Repeated domains of leptospira immunoglobulin-like proteins interact with elastin and tropoelastin

Abstract

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7'-8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17-27HTE) as determined by ELISA (LigBCon4, K(D) = 0.50 microm; LigBCen7'-8, K(D) = 0.82 microm; LigBCen9, K(D) = 1.54 microm; and LigBCen12, K(D) = 0.73 microm). The interaction of LigBCon4 and 17-27HTE was further confirmed by steady state fluorescence spectroscopy (K(D) = 0.49 microm) and ITC (K(D) = 0.54 microm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17-27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.

FIGURE 1.

Binding of L. interrogans serovar Pomona (NVSL 1427-35-093002) to elastin and tropoelastin. A, binding of Leptospira to BSA, elastin, or tropoelastin. Leptospira (10⁷) were added to wells coated with BSA, human lung elastin, or HTE (1 μg in 100 μl of Tris buffer). B, binding of Leptospira to immobilized elastin or HTE. Leptospira (10⁸) were cultured in human lung elastin-, THE-, or BSA-coated (negative control) (1 μg in 100 Tris buffer) or -uncoated wells (negative control). Leptospiral immobilization was assayed by immunofluorescence microscopy. C, binding of Leptospira (10⁷) to various concentrations of human lung elastin, human aortic elastin, human skin elastin, HTE, or BSA (0, 0.25, 0.5, 1, 2, 4, 8, and 16 μg/ml in 100 μl of Tris buffer). BSA serves as negative control. A and C, the binding of Leptospira was estimated by ELISA, and each value represents the mean ± S.E. of three trials performed in triplicate samples. Statistically significant (p < 0.05) differences compared with the negative reference are indicated by an asterisk.

FIGURE 2.

A schematic diagram showing the structure of Lig proteins and the truncated Lig proteins used in this study.

FIGURE 3.

Localization of the elastin or HTE binding domains on Lig proteins. Various concentrations (0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 μm) of biotin (negative control), biotinylated rAFnBPA-(194–511) are as follows: A, LigBCon, LigAVar, LigBCen, and LigBCtv; B, LigBCon, LigBCon1–3, and LigBCon4–7′; C, LigBCen, LigBCen1, LigBCen2, and LigBCen3; D, LigBCon4′-7, LigBCon4, LigBCon5, and LigBCon6–7′; E, LigBCen1, LigBCen7′–8, LigBCen9, LigBCen10, and LigBCen11; F, LigBCen2, LigBCen12, or LigBCen2NR; and G, LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 were added to wells coated with 1 μg of BSA (negative control and data not shown). A–F, human lung elastin; G, HTE in Tris buffer. The binding of biotinylated proteins to elastin or HTE was measured by ELISA. For all experiments, each value represents the mean ± S.E. of three trials in triplicate samples. Statistically significant (p < 0.05) differences compared with negative control are indicated by an asterisk.

FIGURE 4.

Soluble elastin peptide or HTE inhibited LigBCon4, LigBCen7′-8, LigBCen9, LigBCen12 binding to immobilized elastin or HTE. One μm of LigBCon1–3 (negative control), biotinylated rAFnBPA-(194–511) (positive control) LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 treated with various concentrations (3.90, 7.81, 15.62, 31.25, 62.5, 125, and 250 μg/ml in 100 μl of Tris buffer) of soluble elastin peptide or HTE were added to each well coated with 1 μg of BSA (negative control and data not shown). A, human lung elastin; B, HTE in Tris buffer. The binding of biotinylated proteins to wells was measured by ELISA. The percentage of binding was determined relative to the binding of biotinylated proteins in the untreated well. For all experiments, each value represents the mean ± S.E. of three trials in triplicate samples. Statistically significant (p < 0.05) differences compared with the negative reference are indicated by an asterisk.

FIGURE 5.

Mapping the binding site of LigBCon4, LigBCen7′-8, LigBCen9, and LigBCen12 on HTE. A, chart presenting the location of HTE and truncated HTE used in this study. B–E, binding of LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 to various concentrations of immobilized truncated HTE. Various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 μm) of biotin (negative control and data not shown), biotinylated (B) LigBCon4, (C) LigBCen7′–8, (D) LigBCen9, or (E) LigBCen12 were added to 1 μm of full-length HTE, 1–18HTE, 17–27HTE, 27–36HTE, or BSA (negative control) in 100 μl of phosphate-buffered saline-coated microtiter plate wells. Bound proteins were measured by ELISA. For all experiments, each value represents the mean ± S.E. of three trials in triplicate samples. Statistically significant (p < 0.05) differences compared with the negative control are indicated by an asterisk.

FIGURE 6.

Interaction of 17–27HTE and LigBCon4 by steady state fluorescence spectroscopy and ITC. A, intrinsic fluorescence spectrum of LigBCon4 in the presence and absence of 17–27HTE. One μm of LigBCon4in Tris buffer was excited at 295 nm. Aliquots of 17–27HTE from respective stock solutions were added. The figure shows Trp fluorescence in the presence of 0, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 μm 17–27HTE. B, determination of K_D value of LigBCon4 and truncated HTE by monitoring the quenching fluorescence intensities of LigBCon4 titrated by 1–18HTE, 27–36HTE (data not shown), or 17–27HTE shown in A. The emission wavelength recorded in this figure was 327 nm; only the titration of 17–27HTE can quench the spectrum of LigBCon4. K_D value was determined by fitting the data point into Equation 2 as described under “Materials and Methods” (K_D = 0.49 ± 0.07 μm). C, ITC profile of LigBCon4 with 17–27HTE as a typical ITC profile in this study. Upper panel, heat difference obtained from 25 injections. Lower panel, integrated curve with experimental point (◇) and the best fit (). The thermodynamic parameters are shown in Table 3.

FIGURE 7.

Effect of different pH values on the binding of LigBCon4 to 17–27HTE. A, titration curve for LigBCon4 and 17–27HTE. LigBCon4 (broken line) undergoes two-step charge transition at pH 4 and 11. 17–27 HTE (solid line) undergoes a charge transition at pH 9.5. B, determination of K_D value of LigBCon4 and 17–27HTE by monitoring the quenching fluorescence intensities of LigBCon4 at various concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 μm) of 17–27HTE in Tris buffer at different pH values (4.5, 5.5, 6.5, 7.5, and 8.5). One μm LigBCon4 in Tris buffer was excited at 295 nm, and the emission wavelength recorded in this figure was 327 nm. The K_D value was determined by fitting the data point into Equation 1 as described under “Materials and Methods.” C, LigBCon4 binding to 17–27HTE from pH 4.5 to 8.5. A plot of K_D against pH indicates that the interaction is strongly dependent on pH.

FIGURE 8.

Asp-341 is one of the important residues contributing to the LigBCon4-17-27HTE interaction. A, sequence alignment of LigBCon4 (Asp-341), LigBCen7′–8 (Asp-703), LigBCen9 (Asp-789), and LigBCen12 (Asp-1061) shows that an aspartate is conserved in these four domains as indicated by an asterisk. The gaps were introduced to maximize the alignment. Black and gray colored residues indicate the conserved residues, and the homology analysis was performed with EMBL-EBI ClustalW. B, intrinsic fluorescence spectrum of LigBCon4D341N in the presence and absence of 17–27HTE. One μm LigBCon4D341N in Tris buffer was excited at 295 nm. Aliquots of 17–27HTE from the respective stock solutions were added. The figure shows Trp fluorescence in the presence of 0, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 μm of 17–27HTE (inner plot). The determination of the K_D value of LigBCon4D35N and 17–27HTE by monitoring the quenching fluorescence intensities of LigBCon4D35N was titrated by 17–27HTE. The emission wavelength recorded in this figure was 327 nm, and K_D value was revealed by fitting the data point into Equation 2 as described under “Materials and Methods.” (K_D = 2.44 ± 0.21 μm). C, ITC profile of LigBCon4D35N with 17–27HTE as a typical ITC profile in this study. Upper panel, heat difference obtained from 25 injections. Lower panel, integrated curve with experimental point (◇) and the best fit (). The thermodynamic parameters are shown in Table 3.