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.

The Borrelia burgdorferi flgB promoter was amplified with PflgA (5'-TAATACCCGAGCTTCAAGGAAG-3') and PflgB (5'-AACATATGGAAACCTCCCTC-3') and cloned into pCR2.1 (Invitrogen) to generate plasmid pCRPromFlgB. The ligA and ligB genes were amplified with flanking NdeI and XhoI sites, using primer pairs LANF (5'-GGGAATTCCATATGAAGAAAATATTTTGTATTTCG-3') - LAXR (5' CGGCTCGAGTTATTATGGCTCCGTTTTAATAGAGG-5') and LBNF (5'-GGGAATTCCATATGAAGAAAATATTTTGTATTTCG-5') - LBXR (5'-CGGCTCGAGTTATTATTGATTCTGTTGTCTGT-3'), respectively, from genomic DNA of L. interrogans serovar Copenhageni strain Fiocruz L1-130. Amplified lig genes were then digested with NdeI and XhoI restriction enzymes, purified, and inserted between the corresponding restriction sites of pCRPromFlgB to generate pCRPflgBLigA and pCRPflgBLigB, respectively. The DNA fragment containing Prom_flgB ligA (4183 bp) and Prom_flgB ligB (6188 bp) were released from plasmids pCRPflgBLigA and pCRPflgBLigB by SpeI and XbaI digestion, then blunt-ended, and cloned into the PvuII restriction site of the E. coli-L. biflexa shuttle vector pSLe94 [49] to generate pSLePFligA and pSLePFligB (Figure ​(Figure1).1). Plasmid constructs were verified by nucleotide sequencing.

L. biflexa was prepared for transformation as previously described [4]. In brief, L. biflexa was grown at 30°C until the optical density reached 0.4 at 420 nm. Bacteria were collected by centrifugation at room temperature and washed by resuspension in deionized water followed by centrifugation. After removing the supernatant fluid, the bacteria were resuspended with deionized water to a final concentration of around 5 × 10¹⁰ cells/ml (100× concentration). 100 μl of the suspended bacteria were added to the plasmid DNA, and the DNA-bacteria mixture was added to chilled electroporation cuvettes with a 0.2 cm gap. The cuvette was placed in the electroporation unit (Bio-Rad Gene Pulser II) and subjected to electroporation at a setting of 1.8 kV, 25 μF, and 200 Ω. After adding 1 ml of EMJH, the bacteria were transferred to a 15 ml Falcon tube and incubated for 24 hours at 30°C with shaking. The culture (0.2 ml) was plated onto EMJH plates containing 40 μg/ml of spectinomycin and incubated at 30° for 10 days. Colonies were inoculated into liquid EMJH containing 40 μg/ml spectinomycin. L. biflexa transformants were maintained by serial passage in the liquid medium.

Exponential phase cultures of L. biflexa Patoc wild-type, Patoc ligA, Patoc ligB, and L. interrogans Fiocruz strains were washed, resuspended in PBS and solubilized in 62.5 mM Tris hydrochloride (pH 6.8)-10% glycerol-5% 2-mercaptoethanol-2% sodium dodecyl sulfate. A 20 μl volume of crude extracts containing 2 × 10⁸ bacteria/per well was resolved by 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using a discontinuous buffer system. After transfer to nitrocellulose membranes, immunoblots were blocked in 0.05 M Tris-buffered saline (pH 7.4)-0.05% (vol/vol) Tween 20 with 5% (wt/vol) nonfat dry milk. The blots were washed, incubated for 1 h at room temperature with a 1,000-fold dilution of mouse ascites containing MAb to the LigB identical repeat region (LigA/B) [6] and probed with goat anti-mouse conjugated to alkaline phosphatase (Sigma). Immunoblots were developed in a nitroblue tetrazolium--5-bromo-4-chloro-3-indolylphosphate (BCIP) solution (Bio-Rad).

We evaluated the localization of LigA and LigB by performing immunofluorescence labeling according to a modified protocol of Cullen et al. [50]. Suspensions of 10⁷ live leptospires in 10 μl of PBS were placed onto poly-L-lysine-coated slides (Sigma-Aldrich) for 1 h in a humidified chamber for adherence of the leptospires. In experiments in which the bacteria were permeabilized prior to incubation with antibody, slides were incubated with cold methanol for 10 min at -20°C, followed by two washes with PBS. Blocking with 1% bovine serum albumin (Sigma-Aldrich) (PBS-BSA) for 20 min was performed before incubation for 1 h at 37°C with normal rabbit serum, rabbit hyperimmune antisera to whole extracts of L. interrogans serovar Icterohaemorrhagiae strain RGA, LigB non-identical region (LigBNI), and LigA non-identical region (LigANI) [6], and rat antiserum to GroEL, which were diluted 1:100 in PBS-BSA. The slides were washed gently with PBS-BSA and incubated with goat anti-rabbit IgG antibodies conjugated to Alexa dye (Molecular Probes) or goat anti-rat IgG antibodies conjugated to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) for 1 h at 37°C. The slides were washed twice with PBS-BSA and incubated with 1 μg/ml DAPI (Molecular Probes) for 1 h at room temperature. Slides were then washed, then mounted in anti-fading solution (Prolong-Molecular Probes) and visualized by fluorescence microscopy (Olympus BX51).

Madin Darby canine kidney (MDCK) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (Cultilab), 2% sodium bicarbonate, 25 mM HEPES, and 5 mM L-glutamine (Sigma) at 37°C in an atmosphere of 5% CO₂. MDCK cells were harvested by treating cell cultures with 0.05% trypsin and 0.02% EDTA in PBS. For adhesion assays, MDCK cells were plated onto 24-well plates in DMEM, containing 13-mm-diameter glass coverslips at 37°C in an atmosphere of 5% CO₂ until they were confluent. The number of MDCK cells in wells was determined by lysing cells with 0.1 M citric acid containing 0.05% crystal violet (Sigma-Aldrich) and 1% Cetrimide (Sigma) [51], then the nuclei were counted in a hemacytometer. The cells were incubated with a suspension of Patoc wild-type, Patoc ligA, Patoc ligB and Fiocruz L1-130 strains in cell culture medium at the final bacteria: cell ratio of 10:1. Incubations were performed for periods of 30 to 240 min. Prior to staining, the cells were washed three times in PBS to remove nonadherent bacteria and then fixed with cold methanol for 10 min. An immunofluorescence assay was performed to detect adherent leptospires for which rabbit polyclonal antisera against whole extracts of L. interrogans strain RGA and goat anti-rabbit antibodies conjugated with Alexa488 (Molecular Probes) were used as first and second antibodies, respectively. DAPI and Alcian Blue were used to stain the nucleus and cytoplasm, respectively. The number of leptospires and MDCK cells was determined by examining ten high-magnification (1000×) fields during fluorescence microscopy. All incubation points were performed in triplicate. The ANOVA test was used to determine statistically significant (p < 0.05) differences between numbers of adherent leptospires/cell. We performed a translocation assay according to a protocol modified from that described by Barocchi et al [30]. MDCK cells at a concentration of 2 × 10⁵ cells in 500 μl of DMEM were seeded onto 12-mm-diameter Transwell filter units with 3- μm pores. Monolayers were incubated at 37°C in 5% CO₂ for 3 to 4 days with daily changes in media until the transmonolayer electrical resistance (TER) reached a range of 200 and 300 Ω/cm², as measured with an epithelial voltohmmeter (EVOM, World Precision Instruments, Sarasota, Fla.). The TER for polycarbonate filters without cells was approximately 100 Ω/cm². The upper chamber of the transwell apparatus was inoculated with leptospires at a multiplicity of infection (MOI) of 100 by adding 500 μL of bacteria which were resuspended in 1:2 v/v ratio of DMEM and EMJH media. Duplicate transwell chamber assays were performed for each leptospiral strain which were tested. Aliquots were removed from lower chamber (100 μl) at 30, 120 and 240 min and the number of leptospires was counted in triplicate by using the Petroff-Hausser chamber. The ability of leptospires to translocate MDCK polarized monolayers was determined by calculating the proportion of leptospires in the lower chamber in comparison to the initial inoculum for duplicate assays at each time point. The ANOVA test was used to determine significant differences in the proportions of translocating leptospires and TER values obtained during incubations with different leptospiral strains.