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1.  Identification of Immunodominant B- and T-Cell Combined Epitopes in Outer Membrane Lipoproteins LipL32 and LipL21 of Leptospira interrogans▿  
Leptospirosis is a serious infectious disease caused by pathogenic Leptospira. B- and T-cell-mediated immune responses contribute to the mechanisms of Leptospira interrogans infection and immune intervention. LipL32 and LipL21 are the conserved outer membrane lipoproteins of L. interrogans and are considered vaccine candidates. In this study, we identified B- and T-cell combined epitopes within LipL32 and LipL21 to further develop a novel vaccine. By using a computer prediction algorithm, two B- and T-cell combined epitopes of LipL21 and four of LipL32 were predicted. All of the predicted epitopes were expressed in a phage display system. Four epitopes, LipL21 residues 97 to 112 and 176 to 184 (LipL2197-112 and LipL21176-184, respectively) and LipL32133-160 and LipL32221-247 of LipL32 were selected as antigens by Western blotting and enzyme-linked immunosorbent assay. These selected epitopes were also recognized by CD4+ T lymphocytes derived from LipL21- or LipL32-immunized BALB/c (H-2d) mice and mainly polarized the immune response toward a Th1 phenotype. The identification of epitopes that have both B- and T-cell immune reactivities is of value for studying the immune mechanisms in response to leptospiral infection and for designing an effective vaccine for leptospirosis.
doi:10.1128/CVI.00405-09
PMCID: PMC2863375  PMID: 20237196
2.  High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris 
Background
Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.
Results
The vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis.
Conclusions
The expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.
doi:10.1186/1475-2859-9-98
PMCID: PMC3004844  PMID: 21134266
3.  Cloning and Sequence Analysis of LipL32, a Surface–Exposed Lipoprotein of Pathogenic Leptospira Spp 
Background
Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira species. A major challenge of this disease is the application of basic research to improve diagnostic methods and related vaccine development. Outer membrane proteins of Leptospira are potential candidates that may be useful as diagnostic or immunogenic factors in treatment and analysis of the disease.
Objectives
To develop an effective subunit vaccine against prevalent pathogenic Leptospira species, we sequenced and analyzed the LipL32 gene from three different Leptospira interrogans (L.interrogans) vaccinal serovars in Iran.
Materials and Methods
Following DNA extraction from these three serovars, the related LipL32 genes were amplified and cloned in the pTZ57R/T vector. Recombinant clones were confirmed by colony- PCR and DNA sequencing. The related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database.
Results
The LipL32 sequences were >94% homologous among the vaccinal and other pathogenic Leptospira serovars in GenBank. This result indicates the conservation of this gene within the pathogenic Leptospires.
Conclusions
The cloned gene in this study may provide a potentially suitable platform for development of a variety of applications such as serological diagnostic tests or recombinant vaccines against leptospirosis.
doi:10.5812/ircmj.8793
PMCID: PMC3971780  PMID: 24719688
Leptospirosis; Leptospira Interrogans; LipL32
4.  Leptospiral Outer Membrane Proteins OmpL1 and LipL41 Exhibit Synergistic Immunoprotection 
Infection and Immunity  1999;67(12):6572-6582.
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 His6-OmpL1 and His6-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.
PMCID: PMC97069  PMID: 10569777
5.  LipL21 Is a Novel Surface-Exposed Lipoprotein of Pathogenic Leptospira Species  
Infection and Immunity  2003;71(5):2414-2421.
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.
doi:10.1128/IAI.71.5.2414-2421.2003
PMCID: PMC153295  PMID: 12704111
6.  LipL46 is a novel surface-exposed lipoprotein expressed during leptospiral dissemination in the mammalian host 
Microbiology (Reading, England)  2006;152(Pt 12):3777-3786.
Leptospirosis is a widespread zoonosis caused by invasive spirochaetes belonging to the genus Leptospira. Pathogenic leptospires disseminate via the bloodstream to colonize the renal tubules of reservoir hosts. Little is known about leptospiral outer-membrane proteins expressed during the dissemination stage of infection. In this study, a novel surface-exposed lipoprotein is described; it has been designated LipL46 to distinguish it from a previously described 31 kDa peripheral membrane protein, P31LipL45, which is exported as a 45 kDa probable lipoprotein. The lipL46 gene encodes a 412 aa polypeptide with a 21 aa signal peptide. Lipid modification of cysteine at the lipoprotein signal peptidase cleavage site FSISC is supported by the finding that Leptospira interrogans intrinsically labels LipL46 during incubation in medium containing [14C]palmitate. LipL46 appears to be exported to the leptospiral outer membrane as a 46 kDa lipoprotein, based on Triton X-114 solubilization and phase partitioning studies, which included the outer and inner membrane controls LipL32 and LipL31, respectively. Surface immunoprecipitation and whole-cell ELISA experiments indicate that LipL46 is exposed on the leptospiral surface. Immunohistochemistry studies demonstrated expression of LipL46 by leptospires found in the bloodstream of acutely infected hamsters. Leptospires expressing LipL46 were also found in the intercellular spaces of the liver, within splenic phagocytes, and invading the glomerular hilum of the kidney. Infection-associated expression is supported by the finding that LipL46 is a major antigen recognized by sera from infected hamsters. These findings indicate that LipL46 may be important in leptospiral dissemination, and that it may serve as a useful serodiagnostic antigen.
doi:10.1099/mic.0.29162-0
PMCID: PMC2667200  PMID: 17159228
7.  Leptospiral Outer Membrane Protein LipL41 Is Not Essential for Acute Leptospirosis but Requires a Small Chaperone Protein, Lep, for Stable Expression 
Infection and Immunity  2013;81(8):2768-2776.
Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.
doi:10.1128/IAI.00531-13
PMCID: PMC3719587  PMID: 23690405
8.  Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans▿ †  
Infection and Immunity  2008;77(3):952-958.
Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B12. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.
doi:10.1128/IAI.01370-08
PMCID: PMC2643616  PMID: 19103763
9.  Differential In Vivo Gene Expression of Major Leptospira Proteins in Resistant or Susceptible Animal Models 
Applied and Environmental Microbiology  2012;78(17):6372-6376.
Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.
doi:10.1128/AEM.00911-12
PMCID: PMC3416592  PMID: 22729538
10.  Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein  
Infection and Immunity  2002;70(1):323-334.
Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the α, β, and β′ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val−4-Phe−3-Asn−2-Ala−1↓Cys+1 was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31LipL45. Membrane fractionation studies demonstrated that P31LipL45 is a peripheral membrane protein. Finally, we found that P31LipL45 levels increased as Leptospira entered the stationary phase, indicating that P31LipL45 levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.
doi:10.1128/IAI.70.1.323-334.2002
PMCID: PMC127625  PMID: 11748198
11.  Expression and Distribution of Leptospiral Outer Membrane Components during Renal Infection of Hamsters 
Infection and Immunity  1999;67(2):853-861.
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.
PMCID: PMC96396  PMID: 9916100
12.  LipL41, a Hemin Binding Protein from Leptospira santarosai serovar Shermani 
PLoS ONE  2013;8(12):e83246.
Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a Kd = 0.59 ± 0.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at 140Cys-Ser and 220Cys-Pro. The mutation study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly.
doi:10.1371/journal.pone.0083246
PMCID: PMC3861479  PMID: 24349474
13.  Protection against Leptospira interrogans Sensu Lato Challenge by DNA Immunization with the Gene Encoding Hemolysin-Associated Protein 1  
Infection and Immunity  2005;73(7):4062-4069.
The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.
doi:10.1128/IAI.73.7.4062-4069.2005
PMCID: PMC1168576  PMID: 15972494
14.  Evaluation of Immunoprotective Activity of Six Leptospiral Proteins in the Hamster Model of Leptospirosis 
Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of L. interrogans serovar Copenhageni together with bioinformatics tools represent a great opportunity to search for novel antigen candidates that could be used as subunit vaccine against leptospirosis. We focused on six genes encoding for conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from Leptospira interrogans genomic DNA and were cloned and expressed in Escherichia coli. The recombinant proteins tagged with N-terminal hexahistidine were purified by metal-charged chromatography. The immunization of hamsters followed by challenge with lethal dose of virulent strain of Leptospira showed that the recombinant proteins Lsa21, Lsa66 and rLIC11030 elicited partial protection to animals. These proteins could be used combined or in a mixture with novel adjuvants in order to improve their effectiveness.
doi:10.2174/1874285801206010079
PMCID: PMC3502890  PMID: 23173023
Leptospira interrogans; leptospirosis; recombinant protein; vaccine.
15.  Detection and Quantification of Leptospira interrogans in Hamster and Rat Kidney Samples: Immunofluorescent Imprints versus Real-time PCR 
PLoS ONE  2012;7(2):e32712.
A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.
doi:10.1371/journal.pone.0032712
PMCID: PMC3290571  PMID: 22393440
16.  Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity 
PLoS ONE  2011;6(3):e17409.
Background
Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field.
Methodology and Principal Findings
A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain.
Conclusions
The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.
doi:10.1371/journal.pone.0017409
PMCID: PMC3060810  PMID: 21445252
17.  In LipL32, the Major Leptospiral Lipoprotein, the C Terminus Is the Primary Immunogenic Domain and Mediates Interaction with Collagen IV and Plasma Fibronectin ▿  
Infection and Immunity  2008;76(6):2642-2650.
LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.
doi:10.1128/IAI.01639-07
PMCID: PMC2423089  PMID: 18391007
18.  LipL32 Is an Extracellular Matrix-Interacting Protein of Leptospira spp. and Pseudoalteromonas tunicata▿  
Infection and Immunity  2008;76(5):2063-2069.
LipL32 is the major outer membrane protein in pathogenic Leptospira. It is highly conserved throughout pathogenic species and is expressed in vivo during human infection. While these data suggest a role in pathogenesis, a function for LipL32 has not been defined. Outer membrane proteins of gram-negative bacteria are the first line of molecular interaction with the host, and many have been shown to bind host extracellular matrix (ECM). A search for leptospiral ECM-interacting proteins identified the major outer membrane protein, LipL32. To verify this finding, recombinant LipL32 was expressed in Escherichia coli and was found to bind Matrigel ECM and individual components of ECM, including laminin, collagen I, and collagen V. Likewise, an orthologous protein found in the genome of Pseudoalteromonas tunicata strain D2 was expressed and found to be functionally similar and immunologically cross-reactive. Lastly, binding activity was mapped to the C-terminal 72 amino acids. These studies show that LipL32 and an orthologous protein in P. tunicata are immunologically cross-reactive and function as ECM-interacting proteins via a conserved C-terminal region.
doi:10.1128/IAI.01643-07
PMCID: PMC2346718  PMID: 18285490
19.  Characterization of Conserved Combined T and B Cell Epitopes in Leptospira interrogans Major Outer Membrane Proteins OmpL1 and LipL41 
BMC Microbiology  2011;11:21.
Background
Leptospira interrogans are bacterial pathogens of animal that cause zoonotic infections in human. Outer membrane proteins of leptospire are among the most effective antigens which can stimulate remarkable immune responses during the infection processes, and thus are currently considered leading candidate vaccine antigens. The objective of the present study is to predict and confirm major combined B and T cell epitopes of leptospiral outer membrane proteins OmpL1 and LipL41, as well as to evaluate their capacity in the induction of immune responses in BALB/c mice.
Results
In this study, four epitopes from OmpL1 and four from LipL41 conserved regions were evaluated for their potential utilization in leptospire vaccines. Firstly, combined B and T cell epitopes were predicted by softwares and expressed using a phage display system. OmpL1 residues 87-98 and 173-191 (OmpL187-98 and OmpL1173-191) and LipL4130-48, LipL41233-256 of LipL41 were identified as immunodominant B cell epitopes by Western blot. Epitopes OmpL1173-191, OmpL1297-320 of OmpL1 and LipL41233-256, LipL41263-282 of LipL41 were identified as immunodominant CD4+ T cell epitopes through proliferation analysis of splenocytes from recombinant OmpL1 (rOmpL1) or recombinant LipL41 (rLipL41)-immunized BALB/c (H-2d) mice. These epitopes induced responses of CD4+ T cells and Th1 (T helper cells) type cytokine responses during the infection.
Conclusion
This work identified combined T and B cell immunodominant epitopes in outer membrane proteins OmpL1 and LipL41 of Leptospira interrogans. OmpL1173-191 of OmpL1 and LipL41233-256 of LipL41 could be useful in a vaccine against Leptospira. The findings could also contribute to the development of effective cross-protective vaccine strategies for leptospirosis.
doi:10.1186/1471-2180-11-21
PMCID: PMC3038132  PMID: 21269437
20.  Lipase modulator protein (LimL) of Pseudomonas sp. strain 109. 
Journal of Bacteriology  1995;177(5):1254-1258.
Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.
PMCID: PMC176731  PMID: 7868599
21.  Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae▿ 
Clinical and Vaccine Immunology : CVI  2011;18(12):2168-2177.
A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.
doi:10.1128/CVI.05230-11
PMCID: PMC3232707  PMID: 22030372
22.  LipL32 Is a Subsurface Lipoprotein of Leptospira interrogans: Presentation of New Data and Reevaluation of Previous Studies 
PLoS ONE  2013;8(1):e51025.
The agents of leptospirosis, a zoonosis with worldwide distribution, are pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via fresh water and colonization of the renal tubules of their reservoir hosts. Infection of accidental hosts, including humans, may result in life-threatening sequelae. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen virulence mechanisms and adaptation to environmental conditions, including those found in the mammalian host. Therefore, elucidation and characterization of the surface-exposed OMPs of Leptospira spp. is of great interest in the leptospirosis field. A thorough, multi-pronged approach for assessing surface exposure of leptospiral OMPs is essential. Herein, we present evidence for a sub-surface location for most or all of the major leptospiral lipoprotein, LipL32, based on surface immunofluorescence utilizing three different types of antibodies and four different permeabilization methods, as well as surface proteolysis of intact and lysed leptospires. We reevaluate prior evidence presented in support of LipL32 surface-exposure and present a novel perspective on a protein whose location has been misleading researchers, due in large part to its extraordinary abundance in leptospiral cells.
doi:10.1371/journal.pone.0051025
PMCID: PMC3544172  PMID: 23323152
23.  Surfaceome of Leptospira spp.  
Infection and Immunity  2005;73(8):4853-4863.
The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31LipL45, GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<107 cells) offering the potential to analyze bacterial surface expression during infection.
doi:10.1128/IAI.73.8.4853-4863.2005
PMCID: PMC1201201  PMID: 16040999
24.  Oral immunization with an attenuated Salmonella Gallinarum mutant as a fowl typhoid vaccine with a live adjuvant strain secreting the B subunit of Escherichia coli heat-labile enterotoxin 
Background
The Salmonella Gallinarum (SG) lon/cpxR deletion mutant JOL916 was developed as a live vaccine candidate for fowl typhoid (FT), and a SG mutant secreting an Escherichia coli heat-labile enterotoxin B subunit (LTB), designated JOL1229, was recently constructed as an adjuvant strain for oral vaccination against FT. In this study, we evaluated the immunogenicity and protective properties of the SG mutant JOL916 and the LTB adjuvant strain JOL1229 in order to establish a prime and boost immunization strategy for each strain. In addition, we compared the increase in body weight, the immunogenicity, the egg production rates, and the bacteriological egg contamination of these strains with those of SG 9R, a widely used commercial vaccine.
Results
Plasma IgG, intestinal secretory IgA (sIgA), and cell-mediated responses were significantly induced after a boost inoculation with a mixture of JOL916 and JOL1229, and significant reductions in the mortality of chickens challenged with a wild-type SG strain were observed in the immunized groups. There were no significant differences in increases in body weight, cell-mediated immune responses, or systemic IgG responses between our vaccine mixture and the SG 9R vaccine groups. However, there was a significant elevation in intestinal sIgA in chickens immunized with our mixture at 3 weeks post-prime-immunization and at 3 weeks post-boost-immunization, while sIgA levels in SG 9R-immunized chickens were not significantly elevated compared to the control. In addition, the SG strain was not detected in the eggs of chickens immunized with our mixture.
Conclusion
Our results suggest that immunization with the LTB-adjuvant strain JOL1229 can significantly increase the immune response, and provide efficient protection against FT with no side effects on body weight, egg production, or egg contamination.
doi:10.1186/1746-6148-9-96
PMCID: PMC3653815  PMID: 23647814
25.  Evaluation and comparison of native and recombinant LipL21 protein-based ELISAs for diagnosis of bovine leptospirosis 
Journal of Veterinary Science  2012;13(1):99-101.
A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis.
doi:10.4142/jvs.2012.13.1.99
PMCID: PMC3317464  PMID: 22437542
ELISA; leptospirosis; microscopic agglutination test; nLipL21; rLipL21

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