A Newly Identified Leptospiral Adhesin Mediates Attachment to Laminin

ECM components.All macromolecules, including the control proteins fetuin and bovine serum albumin (BSA), were purchased from Sigma Chemical Co. (St. Louis, Mo.). 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.

Leptospira strains and culture.A virulent strain of L. interrogans serovar Pomona and a culture-attenuated strain of L. interrogans serovar Copenhageni (Laboratório de Zoonoses, Faculdade de Medicina Veterinária e Zootecnia, Universidade São Paulo, São Paulo, Brazil) were used in the assays. The virulent strain is maintained by iterative passages in hamsters. Both strains were cultured at 29°C under aerobic conditions in liquid EMJH medium (Difco) with 10% rabbit serum enriched with l-asparagine, sodium pyruvate, calcium chloride, and magnesium chloride. For the ECM-binding assays, cells were harvested by centrifugation at 11,600 × g for 30 min and gently washed in sterile phosphate-buffered saline (PBS) twice. The leptospires were resuspended in PBS, and bacterial density was determined using dark-field microscopy and a Petroff-Hausser chamber.

In silico selection of outer membrane proteins.Cellular localization of the proteins was predicted by the PSORT program (http://psort.nibb.ac.jp ) (27). Sequence motifs, including signal peptides, lipoprotein cleavage sites, and transmembrane domains, were searched for on public web servers (http://www.cbs.dtu.dk/services/SignalP ) (20, 22) and (http://www.cbs.dtu.dk/services/TMHMM ) (3).

Cloning, expression, and purification of recombinant proteins.Predicted coding sequences LIC10009, LIC10191, LIC10494, LIC11947, LIC12730, and LIC12906 were amplified by PCR from total L. interrogans genomic DNA (strain Fiocruz L1-130) using the primer pairs listed in Table 1. The Gateway (Invitrogen) system was used for the cloning and expression of proteins encoded by LIC10191, LIC10494, LIC11947, and LIC12730. PCR products were first cloned into a pENTR TOPO vector. Directional cloning was ensured by the addition of four bases to the forward primers (CACC) which annealed to a complementary overhang in the cloning vector (GTGG). DNA inserts were then transferred by recombination to the Escherichia coli expression vector pDEST17 using LR Clonase (Invitrogen). PCR fragments corresponding to LIC10009 and LIC12906 genes were cloned into the pGEM T-Easy vector (Promega) and transformed into E. coli DH5α. Following digestion with restriction enzymes (XhoI/HindIII for LIC10009 and XhoI/NcoI for LIC12906), fragments were subcloned into the E. coli expression vector pAE, which has been previously described (2, 36). All constructs were verified by DNA sequencing with appropriate vector-specific primers. Expression and purification of the resulting six-His-tagged recombinant proteins were performed as previously described (1). Protein refolding was achieved by multistep dialysis against 20 mM Tris-HCl (pH 8.0) and 0.5 M NaCl, gradually decreasing the urea concentration. Renatured proteins were extensively dialyzed against PBS for 24 to 48 h, and samples were analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All purified recombinant proteins were passed through columns containing immobilized polymyxin B (Detoxi-Gel AffinityPak prepacked columns; Pierce, Rockford, IL) for removal of residual E. coli lipopolysaccharide (LPS).

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, Japan) 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 190 to 260 nm.

Antisera against recombinant proteins.Ten female BALB/c mice (4 to 6 weeks old) were immunized subcutaneously with 10 μg of recombinant proteins. Aluminum hydroxide was used as adjuvant. Two subsequent booster injections were given at 2-week intervals with the same protein preparation. 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.

Binding of recombinant proteins to ECM.Protein attachment to individual macromolecules of the extracellular matrix was analyzed according to a previously published protocol (5) with some modifications. Briefly, ELISA plate wells (Nunc-Immuno plate, MaxiSorp surface) were coated with 1 μg of laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin, BSA (nonglycosylated attachment-negative control protein), and fetuin (highly glycosylated attachment-negative control protein) in 100 μl of PBS for 2 h at 37°C. The wells were washed three times with PBS-0.05% Tween 20 (PBST) and then blocked with 200 μl of 1% BSA for 1 h at 37°C, followed by an overnight incubation at 4°C. One microgram of recombinant protein was added per well in 100 μl of PBS, and protein was allowed to attach to the different substrates for 1 h 30 min at 37°C. After washing six times with PBST, bound proteins were detected by adding an appropriate dilution of mouse antiserum in 100 μl of PBS. Dilutions of mouse antisera against each recombinant protein were as follows: LIC10009, 1:5,000; LIC10191 (Loa22), 1:10,000; LIC10494, 1:5,000; LIC11947, 1:2,000; LIC12730, 1:5,000; LIC12906 (Lsa24), 1:2,000. Incubation proceeded for 1 h, and after three washes with PBST, 100 μl of a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G in PBS was added per well for 1 h. All incubations took place at 37°C. The wells were washed three times, and o-phenylenediamine (0.04%) in citrate phosphate buffer (pH 5.0) plus 0.01% H₂O₂ was added. The reaction was allowed to proceed for 15 min and was then interrupted by the addition of 50 μl of 8 M H₂SO₄. The absorbance at 492 nm was determined in a microplate reader (Multiskan EX; Labsystems Uniscience). For determination of dose-dependent attachment of Lsa24 to laminin, protein concentrations varying from 0 to 1,000 nM in PBS were used. Binding was also evaluated as a function of varied laminin amounts from 0 to 1 μg. For statistical analyses, the attachment of Lsa24 to laminin was compared to its binding to BSA and to all ECM macromolecules by Student's two-tailed t test. A P value less than 0.05 was considered statistically significant.

Metaperiodate treatment of laminin.Microtiter 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 50 mM sodium acetate buffer, pH 5.0, and immobilized laminin was treated with different sodium metaperiodate concentrations (5 to 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 Lsa24 (1 μg in PBS per well) to periodate-treated laminin was assessed as outlined above.

L. interrogans ECM-binding assays.Adhesion of intact L. interrogans to ECM components was performed according to a protocol described for spirochetes (33). Briefly, individual wells of Lab-Tek II chamber slides (Nalge Nunc International) were coated with 4 μg of laminin, cellular and plasma fibronectin, collagens I and IV, and the attachment negative control protein BSA, in PBS. After 16 h of incubation at 4°C, wells were washed with PBS and blocked for 2 h with 1% BSA in PBS. After washing the wells with PBS, 5 × 10⁷ leptospires diluted in PBS were added to each well. Following a 2-h incubation at 29°C, wells were gently washed with PBS (six times for 5 min each time), and the attached spirochetes were visualized by dark-field microscopy. The number of leptospires in six 400× fields was enumerated, and the average number of bacteria per field was estimated.

Inhibition experiments.In vitro inhibition experiments were performed using the same protocol described for L. interrogans ECM-binding assays, except for the addition of 20 μg of the recombinant proteins Lsa24 and Loa22 (used as a negative control) to the blocking solution. Quantification of inhibition was performed essentially as described by Guo and colleagues (16). ELISA plate wells were coated with 1 μg of laminin, blocked and washed as already described, and then incubated with 0.5 μg of Lsa24 diluted in 100 μl PBS. Control wells were incubated with PBS. After washing with PBS, the wells were incubated with 50 μl of a suspension containing 2 × 10⁸ spirochetes ml⁻¹ PBS-0.1% BSA (PBSB) for 1 h at 37°C. Unattached organisms were removed after three washes with PBSB. The wells were then incubated for 1 h with 100 μl of a 1:10,000 dilution of anti-LipL32 serum in PBS. After three washes with PBSB, the wells were incubated for 1 h with 100 μl of a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G in PBS. All incubations were performed at 37°C. After washing, the reactions were developed as described above and the absorbance at 492 nm was taken.

Isolation of leptospiral OMPs by TX-114 extraction.OMPs were extracted according to a previously described method (18). In brief, leptospires cultured as outlined above were washed in PBS-5 mM MgCl₂ and then extracted in the presence of 2% Triton X-114 (TX-114; Sigma-Aldrich), 150 mM NaCl, 10 mM Tris-HCl pH 8.0, and 1 mM EDTA at 4°C for 2 h. The insoluble material was removed by centrifugation at 17,000 × g for 10 min. After centrifugation, 20 mM CaCl₂ was added to the supernatant. Phase separation was performed by warming the supernatant at 37°C and subjecting it to centrifugation for 10 min at 1,000 × g. Three distinct fractions became apparent: the aqueous phase, the TX-114 phase, and the insoluble pellet. The detergent phase was precipitated with acetone. All fractions were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes for immunological analysis with Lsa24 antiserum.

Protein Data Bank accession numbers.The public database accession numbers for each protein sequence analyzed in this work are listed in Table 2. Proteins can also be accessed by their genome nomenclature for the gene locus and LIC numbers (Leptospira interrogans serovar Copenhageni).

Selection of putative surface proteins from genome sequences.With the ECM-binding studies in mind, our rationale for protein selection was mostly based on cellular localization, since surface proteins are potential targets for mediating adhesion to host. We focused on six proteins: four probable outer membrane proteins, according to genome annotation (29), one lipoprotein having a C-terminal OmpA consensus domain (17, 21, 37), and one protein annotated as hypothetical (29). Except for the previously characterized lipoprotein Loa22 (21), the proteins are new, of unknown function, and could be expressed and purified in our laboratory in concentrations and purities suitable to perform the binding assays. Gene locus identification, protein accession number, features, and computational predictions concerning cellular location of these proteins are listed in Table 2.

Expression, purification, and characterization of recombinant proteins.Oligonucleotides for PCR amplifications (Table 1) were designed from the genome, excluding the signal peptide sequences. The amplified coding sequences were cloned and expressed as full-length proteins in E. coli. Recombinant proteins were purified by metal chelation chromatography, and an aliquot of each protein was analyzed by SDS-PAGE (Fig. 1). As we can see from this figure, most of the contaminants were washed away and all proteins are represented as single major bands. Structural integrity of the purified proteins was assessed by CD spectroscopy. As depicted in Fig. 2, the minima at 208 and 222 nm and the maximum at 192 nm in the CD spectrum showed the high α-helical secondary structure content of the recombinant protein encoded by LIC11947. LIC10009-encoded protein and Loa22 also seemed to have a predominance of α-helices, while the protein encoded by LIC10494 may have both α-helices and β-sheets in its secondary structure composition. LIC12730-encoded protein and Lsa24 presented a predominant signal of β-strands, with minimum ellipticities around 215 nm. The experimental data confirmed the secondary structure content previously predicted by computational analysis (http://bioinf.cs.ucl.ac.uk/psipred/ ).

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FIG. 1.

Purification of recombinant proteins: SDS-PAGE (15%) of purified recombinant proteins obtained by metal affinity chromatography. Proteins encoded by LIC10009 (lane 1), LIC10191, known as protein Loa22 (lane 2), LIC11947 (lane 3), LIC12730 (lane 4), LIC10494 (lane 5), LIC12906, named Lsa24 (lane 6) are shown. Lane M, molecular mass standard.

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FIG. 2.

Circular dichroism spectra of the recombinant proteins. CD spectra of proteins encoded by LIC10009, LIC10191 (Loa22), LIC11947 (predominant α-helical secondary structure), LIC10494 (both α-helical and β-strand), and LIC12730 and LIC12906 (Lsa24) (predominant signal of β-strand) are shown. Far-UV CD spectra are presented as an average of five scans recorded from 190 to 260 nm. φ, molar ellipticity.

Adhesion of recombinant proteins to ECM components.Laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin were immobilized on microdilution wells, and recombinant protein attachment was assessed by an ELISA-based method. As shown in Fig. 3, Lsa24 exhibited a significant level of attachment to laminin, 40-fold above the background level seen with immobilized BSA. Binding of Lsa24 to laminin was also significant compared to its binding to collagen IV (P < 0.05) and cellular fibronectin (P < 0.05) (Table 3). No binding was observed when wells were coated with collagen type I or with the highly glycosylated control protein fetuin. A moderate binding to collagen type I, collagen type IV, and plasma fibronectin was observed with Loa22 (Fig. 3), but optical density values registered at 492 nm were too low to prompt further investigation (Table 3). No specific binding to the target ECM macromolecules was detected with proteins encoded by LIC10009, LIC10494, LIC11947, and LIC12730 (Fig. 3). In fact, LIC10009 encoded protein bound to all coating macromolecules, including the control proteins BSA and fetuin. It seems that those interactions are more due to a general stickiness of the protein or, alternatively, due to residual contaminants not fully eliminated during the purification step rather than to specific interactions. Data are presented as the magnitude of attachment over the negative control protein BSA (arbitrarily set equal to 1) to emphasize specificity of attachment to the ECM macromolecules.

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FIG. 3.

Binding of recombinant proteins to ECM components. Wells were coated with 1 μg of laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin, and the control proteins BSA and fetuin. Recombinant protein attachment to those ECM macromolecules was assessed by an ELISA-based assay. One microgram of recombinant protein was added per well. Optical densities were taken at 492 nm. Specific profiles of adhesion to the various coating macromolecules are shown as magnitude of attachment (fold) over the negative control protein BSA, arbitrarily set equal to 1. Data represent the mean ± standard error of three independent experiments.

The interaction between Lsa24 and laminin was also assessed on a quantitative basis as illustrated in Fig. 4. A dose-dependent and saturable binding was observed when increasing concentrations of the recombinant protein (0 to 1,000 nM) were allowed to adhere to a fixed laminin amount (1 μg). The saturation level was reached at a protein concentration of 500 nM (Fig. 4A). Binding was also evaluated as a function of laminin concentration for a given recombinant protein concentration (1 μg). As shown in Fig. 4B, the degree of attachment increased proportionally as a function of laminin concentration and reached its maximum level at 1 μg of laminin. Increasing the laminin or protein concentration had no effect on the Loa22-laminin interaction (Fig. 4). This recombinant protein was included in our experiments as a negative control.

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FIG. 4.

Binding of Lsa24 to laminin as a function of protein and laminin concentration. (A) Binding of laminin (1 μg) with recombinant protein (concentration ranging from 0 to 1,000 nM); (B) recombinant protein (1 μg) with laminin (amount ranging from 0 to 1 μg). Each point represents the mean absorbance value at 492 nm ± the standard deviation of three independent experiments. Loa22 was included as a negative control since it showed no specific attachment to laminin.

All recombinant proteins used in the binding assays presented above were treated with polymyxin B to ensure removal of residual E. coli LPS that could eventually interfere with the assays. In fact, we compared absorbance values before and after polymyxin treatment and found that Loa22, Lsa24, and the protein encoded by LIC11947 showed a partial reduction in their optical density values after treatment, suggesting a possible involvement of LPS in mediating attachment to ECM (data not shown).

Effects of laminin oxidation on protein binding.In order to address the role of laminin sugar moieties in the interaction with Lsa24, laminin was oxidized by increasing concentrations of sodium metaperiodate, ranging from 5 to 100 mM. The effect of periodate concentration on the binding is shown in Fig. 5. Laminin oxidation was performed at 4°C for 15 min. This mild treatment ensures cleavage of vicinal carbohydrate hydroxyl groups while the polypeptide chain structure remains intact (44). The oxidation effect was dose dependent, and a considerable reduction (72%) in Lsa24 attachment to metaperiodate-treated laminin was observed at a 10 mM concentration of periodate (Fig. 5). These results indicate that laminin sugar residues are critical for the interaction of Lsa24 with this major ECM glycoprotein.

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FIG. 5.

Sugar moiety contribution to the laminin-Lsa24 interaction. Immobilized laminin was treated with sodium metaperiodate (5 to 100 mM) for 15 min at 4°C in the dark. Mean absorbance values at 492 nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated laminin (0 mM). The percent reduction in protein attachment to metaperiodate-treated laminin as a function of periodate concentration is indicated above each bar.

L. interrogans attachment to ECM macromolecules.The ability of intact L. interrogans to adhere to various ECM proteins was evaluated by dark-field microscopy. Leptospires attached to chamber slides coated with laminin at an average density of 40 organisms per 400× field (Fig. 6A). Slides coated with collagen I, collagen IV, cellular fibronectin, or plasma fibronectin bound approximately 100 to 140 leptospires per 400× field, as shown in Fig. 6B to E, respectively. In contrast, control slides coated with BSA bound fewer than five organisms per 400× field (Fig. 6F).

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FIG. 6.

L. interrogans attachment to ECM macromolecules. Glass slides coated with laminin (A), collagen I (B), collagen IV (C), cellular fibronectin (D), plasma fibronectin (E), or BSA (F) were incubated with 5 × 10⁷ leptospires (L. interrogans serovar Pomona). Attachment was visualized using dark-field microscopy.

Inhibition of L. interrogans attachment to laminin by Lsa24.The effect of recombinant Lsa24 on leptospiral adherence to laminin was first examined by in vitro inhibition experiments (Fig. 7). Laminin-coated chamber slides were preincubated with Lsa24 for 2 h before whole L. interrogans cells were added. Attached leptospires were visualized by dark-field microscopy. The addition of Lsa24 reduced leptospire binding to laminin-coated slides (Fig. 7B). Attachment was not affected by prior incubation with the negative control protein Loa22 (data not shown). The inhibitory effect exerted by Lsa24 on leptospiral adherence was quantified by an ELISA-like method. Laminin-coated microtiter wells were incubated with 0.5 μg of Lsa24 prior to the addition of 1 × 10⁷L. interrogans. Wells were probed with anti-LipL32 serum, based on the fact that LipL32 is a major outer membrane leptospiral protein (18). Lsa24 caused a modest but significant reduction in the number of leptospires adhering to laminin (P < 0.05) (Fig. 7C). The experiment was repeated three times with similar results.

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FIG. 7.

Inhibition of L. interrogans attachment to laminin by Lsa24. Chamber slides were coated with 4 μg of laminin for 16 h. The slides were blocked with BSA, and 5 × 10⁷ leptospires were added to each chamber. (A) No protein added to the blocking solution; (B) 20 μg of Lsa24 was added to the blocking solution to test for its ability to competitively inhibit attachment of L. interrogans to laminin. Adherence was visualized by dark-field microscopy. (C) Leptospira attachment to the substrate was quantified by ELISA. Laminin-coated microtiter wells were incubated with 0.5 μg of Lsa24 for 1 h 30 min prior to the addition of 1 × 10⁷ leptospires. Wells were probed with anti-LipL32 serum. Data are expressed as the mean A₄₉₂ ± the standard error of three independent experiments, each performed in triplicate. Significance was assessed by comparison with the no-protein wells by Student's two-tailed t test (*, P < 0.05).

Cellular localization of Lsa24.To determine whether Lsa24 is localized at the membrane, we performed an extraction of the L. interrogans outer membrane with the nonionic detergent Triton X-114. The three fractions derived from TX-114-extracted material after incubation at 37°C were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with Lsa24 antiserum. Lsa24 was found to be completely absent from the detergent-insoluble pellet fraction but could be readily detected in the TX-114 detergent phase, indicating that this protein is a component of the leptospiral membrane (Fig. 8).

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FIG. 8.

Cellular localization of Lsa24. Triton X-114 fractions of L. interrogans serovar Copenhageni organisms were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with Lsa24 antiserum. Fractions analyzed were the whole organism (lane 1), Triton X-114-insoluble pellet (lane 2), aqueous phase (lane 3), and detergent phase (lane 4).