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1.  Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily 
Molecular microbiology  2003;49(4):929-945.
Summary
Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.
doi:10.1046/j.1365-2958.2003.03619.x
PMCID: PMC1237129  PMID: 12890019
2.  Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen▿  
Infection and Immunity  2007;75(5):2441-2450.
Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.
doi:10.1128/IAI.01635-06
PMCID: PMC1865782  PMID: 17296754
3.  The Multifunctional LigB Adhesin Binds Homeostatic Proteins with Potential Roles in Cutaneous Infection by Pathogenic Leptospira interrogans 
PLoS ONE  2011;6(2):e16879.
Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9–11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats.
doi:10.1371/journal.pone.0016879
PMCID: PMC3036719  PMID: 21347378
4.  Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin 
BMC Microbiology  2011;11:129.
Background
In 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.
Results
The 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.
Conclusions
This 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.
doi:10.1186/1471-2180-11-129
PMCID: PMC3133549  PMID: 21658265
5.  Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates 
Journal of Medical Microbiology  2009;58(Pt 9):1173-1181.
The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.
doi:10.1099/jmm.0.009175-0
PMCID: PMC2887549  PMID: 19528180
6.  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.
doi:10.1128/CVI.00601-12
PMCID: PMC3647749  PMID: 23486420
7.  Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of the LigB Gene Does Not Affect Virulence in Animal Models of Leptospirosis▿  
Infection and Immunity  2008;76(12):5826-5833.
The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spcr) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.
doi:10.1128/IAI.00989-08
PMCID: PMC2583567  PMID: 18809657
8.  Osmolarity, a Key Environmental Signal Controlling Expression of Leptospiral Proteins LigA and LigB and the Extracellular Release of LigA  
Infection and Immunity  2005;73(1):70-78.
The high-molecular-weight leptospiral immunoglobulin-like repeat (Lig) proteins are expressed only by virulent low-passage forms of pathogenic Leptospira species. We examined the effects of growth phase and environmental signals on the expression, surface exposure, and extracellular release of LigA and LigB. LigA was lost from stationary-phase cells, while LigB expression was maintained. The loss of cell-associated LigA correlated with selective release of a lower-molecular-weight form of LigA into the culture supernatant, while LigB and the outer membrane lipoprotein LipL41 remained associated with cells. Addition of tissue culture medium to leptospiral culture medium induced LigA and LigB expression and caused a substantial increase in released LigA. The sodium chloride component of tissue culture medium was primarily responsible for the enhanced release of LigA. Addition of sodium chloride, potassium chloride, or sodium sulfate to leptospiral medium to physiological osmolarity caused the induction of both cell-associated LigA and LigB, indicating that osmolarity regulates the expression of Lig proteins. Osmotic induction of Lig expression also resulted in enhanced release of LigA and increased surface exposure of LigB, as determined by surface immunofluorescence. Osmolarity appears to be a key environmental signal that controls the expression of LigA and LigB.
doi:10.1128/IAI.73.1.70-78.2005
PMCID: PMC538979  PMID: 15618142
9.  The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells 
PLoS ONE  2010;5(6):e11301.
Leptospira spp. are pathogenic spirochetes that cause the zoonotic disease leptospirosis. Leptospiral immunoglobulin (Ig)-like protein B (LigB) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin, fibrinogen, laminin, elastin, tropoelastin and collagen. A high-affinity Fn-binding region of LigB has been localized to LigBCen2, which contains the partial 11th and full 12th Ig-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, the gelatin binding domain of fibronectin was shown to interact with LigBCen2R (KD = 1.91±0.40 µM). Not only LigBCen2R but also other Ig-like domains of Lig proteins including LigAVar7'-8, LigAVar10, LigAVar11, LigAVar12, LigAVar13, LigBCen7'-8, and LigBCen9 bind to GBD. Interestingly, a large gain in affinity was achieved through an avidity effect, with the terminal domains, 13th (LigA) or 12th (LigB) Ig-like repeat of Lig protein (LigAVar7'-13 and LigBCen7'-12) enhancing binding affinity approximately 51 and 28 fold, respectively, compared to recombinant proteins without this terminal repeat. In addition, the inhibited effect on MDCKs cells can also be promoted by Lig proteins with terminal domains, but these two domains are not required for gelatin binding domain binding and cell adhesion. Interestingly, Lig proteins with the terminal domains could form compact structures with a round shape mediated by multidomain interaction. This is the first report about the interaction of gelatin binding domain of Fn and Lig proteins and provides an example of Lig-gelatin binding domain binding mediating bacterial-host interaction.
doi:10.1371/journal.pone.0011301
PMCID: PMC2892007  PMID: 20585579
10.  Immunoprotection of Recombinant Leptospiral Immunoglobulin-Like Protein A against Leptospira interrogans Serovar Pomona Infection  
Infection and Immunity  2006;74(3):1745-1750.
We previously reported the cloning and characterization of leptospiral immunoglobulin-like proteins LigA and LigB of Leptospira interrogans. LigA and LigB are conserved at the amino-terminal region but are variable at the carboxyl-terminal region. Here, we evaluate the potential of recombinant LigA (rLigA) as a vaccine candidate against infection by L. interrogans serovar Pomona in a hamster model. rLigA was truncated into conserved (rLigAcon) and variable (rLigAvar) regions and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (rLigA). Golden Syrian hamsters were immunized at 3 and 6 weeks of age with rLigA (rLigAcon and rLigAvar) with aluminum hydroxide as an adjuvant. Hamsters given recombinant glutathione-S-transferase (rGST)-adjuvant and phosphate-buffered saline-adjuvant served as nonvaccinated controls. Three weeks after the last vaccination, all animals were challenged intraperitoneally with 108 L. interrogans serovar Pomona bacteria (NVSL 1427-35-093002). All hamsters immunized with recombinant LigA survived after challenge and had no significant histopathological changes. In contrast, nonimmunized and rGST-immunized hamsters were subjected to lethal doses, and the hamsters that survived showed severe tubulointerstitial nephritis. All vaccinated animals showed a rise in antibody titers against rLigA. Results from this study indicate that rLigA is a potential vaccine candidate against L. interrogans serovar Pomona infection.
doi:10.1128/IAI.74.3.1745-1750.2006
PMCID: PMC1418682  PMID: 16495547
11.  Genetic and Biochemical Characterization of 4-Carboxy-2-Hydroxymuconate-6-Semialdehyde Dehydrogenase and Its Role in the Protocatechuate 4,5-Cleavage Pathway in Sphingomonas paucimobilis SYK-6 
Journal of Bacteriology  2000;182(23):6651-6658.
Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase. The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol dehydrogenase of Alcaligenes sp. strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13–56, 1994). The ligC gene was expressed in Escherichia coli and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP+ by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25°C, respectively. The Km for NADP+ was estimated to be 24.6 ± 1.5 μM, which was approximately 10 times lower than that for NAD+ (252 ± 3.9 μM). The Kms for CHMS in the presence of NADP+ and NAD+ are 26.0 ± 0.5 and 20.6 ± 1.0 μM, respectively. Disruption of ligC in S. paucimobilis SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of ligC into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for ligAB gene expression.
PMCID: PMC111406  PMID: 11073908
12.  The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis 
Vaccine  2007;25(33):6277-6286.
Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.
doi:10.1016/j.vaccine.2007.05.053
PMCID: PMC1994161  PMID: 17629368
Leptospirosis; subunit vaccine; Leptospiral immunoglobulin-like protein; recombinant protein; immunity; antibodies; hamsters
13.  Identification of immunodominant antigens in canine leptospirosis by Multi-Antigen Print ImmunoAssay (MAPIA) 
BMC Veterinary Research  2014;10(1):288.
Background
The microscopic agglutination test (MAT), the standard method for serological diagnosis of leptospirosis, may present limitations regarding its sensitivity. Current studies suggest that Leptospira immunoglobulin-like (Lig) proteins and LipL32 are of particular interest as serodiagnostic markers since they are present only in pathogenic species of the Leptospira genus. The purpose of this study was to identify leptospiral immunodominant proteins that are recognized by canine sera from diseased dogs.
Results
A total of 109 dogs were studied, including seroreactive dogs (MAT ≥800) and dogs with no seroreactivity detectable by MAT. Eight recombinant fragments (31–70 kDa) of pathogenic Leptospira were tested for their use as diagnostic markers for canine leptospirosis using the Multi-antigen Print Immunoassay (MAPIA) platform: LigB [582-947aa] from L. interrogans serovar Pomona, L. interrogans serovar Copenhageni and L. kirschneri serovar Gryppotyphosa, LigB [131-649aa] from L. interrogans serovar Copenhageni, L. interrogans serovar Canicola and L. kirschneri serovar Gryppotyphosa, LigA [625-1224aa] L. interrogans serovar Copenhageni and LipL32 from L. interrogans serovar Copenhageni. The data were analyzed and ROC curves were generated. Altogether, LigB [131-649aa] L. interrogans Canicola, LigB [131-649aa] L. kirschneri Gryppotyphosa and LipL32 L. interrogans Copenhageni showed best accuracy (AUC = 0.826 to 0.869), with 70% specificity and sensitivity ranging from 89% to 95%.
Conclusions
These results reinforce their potential as diagnostic candidates for the development of new methods for the serological diagnosis of canine leptospirosis.
doi:10.1186/s12917-014-0288-2
PMCID: PMC4269070  PMID: 25466383
Leptospirosis; Dogs; MAPIA; LipL32; Lig proteins
14.  Multiple Activities of LigB Potentiate Virulence of Leptospira interrogans: Inhibition of Alternative and Classical Pathways of Complement 
PLoS ONE  2012;7(7):e41566.
Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7′-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective Kds of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans to efficiently counteract the multiple homeostatic processes of the host.
doi:10.1371/journal.pone.0041566
PMCID: PMC3402383  PMID: 22911815
15.  Role for cis-Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans 
Journal of Bacteriology  2013;195(22):5092-5101.
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-tRNAfMet-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.
doi:10.1128/JB.00663-13
PMCID: PMC3811586  PMID: 24013626
16.  Leptospira Immunoglobulin-Like Proteins as a Serodiagnostic Marker for Acute Leptospirosis▿  
Journal of Clinical Microbiology  2007;45(5):1528-1534.
There is an urgent need for improved diagnosis of leptospirosis, an emerging infectious disease which imparts a large disease burden in developing countries. We evaluated the use of Leptospira immunoglobulin (Ig)-like (Lig) proteins as a serodiagnostic marker for leptospirosis. Lig proteins have bacterial immunoglobulin-like (Big) tandem repeat domains, a moiety found in virulence factors in other pathogens. Sera from patients identified during urban outbreaks in Brazil reacted strongly with immunoblots of a recombinant fragment comprised of the second to sixth Big domains of LigB from L. interrogans serovar Copenhageni, the principal agent for transmission in this setting. Furthermore, the sera recognized an analogous LigB fragment derived from L. kirschneri serovar Grippotyphosa, a pathogenic serovar which is not endemic to the study area. The immunoblot assay detected anti-LigB IgM antibodies in sera from 92% (95% confidence interval, 85 to 96%) of patients during acute-phase leptospirosis. The assay had a sensitivity of 81% for sera from patients with less than 7 days of illness. Anti-LigB antibodies were found in sera from 57% of the patients who did not have detectable anti-whole-Leptospira responses as detected by IgM enzyme-linked immunosorbent assay and microagglutination test. The specificities of the assay were 93 to 100% and 90 to 97% among sera from healthy individuals and patients with diseases that have clinical presentations that overlap with those of leptospirosis, respectively. These findings indicate that the antibody response to this putative virulence determinant is a sensitive and specific marker for acute infection. The use of this marker may aid the prompt and timely diagnosis required to reduce the high mortality associated with severe forms of the disease.
doi:10.1128/JCM.02344-06
PMCID: PMC1865864  PMID: 17360842
17.  Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in Sphingomonas paucimobilis SYK-6 
Journal of Bacteriology  2003;185(1):41-50.
The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6. In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI. We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK. The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp. strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1. The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO2. LigK is a hexamer, and its isoelectric point is 5.1. The Km for CHA and oxaloacetate are 11.2 and 136 μM, respectively. Inactivation of ligK in S. paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.
doi:10.1128/JB.185.1.41-50.2003
PMCID: PMC141877  PMID: 12486039
18.  A LigA Three-Domain Region Protects Hamsters from Lethal Infection by Leptospira interrogans 
The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×103 to 8×105 copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences.
Author Summary
Leptospirosis is the most widespread bacterial infection transmitted to humans from host animals that harbor the bacteria in their kidneys. Human infections caused by the bacterium, Leptospira interrogans, frequently result in a life-threatening illness characterized by jaundice and kidney failure. Vaccines are urgently needed to prevent leptospirosis in populations at risk. The leptospiral protein, LigA, is a promising vaccine candidate because it is the first purified protein to be shown to protect animals from fatal leptospirosis. The goal of this study was to determine which of LigA's 13 domains are required for the protective effect. Immunization with domains 11 and 12 was found to be required, but was insufficient, for protection. A third domain, either 10 or 13, was required for 100% survival. As in previous studies, residual bacteria were cultured from the kidneys of survivors. However, in contrast to previous studies, we determined the amount of bacterial DNA in the kidneys as a measure of vaccine efficacy. We also examined the kidneys microscopically for signs of damage and measured blood chemistries to assess kidney function. These are important steps towards developing vaccines that provide protection from kidney damage and infection.
doi:10.1371/journal.pntd.0001422
PMCID: PMC3236721  PMID: 22180800
19.  Bioinformatics Describes Novel Loci for High Resolution Discrimination of Leptospira Isolates 
PLoS ONE  2010;5(10):e15335.
Background
Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.
Methodology and Principal Findings
We used bioinformatics to evaluate pre-existing loci with the potential to increase the discrimination of outbreak strains. Previously deposited sequence data were evaluated by phylogenetic analyses using either single or concatenated sequences. We identified and evaluated the applicability of the ligB, secY, rpoB and lipL41 loci, individually and in combination, to discriminate between 38 pathogenic Leptospira strains and to cluster them according to the species they belonged to. Pairwise identity among the loci ranged from 82.0–92.0%, while interspecies identity was 97.7–98.5%. Using the ligB-secY-rpoB-lipL41 superlocus it was possible to discriminate 34/38 strains, which belong to six pathogenic Leptospira species. In addition, the sequences were concatenated with the superloci from 16 sequence types from a previous MLST scheme employed to study the association of a leptospiral clone with an outbreak of human leptospirosis in Thailand. Their use enhanced the discriminative power of the existing scheme. The lipL41 and rpoB loci raised the resolution from 81.0–100%, but the enhanced scheme still remains limited to the L. interrogans and L. kirschneri species.
Conclusions
As the first aim of our study, the ligB-secY-rpoB-lipL41 superlocus demonstrated a satisfactory level of discrimination among the strains evaluated. Second, the inclusion of the rpoB and lipL41 loci to a MLST scheme provided high resolution for discrimination of strains within L. interrogans and L. kirschneri and might be useful in future epidemiological studies.
doi:10.1371/journal.pone.0015335
PMCID: PMC2955542  PMID: 21124728
20.  The C-terminal variable domain of LigB from Leptospira mediates binding to fibronectin 
Journal of Veterinary Science  2008;9(2):133-144.
Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection.
doi:10.4142/jvs.2008.9.2.133
PMCID: PMC2839090  PMID: 18487934
adhesion; Fn; Leptospira; LigB; MDCK cell; siRNA
21.  Response of Leptospira interrogans to Physiologic Osmolarity: Relevance in Signaling the Environment-to-Host Transition▿ † 
Infection and Immunity  2007;75(6):2864-2874.
Transmission of pathogenic Leptospira between mammalian hosts usually involves dissemination via soil or water contaminated by the urine of carrier animals. The ability of Leptospira to adapt to the diverse conditions found inside and outside the host is reflected in its relatively large genome size and high percentage of signal transduction genes. An exception is Leptospira borgpetersenii serovar Hardjo, which is transmitted by direct contact and appears to have lost genes necessary for survival outside the mammalian host. Invasion of host tissues by Leptospira interrogans involves a transition from a low osmolar environment outside the host to a higher physiologic osmolar environment within the host. Expression of the lipoprotein LigA and LigB adhesins is strongly induced by an upshift in osmolarity to the level found in mammalian host tissues. These data suggest that Leptospira utilizes changes in osmolarity to regulate virulence characteristics. To better understand how L. interrogans serovar Copenhageni adapts to osmolar conditions that correspond with invasion of a mammalian host, we quantified alterations in transcript levels using whole-genome microarrays. Overnight exposure in leptospiral culture medium supplemented with sodium chloride to physiologic osmolarity significantly altered the transcript levels of 6% of L. interrogans genes. Repressed genes were significantly more likely to be absent or pseudogenes in L. borgpetersenii, suggesting that osmolarity is relevant in studying the adaptation of L. interrogans to host conditions. Genes induced by physiologic osmolarity encoded a higher than expected number of proteins involved in signal transduction. Further, genes predicted to encode lipoproteins and those coregulated by temperature were overrepresented among both salt-induced and salt-repressed genes. In contrast, leptospiral homologues of hyperosmotic or general stress genes were not induced at physiologic osmolarity. These findings suggest that physiologic osmolarity is an important signal for regulation of gene expression by pathogenic leptospires during transition from ambient conditions to the host tissue environment.
doi:10.1128/IAI.01619-06
PMCID: PMC1932867  PMID: 17371863
22.  Cloning and Molecular Characterization of an Immunogenic LigA Protein of Leptospira interrogans  
Infection and Immunity  2002;70(11):5924-5930.
A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30°C or when cultures were shifted to 37°C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.
doi:10.1128/IAI.70.11.5924-5930.2002
PMCID: PMC130282  PMID: 12379666
23.  Xanthan Gum as an Adjuvant in a Subunit Vaccine Preparation against Leptospirosis 
BioMed Research International  2014;2014:636491.
Leptospiral immunoglobulin-like (Lig) proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106), aluminium hydroxide (alhydrogel), and CpG ODN as adjuvants in a LigA subunit vaccine preparation. Xanthan gum is a high molecular weight extracellular polysaccharide produced by fermentation of Xanthomonas spp., a plant-pathogenic bacterium genus. Preparations containing xanthan induced a strong antibody response comparable to that observed when alhydrogel was used. Upon challenge with a virulent strain of L. interrogans serovar Copenhageni, significant protection (Fisher test, P < 0.05) was observed in 100%, 100%, and 67% of hamsters immunized with rLigANI-xanthan, LigA-CpG-xanthan, and rLigANI-alhydrogel, respectively. Furthermore, xanthan did not cause cytotoxicity in Chinese hamster ovary (CHO) cells in vitro. The use of xanthan as an adjuvant is a novel alternative for enhancing the immunogenicity of vaccines against leptospirosis and possibly against other pathogens.
doi:10.1155/2014/636491
PMCID: PMC4033433  PMID: 24895594
24.  Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins 
PLoS ONE  2010;5(12):e14377.
Background
Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold.
Principal Findings
We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold.
Conclusions
We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding.
doi:10.1371/journal.pone.0014377
PMCID: PMC3012076  PMID: 21206924
25.  The 4-Oxalomesaconate Hydratase Gene, Involved in the Protocatechuate 4,5-Cleavage Pathway, Is Essential to Vanillate and Syringate Degradation in Sphingomonas paucimobilis SYK-6 
Journal of Bacteriology  2000;182(24):6950-6957.
Sphingomonas paucimobilis SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by the O-demethylase and converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The ligJ gene is transcribed in the same direction as ligABC genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream from ligA. The ligJ gene product (LigJ), expressed in Escherichia coli, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30°C. The Km for OMA and the Vmax were determined to be 138 μM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The ligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the ligJ gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.
PMCID: PMC94820  PMID: 11092855

Results 1-25 (851992)