位均成功地展示于M13噬菌体PⅢ蛋白表面.Western blot、ELISA和淋巴细胞增殖试验结果 均显示,HpaA116是优势抗原表位,HpaA37和HpaA79为有效抗原表位,HpaA10和HpaA143为无效抗原表位.结论 本研究成功地构建了幽门螺杆菌HpaA的T/B联合表位肽噬菌体展示系统.HpaA37和HpaA79,尤其是HpaA116是HpaA有效T/B联合抗原表位.     

问号钩端螺旋体血清群LipL32基因型分析及其重组蛋白的免疫学鉴定

目的　探讨 15群 15型问号钩端螺旋体 (简称钩体 )中国参考标准株及 2群 2型双曲钩体国际参考标准株是否均存在主要外膜蛋白 (MOMP)基因 (LipL32 ) ,克隆并构建该基因的原核表达系统 ,鉴定表达产物的免疫性。方法　常规酚 氯仿法提取上述钩体株基因组DNA ,高保真PCR扩增全长LipL32基因片段 ,T A克隆后测定核苷酸序列并构建表达系统 ,不同浓度IPTG诱导后用SDS PAGE检测rMOMP表达情况。分别用兔抗TR patocⅠ钩体全菌抗血清、rMOMPs免疫兔血清的Westernblot鉴定其免疫反应性和免疫原性 ,显微镜凝集试验 (MAT)检测rMOMPs免疫兔血清的交叉凝集效价 ,钩体细胞黏附模型检测抗体阻断效果。结果　上述 17株钩体均有LipL32基因 ,但可分LipL32 1和LipL32 2两种基因型。 13个LipL32 1和 4个Li pL32 2基因型之间核苷酸和氨基酸序列同源性分别为 95 .12 %～ 96 .6 0 %和 97.79%～ 98.16 %。IPTG诱导后rMOMP1和rMOMP2表达量分别占细菌总蛋白的 4 0 %和 10 %。rMOMP1和rMOMP2均能与兔抗钩体TR patocⅠ血清发生结合反应 ,免疫家兔可对上述 17株钩体产生 1∶2～ 1∶6 4MAT效价的凝集抗体。 1∶2～ 1∶16稀释的兔抗rMOMP1和rMOMP2血清均能有效地阻断钩体的黏附。结论　所有检测的钩体具有LipL32 1或LipL32 2基因。所     

问号钩端螺旋体rLTB/rCTB-LipL32/1免疫保护作用及野生株LipL32基因表达和患者抗体检测

目的构建LTB—LipL32／1和CTB—LipL32／1融合基因原核表达系统并鉴定其表达产物的免疫和佐剂活性及保护作用，了解问号钩端螺旋体(简称钩体)野生株LipL32基因携带、表达及钩体病人血清LipL32基因产物抗体产生频率。方法采用常规分子生物学技术构建LTB—LipL32／1和CTB—LipL32／1融合基因及其原核表达系统。采用SDS-PAGE检测目的重组蛋白rLTB-LipL32／1及rCTB-LipL32／1表达情况。分别采用Western blot和GM1-ELISA检测rLTB-LipL32／1及rCTB—LipL32／1的免疫反应性和佐剂活性。采用PCR和MAT分别检测97株问号钩体野生株LipL32基因及其表达情况。采用ELISA检测228例钩体病人血清LipL32基因产物的抗体。采用豚鼠保护试验检测重组蛋白的免疫保护作用。结果与报道的相关序列比较，LTB—LipL32／1和CTB-LipL32／1融合基因核苷酸序列相似性分别为99．12％～99．71％和98．54％～99．42％，氨基酸序列相似性分别为97．58％-99．63％和96．77％～99．63％。rLTB-LipL32／1和rCTB—LipL32／1表达产量均约为细菌总蛋白的10％，并主要以包涵体形式存在。rLTB—LipL32／1和rCTB-LipL32／1均分别能与LipL32／1兔抗血清和牛GMI结合。97．9％(95／97)问号钩体野生株含有LipL32基因，95．9％(93／97)问号钩体野生株与rLipL32／1和rLipL32／2兔抗血清的MAT结果阳性。97．4％(222／228)和94．7％(216／228)的病人血清rLipL32／1和rLipL32／2抗体阳性。rLTB-LipL32／1、rCTB-LipL32／1和rLipL32／1豚鼠保护率分别为75．0％、75．0％～87．5％、50．O％～62．5％。结论成功构建了LTB—LipL32／1和CTB—LipL32／1融合基因及其原核表达系统。rLTB-LipL32／1和rCTB-LipL32／1融合蛋白有良好的抗原性和佐剂活性及一定的免疫保护作用，具有成为问号钩体属特异性疫苗的良好前景。LipL32／1是不同问号钩体血清群中广泛存在、序列保守、高频率表达的基因。     

我国主要问号钩端螺旋体血清群lipL21基因序列及其产物免疫反应性分析

目的 分析我国15群15型问号钩端螺旋体（简称钩体）参考标准株外膜脂蛋白lipL21基因序列，构建该基因原核表达系统并鉴定表达产物的免疫原性，了解lipL21基因自然表达状况。方法 高保真PCR扩增上述问号钩体株及双曲钩体Paloc型PalocⅠ株基因组DNA中全长lipL21基因片段，T-A克隆后测序并构建其原核表达系统。分别用钩体TR/PatocⅠ和rLipL21兔抗血清为一抗的Western blot鉴定目的重组蛋白rLipL21的免疫原性，显微镜凝集试验（MAT）检测rLipL21兔抗血清的交叉凝集效价。以盐变-去垢剂处理法提取钩体外膜蛋白，用SDS-PAGE和免疫印迹法检测上述钩体株lipL21基因自然表达情况。结果 上述钩体株均存在序列高度保守的lipL21基因，其核苷酸和氨基酸序列相似性分别为98．75％～99．82％和99．46％～100％。rLipL21能与TR/PatocⅠ及rLipL21兔抗血清发生结合反应。rLipL21免疫家兔能产生抗体，该抗体对上述15株问号钩体MAT效价为1：16～1：128。问号钩体外膜标本中均可检出LipL21，双曲钩体则否。结论 我国钩体群参考标准株均含有序列保守的lipL21基因并自然表达于外膜，双曲钩体PatocⅠ株虽含有lipL21基因但未表达。rLipL21具有良好的抗原性和免疫反应性，有可能作为新型钩体疫苗或检测试剂盒的候选属特异性表面抗原之一。     

Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His(6)-OmpL1 and His(6)-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.     

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.     

Recombinant multiepitope protein for diagnosis of leptospirosis

Leptospirosis is an emerging infectious disease and is considered to be the most widespread zoonotic disease in the world. It can be misdiagnosed because manifestations of this febrile disease vary from mild flu-like symptoms to severe illness involving vital organs such as the liver and lungs. Therefore, accurate diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality is imperative for proper treatment. Here, we report a customized recombinant leptospirosis multiepitope protein (r-LMP) that can specifically detect the immunoglobulin class of anti-leptospirosis antibodies in patient sera. Immunodominant epitopes from leptospire outer membrane proteins OmpL1, LipL21, and LipL32 were predicted and confirmed using phage display and immunity reaction. On the basis of the sequences of the identified epitopes, five major immunodominant epitopes were selected to construct a synthetic gene, recombinant lmp. The recombinant lmp gene was doubled and expressed in Escherichia coli. The recombinant protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay for detection of special immunoglobulin M (IgM) or IgG in sera from patients with leptospirosis or other febrile illnesses and healthy subjects. The results showed that the r-LMP protein recognized IgG and IgM in all the sera that were microscope agglutination test positive, and there were no cross-reactions with other patient sera. This approach of creating customized antigens coupled to overexpression and simple purification offers a promising alternative option for leptospirosis diagnosis, with the potential to circumvent the drawbacks of whole-leptospirosis-antigen-based assays.     

Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.     

Immunodominance of LipL3293–272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies

Background/PurposeLeptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL32_93–147 and LipL32_148–184 middle domain of LipL32_93–184, and LipL32_171–214, and LipL32_215–272 of c-terminal LipL32_171–272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera.ResultsIgM-dominant was directed to highly surface accessible LipL32_148–184 and Lipl32_171–214. IgG dominance of LipL32_148–184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32_148–184 truncation whereas two LipL32_148–184 and LipL32_215–272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32_148–214 and IgG-dominant LipL32_148–184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32_243–253 and LipL32_122–130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera.ConclusionThe IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.     

Expression and Distribution of Leptospiral Outer Membrane Components during Renal Infection of Hamsters

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection. Hamsters were challenged with host-derived Leptospira kirschneri to generate sera which contained antibodies to antigens expressed in vivo. Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36. Although LipL36 is a prominent outer membrane antigen of cultivated L. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection. Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS. When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes. These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.