Leptospira strains and sera
The pathogenic Leptospira strains used were: L. interrogans serovar Canicola strain Hound Utrech IV, L. interrogans serovar Copenhageni strain M 20, L. interrogans serovar Hardjo strain Hardjoprajitno, L. interrogans serovar Icterohaemorrhagiae strain RGA, L. interrogans serovar Pomona strain Pomona, L. borgpetersenii serovar Whitcombi strain Whitcomb and serovar Grippothyphosa strain Moskva V, L. kirshneri serovar Cynoptery strain 3522 C, L. santarosai serovar Shermani strain 1342 K, L. noguchi serovar Panama strain CZ 214 and L. biflexa serovar Patoc strain Patoc, were cultured at 28°C under aerobic conditions in liquid EMJH medium (Difco®) with 10% rabbit serum, enriched with L - asparagine (wt/vol: 0.015%), sodium pyruvate (wt/vol: 0.001%), calcium chloride (wt/vol: 0,001%), magnesium chloride (wt/vol: 0.001%), peptone (wt/vol:0.03%) and meat extract (wt/vol: 0.02%) (Turner LH. Leptospirosis. 3. Maintenance, isolation and demonstration of leptospires. Trans R Soc Trop Med Hyg 1970; 64: 623–646). Leptospira cultures are maintained in Faculdade de Medicina Veterinária e Zootecnia, USP, São Paulo, SP, Brazil. Confirmed - leptospirosis serum samples were from Instituto Adolfo Lutz collection, São Paulo, Brazil.
Microscopic agglutination test (MAT)
The microscopic agglutination test was performed according to . In brief, an array of 22 serovars of Leptospira spp. as antigens were employed: Australis, Autumnalis, Bataviae, Canicola, Castellonis, Celledoni, Copenhageni, Cynopteri, Djasiman, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Javanica, Panama, Patoc, Pomona, Pyrogenes, Sejroe, Shermani, Tarassovi and Wolffi. All the strains were maintained in EMJH liquid medium (Difco, USA) at 29°C. A laboratory - confirmed case of leptospirosis was defined by the demonstration of a four - fold microagglutination titer rise between paired serum samples. The probable predominant serovar was considered to be the one with the highest dilution that could cause 50% of agglutination. MAT was considered negative when the titer was below 100.
DNA isolation and PCR analysis
Leptospira cultures were harvested by centrifugation at 11,500 g for 30 min and gently washed in sterile PBS twice. Genomic DNA was isolated from the pellets by guanidine - detergent lysing method using DNAzol® Reagent (Invitrogen), according to the manufacturer’s instructions. Primers were designed according to L. interrogans serovar Copenhageni genome sequences (GenBank accession AE016823) and are listed in Table 1. PCR was performed in a reaction volume of 25 μl containing 100 ng of genomic DNA, 1 × PCR buffer (20 mM Tris - HCl, pH 8.4, 50 mM KCl), 2 mM MgCl2, 20 pmol of each specific primer, 200 μM of each dNTP, and 2.5 U Taq DNA Polymerase (Invitrogen). Cycling conditions were: 94 ° C - 4 min, followed by 40 cycles at 94°C - 50 sec, 57°C (LIC11834) or 56°C (LIC12253) - 50 sec, 72°C - 90 sec, and a final extension cycle of 7 min at 72°C. PCR amplified products were loaded on a 1% agarose gel for electrophoresis and visualization with ethidium bromide.
RNA extraction and RT-PCR analysis
For reverse transcription RT - PCR, total RNA was isolated by the acid guanidinium thiocyanate phenol - chloroform method using TRIzol® Reagent (Invitrogen) according to the manufacturer’s recommendations. 1 μg of RNA from each sample was treated with 1 U of DNAse I Amplification Grade (Invitrogen) for 15 min at room temperature. DNAse I was inactivated by the addition of 1 μl of 25 mM EDTA solution followed by an incubation at 65 ° C for 10 min. DNAse - treated RNAs were reversely transcribed using the SuperScript™ III First - Strand Synthesis System for RT-PCR (Invitrogen). One tenth of RT products were amplified in a 25 μl reaction mix using oligonucleotides LIC11834 - F/LIC11834 - R or LIC12253 - F/LIC12253 - R, as described above. Samples quantity and integrity were verified by amplification of a 1,042 bp 16 S ribosomal cDNA fragment using oligomers:
16S - F 5′CAAGTCAAGCGGAGTAGCAATACTCAGC 3′ and 16S - R 5′GATGGCAACATAAGGTGAGGGTTGC 3′.
DNA recombinant techniques, protein expression and purification
Predicted CDSs LIC11834 and LIC12253, without signal peptides, were amplified by the PCR from L. interrogans serovar Copenhageni strain Fiocruz L1 - 130 genomic DNA using the primer pairs depicted in Table 1. The PCR products obtained for each corresponding gene were cloned into pGEM-T easy vector (Promega) and subcloned into the pAE expression vector  at the restriction sites shown in Table 1. The pAE vector allows the expression of recombinant proteins with a minimal 6X His - tag at the N - terminus. All cloned sequences were confirmed by DNA sequencing with an ABI 3100 automatic sequencer (PE Applied Biosystems, Foster city, CA). Protein expression of rLIC11834 and rLIC12253 was achieved in E. coli BL21 (SI) strain by the action of T7 DNA polymerase under control of the osmotically induced promoter proU . E. coli BL21 (SI) containing recombinant plasmids were grown at 30°C in Luria - Bertani broth without NaCl and with 100 μg/ml ampicillin with continuous shaking until an optical density at 600 nm of 0.6 to 0.8 was reached. Recombinant protein synthesis was induced by the addition of 300 mM NaCl. After three hours, the cells were harvested by centrifugation and the bacterial pellets resuspended in lysis buffer (200 μg/ml of lysozyme, 1% Triton X - 100, 2 mM phenylmethylsulfonyl fluoride [PMSF]). The bacterial cell pellets were lysed on ice with the aid of a sonicator (Ultrasonic Processor; GE Healthcare). The insoluble fractions were washed with 20 ml of buffer (20 mM Tris - HCl, pH 8.0, 500 mM NaCl, 1 M urea and 1% Triton X-100) and resuspended in a buffer containing 20 mM Tris - HCl, pH 8.0, 500 mM NaCl, 5 mM Imidazole, 1 mM β - mercaptoethanol and 8 M urea. The proteins were then purified through metal chelating chromatography in a Sepharose fast flow column (GE Healthcare) and fractions were analyzed in 12% SDS-PAGE. The rLIC12253 protein was refolded by 500 times dilution with 20 mM Tris - HCL, pH 8.0, and 500 mM NaCl before chromatographic purification. The purified recombinant proteins were extensively dialyzed against phosphate - buffered saline (PBS), pH 7.4, 0.1% (wt/vol) glycine solution (1:100), pooled and stored at −20°C.
Circular dichroism spectroscopy
Purified recombinant proteins were dialyzed against sodium phosphate buffer (pH 7.4). Circular dichroism (CD) spectroscopy measurements were performed at 20°C using a Jasco J-810 spectropolarimeter (Japan Spectroscopic, Tokyo) equipped with a Peltier unit for temperature control. Far-UV CD spectra were measured using a 1 mm - path - length cell at 0.5 nm intervals. The spectra were presented as an average of five scans recorded from 185 to 260 nm. The molar ellipticity (Φ) is expressed in deg.cm.dmol1.
Five female BALB/c mice (4–6 weeks old) were immunized subcutaneously with 10 μg of the recombinant proteins. The recombinant proteins were adsorbed in 10% (vol/vol) of Alhydrogel (2% Al(OH)3, Brenntag Biosector, Denmark), used as adjuvant. Two subsequent booster injections were given at two - week intervals with the same preparation of 10 μg of the proteins. Negative - control mice were injected with PBS. One week after each immunization, the mice were bled from the retro - orbital plexus and the pooled sera were analyzed by enzyme -linked immunosorbent assay (ELISA) for determination of antibody titers. All animal studies were approved by the Ethics Committee of the Instituto Butantan, São Paulo, SP, Brazil. The Committee in Animal Research in Instituto Butantan adopts the guidelines of the Brazilian College of Animal Experimentation.
The purified recombinant proteins were loaded into 12% SDS - PAGE and transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare) in semi - dry equipment. Membranes were blocked with 5% non-fat dried milk and 2.5% BSA in PBS containing 0.05% Tween 20 (PBS - T) and then incubated with anti - rLIC11834 (1:500), anti - rLIC12253 (1:500) mouse serum or anti - his antibody (1:1,000) (GE Healthcare) for 2 h at room temperature. After washing, the membranes were incubated with horseradish peroxidase (HRP) - conjugated anti - mouse IgG (1:5,000; Sigma) in PBS - T for 1 h. The protein’s reactivity was revealed by ECL reagent kit chemiluminescence substrate (GE Healthcare) with subsequent exposition to X - Ray film.
ELISA for detection of human antibodies
Human IgG antibodies against Lsa33 or Lsa25 were detected by ELISA as previously described . In brief, serum samples of negative (24) and positive (33) MAT from confirmed - leptospirosis patients were diluted 1:400 and evaluated for total IgG using goat HRP - conjugated anti-human IgG antibodies (1:5,000, Sigma). Cutoff values were set at three standard deviations above the mean OD492nm of sera from 11 health individuals, unexposed to leptospirosis, from the city of São Paulo, Brazil and one pool of normal serum samples from USA (Sigma).
Proteinase K accessibility assay
Suspensions of 5 ml live leptospires/per treatment (~108cells/ml) were harvested at 10,000 rpm for 15 min at room temperature, washed once with PBS (with 50 mM NaCl), resuspended in 5 ml of PBS (with 50 mM NaCl) plus with 25 μg/ml of proteinase K (PK) (Sigma Aldrich). Four similar test tubes were then incubated for 0 to 5 h at 37°C and aliquots were taken at 0, 1, 3 and 5 h before the addition of 100 mM of phenylmethylsulfonyl fluoride (PMSF) to stop PK activity. The suspensions were subsequently pelleted by centrifugation at 10,000 rpm for 5 min, washed twice with PBS (with 50 mM NaCl) and resuspended in 1 ml PBS (with 50 mM NaCl) for ELISA analysis using antibodies against Lsa33, Lsa25, Lip32 and DnaK, as described below. LipL32 and DnaK are membrane and cytoplasmic leptospiral proteins that were employed in our experiment as positive and negative control, respectively.
ELISA for detection cellular localization of the proteins
Leptospires were coated onto microplates and allowed to stand at room temperature for 16 h. The plates were washed three times with PBS (with 50 mM NaCl) and blocked with 5% non-fat dry milk and 1% BSA for 2 h at 37°C. After incubated for 2 h at 37°C with polyclonal mouse anti - serum against Lsa33, Lsa25, LipL32 or DnaK (dilution of an OD equal to 1). The leptospires were washed three times with PBS (with 50 mM NaCl) and incubated with 50 μL of a 1:5,000 dilution of HRP - conjugated goat anti - mouse IgG (Sigma) in PBS (with 50 mM NaCl) for 1 h at 37°C. The wells were washed three times with PBS (with 50 mM NaCl), and o - phenylenediamine (OPD) (1 mg/mL) in citrate phosphate buffer (pH 5.0) plus 1 μL/mL H2O2 was added (100 μL per well). The reaction proceeded for 5 min and was interrupted by the addition of 50 μL of 4 N H2SO4. The absorbance at 492 nm was determined in a microplate reader (TP - reader, Thermo) against the O.D. of blanks, containing all the reaction mixture but antibodies against the proteins. For statistical analyses, the binding of polyclonal mouse anti - serum against Lsa33, Lsa25, LipL32 or DnaK at 0 h incubation was compared with other incubations by Student’s two - tailed t test.
Binding of recombinant proteins to ECM and to serum components
Protein attachment to individual macromolecules of the extracellular matrix was analyzed according to a previously published protocol  with some modifications. Briefly, 96 - well plates (Costar High Binding, Corning) were coated with 1 μg of laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin, human PLG, factor H, C4bp, or gelatin (negative control) and fetuin (highly glycosylated attachment - negative control protein) in 100 μL of PBS for 3 h at 37°C. The wells were washed three times with PBS - T and then blocked with 200 μL of 10% (wt/vol) non-fat dry milk (overnight at 4°C). One microgram of each recombinant protein was added per well in 100 μL of PBS, and protein was allowed to attach to the different substrates for 2 h at 37°C. After washing six times with PBS - T, bound Lsa33 or Lsa25 was detected by adding mouse anti - recombinant proteins (1:750) in 100 μL of PBS or anti - His tag monoclonal (1:200) in 100 μL of PBS. Incubation proceeded for 1 h at 37°C. After three washings with PBS - T, 100 μL of a 1:5,000 dilution of HRP - conjugated goat anti - mouse IgG (Sigma) in PBS was added per well for 1 h at 37°C. The wells were washed three times, and o - phenylenediamine (OPD) (1 mg/mL) in citrate phosphate buffer (pH 5.0) plus 1 μL/mL H2O2 was added (100 μL per well). The reaction proceeded for 10 min and was interrupted by the addition of 50 μL of 4 N H2SO4. The absorbance at 492 nm was determined in a microplate reader (TP - reader, Thermo). For statistical analyses, the binding of recombinant proteins to ECM macromolecules and to serum components were compared to its binding to gelatin and by Student’s two - tailed t test.
Metaperiodate treatment of laminin
Microtitre wells were coated with 1 μg of laminin in 50 mM sodium acetate buffer, pH 5.0, and incubated for 16 h at 4°C. Wells were washed three times with the same buffer, and laminin was treated with different sodium metaperiodate concentrations (0–100 mM) in the same buffer for 15 min at 4°C in the dark. After three washes with 50 mM sodium acetate buffer, wells were blocked with 200 μl of 1% BSA for 1 h at 37°C. Binding of recombinant proteins (1 μg in PBS per well) to periodate - treated laminin was evaluated as described above.
First, 96 - well plates were coated overnight in PBS at 4°C with 100 μl of 10 μg/ml PLG, laminin or C4bp. Plates were then blocked and increasing concentrations of the purified recombinant proteins (0–6 μM) were added (100 μl/well in PBS). The assessment of bound proteins was performed by incubation for 1 h at 37°C with the antiserum raised against each protein at the dilution of 1:750, followed by HRP - conjugated goat anti-mouse IgG (Sigma) (1:5,000 in PBS). The reactions were detected with OPD as describe above. The ELISA data were used to calculate the dissociation constant (KD) according to the method described by Pathirana et al.  and Lin et al. , based on the equation:
, where A is the absorbance at a given protein concentration, Amax is the maximum absorbance for the ELISA plate reader (equilibrium), [protein] is the protein concentration and KD is the dissociation equilibrium constant for a given absorbance at a given protein concentration (ELISA data point).
Plasmin enzymatic activity assay
96 - well ELISA plates were coated overnight with 10 μg/ml recombinant proteins in PBS at 4°C. Lsa63, which does not bind PLG  and BSA were employed as negative control. Plates were washed once with PBS - T and blocked for 2 h at 37°C with PBS with 10% (wt/vol) non - fat dry milk. The blocking solution was discarded and 100 μl/well of 10 μg/ml human PLG was added, followed by incubation for 2 h at 37°C. Wells were washed three times with PBS - T, and then 4 ng/well of human uPA (Sigma - Aldrich) were added. Subsequently, 100 μl/well of plasmin - specific substrate D - valyl - leucyl – lysine - p - nitroanilide dihydrochloride (Sigma - Aldrich) were added at a final concentration of 0.4 mM in PBS. Plates were incubated overnight at 37°C and substrate degradation was measured by readings at 405 nm.
Inhibition of live leptospires binding to laminin or to PLG by recombinant proteins
ELISA plates were coated with laminin or PLG (1 μg/well). The plates were washed and blocked with 10% non - fat dry milk in PBS - T for 2 h at 37°C. The blocking solution was discarded, and the wells were incubated for 90 min at 37°C with increasing concentrations of proteins (0 to 10 μg). After three washings, 50 μL/well of 4 × 107 live low - passage virulent L. interrogans serovar Copenhageni strain M20 were added for 90 min at 37°C. The unbound leptospires were washed and the quantification of bound leptospires was performed indirectly by anti - LipL32 antibodies produced in mice (1:4,000), given the fact that LipL32 is a major outer membrane leptospiral protein ; the procedure was followed by horseradish peroxidase - conjugated anti - mouse IgG antibodies, essentially as described in Barbosa et al. . The detection was performed by OPD as previously described.
Liquid-phase immunofluorescence assay (L - IFA)
The localization of LIC11834 and LIC12253 encoded proteins by L - IFA was performed as described by Oliveira et al. . In brief, suspensions of 2.5 ml live leptospires (~109cells/ml) were harvested at 10,000 rpm for 15 min, washed twice with PBS (with 50 mM NaCl), resuspended in 200 μl of PBS with 6 μg/ml propidium iodide to stain the nuclei, and incubated for 45 min at 37°C. After incubation, the leptospires were washed gently with PBS and incubated for 30 min at 4°C with polyclonal mouse anti - serum against Lsa33, Lsa25, LipL32 or GroEL at a 1:50 dilution. The leptospires were washed and incubated with goat anti - mouse IgG antibodies conjugated to fluorescein isothiocyante (FITC, Sigma) at a dilution 1:50 for 30 min at 4°C. After incubation with secondary antibody, the leptospires were washed and resuspended in PBS - antifading solution (ProLong Gold, Molecular Probes). The immunofluorescence - labeled leptospires were examined by employ of a confocal LSM 510 META immunofluorescence microscope (Zeiss, Germany).
Nucleotide sequence accession numbers
GenBank accession numbers for protein sequences LIC11834 and LIC12235 are AAS70420 and AAS70825, respectively. The protein can also be accessed by the genome nomenclature for the gene locus, LIC number (Leptospira interrogans serovar Copenhageni).
ECM and biological components
The control proteins fetuin and gelatin, were purchased from Sigma Chemical Co. (St. Louis, Mo.) and Difco®, respectively. Laminin - 1 and collagen Type IV were derived from the basement membrane of Engelbreth - Holm-Swarm mouse sarcoma, cellular fibronectin was derived from human foreskin fibroblasts, plasma fibronectin was isolated from human plasma and collagen Type I was isolated from rat tail. PLG, purified from human plasma, was purchased from Merck. Human Factor H was from Calbiochem. C4bp was from Complement Technology, INC.