LigB Protein Research Articles

LipL41 and LigA/LigB Gene Silencing on a LipL32 Knockout Leptospira interrogans Reveals the Impact of Multiple Mutations on Virulence.

Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. The application of the CRISPR/Cas9 system has facilitated the generation of mutants and subsequent evaluation of phenotypes. Since DNA breaks induced by RNA-guided Cas9 nuclease are lethal to Leptospira, different methodologies were implemented to overcome this limitation. Initially, CRISPR interference (CRISPRi) was employed to create knockdown mutants, utilizing a catalytically inactive Cas9 (dCas9). Subsequently, the co-expression of CRISPR/Cas9 and a DNA repair system from Mycobacterium smegmatis enabled the generation of scarless knockout mutants. We eliminated plasmids from the lipL32 knockout L. interrogans strain and further achieved multiple gene mutations via gene silencing in this knockout background. Strains lacking both LipL41 and LipL32 and LigA, LigB, and LipL32, were evaluated. The absence of proteins LipL32 and LipL41 had no effect on leptospiral virulence. On the other hand, mutants lacking LigA, LigB, and LipL32 were unable to cause acute disease. The expanded apparatus for genetic manipulation of pathogenic leptospires via the CRISPR/Cas9 system has allowed the evaluation of multiple mutations upon leptospiral virulence. This work shows that LipL32 and LipL41 are not required for acute disease and consolidates LigA and LigB proteins as virulence factors.

Preliminary evaluation of Brazilian green propolis as coadjuvant of a subunit vaccine against lethal leptospirosis

Vaccination would be preferred to leptospirosis control measures. However, current vaccines are heat killed whole-cell bacterins that generate serovar specific protection and several side effects. Modern molecular assays have revealed antigens that may replace traditional whole-cell vaccines. Among these, LigB protein is surface-exposed outer membrane protein of virulent leptospires and therefore potential target of a protective immune response. Some unsuccessful attempts at using these antigens as vaccines have been reported. However, we believe that immune modulation through alternative adjuvants and co-adjuvants may overcome previous setbacks. In this light, our study aimed to evaluate the protective immune response in hamsters vaccinated with 40 µg of rLigBNI using oil adjuvant (OA), with or without green propolis (GP) as co-adjuvant. Upon a challenge, all groups immunized with rLigBNI, coupled or not with GP, were highly immunogenic and revealed statistically significant (p<0.05) protection of hamsters from lethal leptospirosis. Additional studies are being carried out to assess the optimum dose, protection against heterologous challenge, and vaccine dynamics.

Leptospiral Immunoglobulin-Like Domain Proteins: Roles in Virulence and Immunity.

The virulence mechanisms required for infection and evasion of immunity by pathogenic Leptospira species remain poorly understood. A number of L. interrogans surface proteins have been discovered, lying at the interface between the pathogen and host. Among these proteins, the functional properties of the Lig (leptospiral immunoglobulin-like domain) proteins have been examined most thoroughly. LigA, LigB, and LigC contain a series of, 13, 12, and 12 closely related domains, respectively, each containing a bacterial immunoglobulin (Big) -like fold. The multidomain region forms a mostly elongated structure that exposes a large surface area. Leptospires wield the Lig proteins to promote interactions with a range of specific host proteins, including those that aid evasion of innate immune mechanisms. These diverse binding events mediate adhesion of L. interrogans to the extracellular matrix, inhibit hemostasis, and inactivate key complement proteins. These interactions may help L. interrogans overcome the physical, hematological, and immunological barriers that would otherwise prevent the spirochete from establishing a systemic infection. Despite significant differences in the affinities of the LigA and LigB proteins for host targets, their functions overlap during lethal infection of hamsters; virulence is lost only when both ligA and ligB transcription is knocked down simultaneously. Lig proteins have been shown to be promising vaccine antigens through evaluation of a variety of different adjuvant strategies. This review serves to summarize current knowledge of Lig protein roles in virulence and immunity and to identify directions needed to better understand the precise functions of the Lig proteins during infection.

Cloning, expression and purification of Leptospira LigB antigen in Escherichia coli

Leptospira is one of the most common zoonotic diseases in the tropics and subtropics. Humans are infected by exposure to Leptospira contained water or food sources. Leptospirosis usually breaks out after the flood and causes several consequences for people and economy. Leptospirosis disease, if not rapidly detected and treated promptly, it causes serious consequences such as acute hepatitis-kidneys, meningitis and bleeding, heart and nerve complications, and severe illness can lead to death. Therefore, quick and accurate detection of Leptospira pathogen plays a very important role in Leptospirosis disease treatment. Among antigens of Leptospira, a conserved domain of LigB antigen (Leptospiral immunoglobulin-like protein) was reported that is present in the most of pathogenic serovars of Leptospira, but not in the non-pathogenic Leptospira biflexa, thus this conserved domain was used for production of Leptospirosis detection kits as well as vaccine for Leptospirosis. In order to create a kit for Leptospirosis diagonostic, especially detect anti-Leptospira antibodies in Leptospira infected serum and plasma samples, about 1kb gene fragment encoding for conserved domain of LigB (about 36 kb in molecular weight) was used as the material for producing of LigB protein by DNA recombinant technology. In this study, we present the results for cloning, expressing a conserved domain of LigB antigen in E. coli cells and purifying protein by affinity chromatography collumn. The result indicates that recombinant LigB protein was successfully expressed in E. coli Rosetta 1 and purified by Hitrap chealating collumn. The LigB protein concentration after purification reached 60 mg/L medium with 98% purity. This purified protein will be used as the materials for creating Leptospirosis kit.

Comparative evaluation of recombinant LigB based latex agglutination test with microscopic agglutination test for the diagnosis of wildlife leptospirosis

In the present study a comparative evaluation microscopic agglutination test with rLigB protein based latex agglutination test was carried out, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiralserovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected wildlife field sera. A total of 80 wildlife sera samples were collec ted, including 27 wild feline sera samples (18 tigers, 8 lions, and 1 jaguar) obtained from Chhatbir zoo, Chandigarh, 42 sera samples (8 tigers, 4 lions and 6 leopards, 2 cheethals, 1 black buck, 12 buffaloes and 9 zoo staff) were received from Jodhpur zoo Rajasthan, 8 sera samples (4 tigers, 3 leopards, 1 lion) from Van Vihar National park, Bohpal, Madhya Pradesh and 3 sera samples (2 lions and 1 tiger) received from Biwani Mini zoo, Haryana, India. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity, under laboratory and field conditions, for the detection of Leptospirosis.
 Asian J. Med. Biol. Res. September 2020, 6(3): 440-448

Recombinant leptospiral immunoglobulin like B protein based latex agglutination test for serodiagnosis of human leptospirosis

Humans get leptospirosis by contact with fresh water, damp soil, or vegetation contaminated by the urine of infected animals, swallowing contaminated food or water or while working in contaminated flood plains or at wet agricultural settings. The bacteria enter the body through abrasions in the skin and mucous membranes. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against leptospirosis suspected human sera. A total of 28 human sera samples were received from Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh India, which tested positive by IgM ELISA test kit were subjected to both rLigB based LAT and MAT. All the 28 sera showed seropositivity by both the tests. Icterohaemorrahigae was the predominant serovar followed by Javanica and Grippotyphosa. Six out seven sera samples received from Indian Veterinary research Institute, Human Hospital and City Hospital, Bareilly were tested positive both by rLAT and MAT. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, reliable diagnostic tool at resource poor and remote diagnostic centers with high sensitivity and specificity, under laboratory and field conditions, for the detection of leptospirosis.
 Asian J. Med. Biol. Res. June 2020, 6(2): 229-236

Cloning and Expression of Leptospiral Immunoglobulin Like B Gene and Use of The Recombinant Antigen for The Diagnosis of Bovine and Caprine Leptospirosis

In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected field sea. Western blot confirmed that rLigB is an immunodominant protein against which antibodies are produced during active infection. A total of 453 field sera, including 432 bovine sera, 18 caprine sera and three sera samples of buffalo bull collected post-mortem following death the animal from Indian Veterinary Research institute (IVRI) were tested using rLigB based LAT. The result showed that 300 sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, under laboratory and field conditions, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity for the detection of Leptospirosis. 
 Int. J. Appl. Sci. Biotechnol. Vol 8(2): 146-153

Serodiagnosis of wildlife leptospirosis employing recombinant leptospiral immunoglobulin like b protein antigen

Leptospirosis is a major threat in tropical and subtropical countries as well as temperate countries. The disease is caused by pathogenic Leptospira species and considered to be an emerging or re-emerging disease in many countries of the world. Infection in domestic animals and wildlife can lead to economic loss and pose a potential spread to the communities. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in P QE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected wildlife field sera. A total of 80 wildlife sera samples were collected, including 27 wild feline sera samples (18 tigers, 8 lions, and 1 jaguar) obtained from Chhatbir zoo, Chandigarh, 42 serasamples ( 8 tigers, 4 lions and 6 leopards, 2 cheethals, 1 black buck, 12 buffaloes and 9 zoo staff) sera and 3 live rodents ) were received from Jodhpur zoo Rajasthan, 8 sera samples (4 tigers, 3 leopards, 1 lion) sera from Van Vihar National park, Bohpal, Madhya Pradesh and 3 sera samples (2 lions,and 1 tiger) received from Biwani Mini zoo, Haryana, India. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity, under laboratory and field conditions, for the detection of Leptospirosis .

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum.

Diversidad molecular de candidatos vacunales en Leptospira spp.

El objetivo de este estudio fue determinar la diversidad molecular de las proteínas OmpL1, LipL32, LipL41, LigA y LigB y de los genes que las codifican mediante análisis bioinformáticos en diferentes cepas patógenas de Leptospira spp., a partir de la información disponible en las bases de datos. Se utilizaron las secuencias de aminoácidos de las proteínas OmpL1, LipL32, LipL41, LigA y LigB, así como las de los genes que las codifican en las cepas de Leptospira spp. registradas en The National Center for Biotechnology Information (NCBI). Los análisis de las proteínas y los genes se realizaron mediante los recursos Protein, Nucleotide y Gene del NCBI. La alineación de las secuencias consenso se realizó con las herramientas PSI-BLAST y BLASTn. El porcentaje de cobertura de las secuencias seleccionadas de los genes ompL1, lipL32, lipL41, ligA y ligB en cepas patógenas de Leptospira spp. es de 100 % para ompL1, lipL32 y lipL41, 75 % para ligA y 99 % para ligB con porcentajes de identidad de 85, 98, 88, 90 y 80 % respectivamente; el porcentaje de cobertura de las secuencias seleccionadas de las proteínas es de 100, 77, 99, 100 y 100 % con porcentajes de identidad de 90, 99, 92, 63 y 60 % respectivamente, lo cual indica que los genes y las proteínas, excepto las proteínas LigA y LigB, son bastante conservadas en los diferentes serovares patógenos de Leptospira spp. Según dichos resultados, se recomienda realizar análisis complementarios de estas proteínas con el fin de determinar si es viable su uso como candidatos vacunales.

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis

This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

NMR solution structure of the terminal immunoglobulin-like domain from the leptospira host-interacting outer membrane protein, LigB.

A number of surface proteins specific to pathogenic strains of Leptospira have been identified. The Lig protein family has shown promise as a marker in typing leptospiral isolates for pathogenesis and as an antigen in vaccines. We used NMR spectroscopy to solve the solution structure of the twelfth immunoglobulin-like (Ig-like) repeat domain from LigB (LigB-12). The fold is similar to that of other bacterial Ig-like domains and comprised mainly of β-strands that form a β-sandwich based on a Greek-key folding arrangement. Based on sequence analysis and conservation of structurally important residues, homology models for the other LigB Ig-like domains were generated. The set of LigB models illustrates the electrostatic differences between the domains as well as the possible interactions between neighboring domains. Understanding the structure of the extracellular portion of LigB and related proteins is important for developing diagnostic methods and new therapeutics directed toward leptospirosis.

Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life.

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

A Conserved Region of Leptospiral Immunoglobulin-Like A and B Proteins as a DNA Vaccine Elicits a Prophylactic Immune Response against Leptospirosis

The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.

Lepstospira interrogans shotgun phage display identified LigB as a heparin-binding protein

LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexaconfers enhanced adhesion to cultured cells and fibronectin

BackgroundIn comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc.ResultsThe genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion.ConclusionsThis work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis.

A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB

Infection by pathogenic strains of Leptospira hinges on the pathogen’s ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708–1712 containing sequence LIPAD with a β-strand and nascent helical structure. This motif binds to 15th type III modules (15F3) (KD=10.70μM), and association (kon=600M−1s−1) and dissociation (koff=0.0129s−1) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB1706–1716 blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB1706–1716 in cellular adhesion. These data indicate that the LIPAD residues (LigB1708–1712) of the Leptospira interrogans LigB protein bind 15F3 of Fn at a novel binding site, and this interaction contributes to adhesion to host cells.

Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.