J Vet Sci. 2008 Jun;9(2):133-144. English.
Published online Jun 30, 2008.
Copyright © 2008 The Korean Society of Veterinary Science
Original Article

The C-terminal variable domain of LigB from Leptospira mediates binding to fibronectin

Yi-Pin Lin and Yung-Fu Chang
    • Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Abstract

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.

Keywords
adhesion; Fn; Leptospira; LigB; MDCK cell; siRNA

Introduction

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes in the genus Leptospira [22]. The disease occurs widely in developing countries and is reemerging in the United States [29]. The clinical features are variable and include subclinical infection, a self-limited anicteric febrile illness and severe, potentially fatal disease [22]. In the severe form of leptospirosis (Weil's syndrome), the symptoms include an acute febrile illness associated with multi-organ damage with liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis [10]. If not treated, the mortality rate may exceed 15% [49]. Furthermore, Leptospira infection can trigger autoimmune diseases in horses as well as humans [36, 41]. Several factors associated with virulence have been proposed for Leptospira spp., including the sphingomyelinases, serine proteases, zinc-dependent proteases, collagenase [3], LipL32 [59], the novel factor H-binding protein LfhA [54], and lipopolysaccarides [56].

Pathogenic spirochetes have evolved a variety of strategies to infect host cells such as evasion of the innate as well as adaptive immunity [54]. Attachment to host cells is an essential step for colonization by bacterial pathogens. Leptospira has been shown to bind to mammalian cells, such as Madin-Darby canine kidney (MDCK) cells [2] via the extracellular matrix (ECM) [15]. Several adhesion molecules in the pathogenic spirochetes have been identified including a Fn binding protein (36 kDa protein) [30], a laminin binding protein (Lsa24) [1], and Lig proteins [25, 33, 34] from Leptospira spp., decorin-binding proteins (Dbp A and B) [37] and Fn-binding proteins (BBK 32 and 47 kDa) [21, 38] from Borrelia spp. and MSP, Tp0155, Tp0483, Tp0751 from Treponema spp. [4, 5, 9]. Lig proteins (Lig A, B and C) possess immunoglobulin-like domains with 90 amino acid repeats that have been identified in other adhesion molecules, such as the intimin of Escherichia coli and the invasin of Yersinia pseudotuberculosis[14, 17]. Interestingly, the N-terminal 630 amino acid sequences of LigA and B are identical, but the C-terminal amino acid sequences are variable with only 34% identitify [33]. ligB also encodes a C-terminal, non-repeat domain of 771 amino acid residues [33]. On the other hand, the ligA-ligB intergenic regions from L. kirschneri and L. interrogans are 943 bp and 1347 bp in length respectively, and ligC is not linked to the ligA-ligB locus [25]. The expression of LigA and LigB is controlled by a key environmental signal, osmolarity, to enhance the binding of Leptospira to host cells [26, 27].

It has been shown that the lig genes are present exclusively in pathogenic Leptospira spp [25, 33]. LigA and LigB are weakly expressed in low passage, but not in high passage cultures of this organism [25, 33]. Importantly, we have shown that LigA and LigB expression is upregulated in vivo in the kidneys of Leptospira-infected hamsters [34]. Recently, LigA and LigB have been reported to bind to extracellular matrix proteins including collagens type I and IV, laminin, fibronectin, and fibrinogen [6, 24]. These data indicate that Lig proteins may play an important role in attachment of pathogenic leptospires to host cells.

Although there are three copies of lig genes (ligA, B and C) in L. interrogans serovar Pomona and L. interrogans serovar Copenhageni [31, 33, 34], only ligB is present in most pathogenic Leptospira spp. ligA is absent in L. interrogans serovar Lai [42], ligC is truncated (a pseudogene) in L. kirschneri serovar Grippotyphosa [25] and both ligA and ligC are absent in L. borgpetersenii serovar Harjo [3]. Therefore, we focused on LigB in this study and report that the variable region of LigB binds with high affinity to Fn, suggesting that this fragment is crucial for bacterial adhesion to host cells.

Materials and Methods

Bacterial strains and cell culture

L. interrogans serovar Pomona (NVSL1427-35-093002) was used in this study [35]. All experiments were performed with virulent, low-passage strains obtained by infecting golden syrian hamsters as previously described [35]. Leptospires were grown in EMJH medium at 30℃ for less than 5 passages and growth was monitored by darkfield microscopy. The MDCK cells (ATCC CCL34) were cultured in Dulbecco minimum essential medium containing 10% fetal bovine serum (GIBCO, USA) and were grown at 37℃ in a humidified atmosphere with 5% CO2.

Reagents and antibodies

Horseradish peroxidase (HRP)-conjugated goat anti-hamster antibody, HRP-conjugated goat anti-mouse antibody and HRP-conjugated goat anti-rabbit antibody were purchased from Zymed (USA). Rabbit anti-glutathione S-transferase (GST) antibody, Alexa 594-conjugated goat anti-hamster antibody, Alexa 488-conjugated goat anti-hamster antibody, and FITC-conjugated goat anti-mouse antibody were purchased from Molecular Probe (USA). Anti-Fn (MAB1932) and anti-actin mouse antibodies (MAB1501) were purchased from Chemicon International (USA). Human plasma Fn was purchased from GIBCO (USA). Anti-L. interrogans antibodies were prepared in hamsters as previously described [35].

Plasmid construction and protein purification

Constructs for the expression of GST, GST fused with the conserved region of LigB (LigBCon; amino acids 1-630) and GST fused with the central variable region of LigB (LigBCen; amino acids 631-1417) were previously generated using the vector pGEX-4T-2 (Amersham Pharmacia Biotech, USA) [33]. GST fused with the C-terminal variable region of LigB (LigBCtv; amino acids 1418-1889) was generated using the vector pET41A (Novogen, USA). Relevant fragments of DNA were amplified by PCR using primers based on the ligB sequence [33]. Primers were designed to introduce a SalI site at the 5' end of each fragment and a stop codon followed by a NotI site at the 3' end of each fragment. The PCR products were digested sequentially with SalI and NotI and then ligated into pGEX-4T-2 or pET41A cut with SalI and NotI. We purified the soluble form of GST-LigBCon, GST-LigBCen and GST-LigBCtv from E. coli as previously described [34, 35].

Binding assays by ELISA

To measure the binding of Leptospira to the ECM components, 1 mg of each ECM component (as indicated in Fig. 1A) in 100 µl PBS (pH 7.2) was coated onto microtiter plate wells. For the dose-dependent binding experiments, different concentrations of Fn (as indicated in Fig. 1B) were coated onto the microtiter plate wells. The plates were incubated at 4℃ for 16 h and subsequently blocked with blocking buffer (50 µl/well) containing 3.5% BSA in 50 mM Tris (pH 7.5)-100 mM NaCl-1 mM MgCl2, MnCl2, and CaCl2 at room temperature (RT) for 2 h. Then, the Leptospira (107) were added to each well and further incubated at 37℃ for 6 h. To determine the inhibition of Leptospira binding to the MDCK cells by Fn, the Leptospira (107) were pre-incubated at 37℃ for 1 h with various concentrations of Fn (as indicated in Fig. 1C) prior to the addition of the MDCK cells (105) and finally incubated for 6 h at 37℃. The percentage of adhesions was determined relative to the attachment of the untreated Leptospira binding to the MDCK cells. For all experiments, the same concentration of BSA was used as a negative control. To determine the binding of LigBCen or LigBCtv to Fn, 10 nM of GST-LigBCen, GST-LigBCtv or GST (negative control) was added to 96 well microtiter plates coated with various concentrations of Fn (as indicated in Fig. 3A) or BSA (negative control and data not shown) in 100 µl PBS for 1 h at 37℃.

Fig. 1
The binding of L. interrogans serovar Pomona (NVSL 1427-35-093002) to Fn (A). Binding of Leptopsira to various immobilized ECM components. Leptospira (107) were added to wells coated with each ECM (1 mg in 100 µl PBS) including Fn, chondroitin-6-sulfate (C6S), chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), gelatin A (GA), gelatin B (GB), heparin (HP), keratin (KR), or BSA (negative control). (B). Binding of Leptospira (107) to various concentrations of Fn (0, 10, 20, 100 or 1,000 µg in 100 µl PBS). BSA served as a negative control. (C). Fn inhibits the binding of Leptospira to the MDCK cells. Leptospira (107) were treated with various concentrations of Fn (0, 0.01, 0.1, 0.2, 1, 2, or 10 µg) or BSA (negative control) prior to addition to the MDCK cells (105). The percentage adhesion was determined relative to the attachment of untreated Leptospira onto the MDCK cells. (D). Binding of Leptospira to immobilize Fn. Leptospira (108) were cultured in Fn or BSA (negative control) coated (1 mg in 100 µl PBS) or un-coated wells (negative control). (E). Fn inhibited the binding of Leptospira to the MDCK cells. Leptospira (108) were pre-treated with 10 µg of Fn or BSA (negative control) prior to addition to the MDCK cells (106). Un-treated Leptospira was used as a negative control. The binding of Leptospira to ECMs or Fn or the adhesion of Leptospira to the MDCK cells was measured by ELISA (A, B, and C) or EPM (D and E). For all experiments, each value represents the mean ± SE of three trials performed in triplicate samples. Statistically significant (p < 0.05) differences are indicted by an asterisk. The EPM settings were identical for all captured images (D and E).

Fig. 3
LigBCen or LigBCtv binds to Fn and inhibits the binding of Leptospira to Fn (A). Binding of LigBCen or LigBCtv to various concentrations of immobilized Fn. Ten nM of GST-LigBCen, GST-LigBCtv or GST (negative control) was added to wells coated with various concentrations of Fn (0, 0.27 µM, 0.45 µM, 2.7 µM, 4.5 µM, 27 µM, or 45 µM) in 100 µl PBS. The binding of each of these proteins to Fn was measured by ELISA. (B) LigBCen or LigBCtv inhibited the binding of Leptospira to immobilized Fn. Various concentrations (0, 2, 4, 6, or 8 nM) of GST-LigBCen, GST-LigBCtv, or GST (negative control) were added to each well coated with Fn (1 mg in 100 µl PBS) prior to the addition of Leptospira (107). The attachment of Leptopsira to wells was measured by ELISA. The percentage of attachment was determined relative to the attachment of Leptopsira in the untreated Fn. (C) LigBCen or LigBCtv inhibited the binding of Leptospira to Fn. Fifty nM of GST-LigBCen, GST-LigBCtv or GST (negative control) was added to wells coated with Fn (1 mg in 100 µl PBS) prior to the addition of Leptospira (108). The binding of Leptospira to wells was detected by EPM. In (A) and (B), each value represents the mean ± SE of three trials performed in triplicate samples. Statistically significant differences (p < 0.05) are indicted by *. In (C), The EPM settings were identical for all captured images. Images were processed using Adobe Photoshop CS2.

To measure the binding inhibition of Leptospira to Fn, various concentrations of GST-LigBCen, GST-LigBCtv (as indicated in Fig. 3B) or GST (negative control) in 100 µl PBS was added to Fn or BSA (negative control and data not shown) (1 mg in 100 µl PBS) coated wells at 37℃ for 1 h, then the Leptospira (107) were added to each well and incubated at 37℃ for 6 h. To measure the binding of LigBCen or LigBCtv to the MDCK cells, the MDCK cells (105) were incubated with various concentrations (as indicated in Fig. 4A) of GST-LigBCen, GST-LigBCtv or GST (negative control) in 100 µl PBS for 1 h at 37℃. To measure the binding inhibition of Leptospira to the MDCK cells treated with LigBCen or LigBCtv, the MDCK cells (105) were pretreated with various concentrations (as indicated in Fig. 4B) of GST-LigBCen, GST-LigBCtv or GST (negative control) in 100 µl PBS for 1 h at 37℃. Then, the Leptospira (107) were added to each well and incubated for 6 h at 37℃. Following the incubation, the plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST). To measure the binding of the Leptospira, hamster anti-Leptospira (1:200) and HRP-conjugated goat anti-hamster IgG (1:1,000) were used as primary and secondary antibodies, respectively. To detect the binding of GST-LigBCen, GST-LigBCtv, or GST to Fn or the MDCK cells, rabbit anti-GST (1:200) and HRP-conjugated goat anti-rabbit IgG (1:1,000) were used as primary and secondary antibodies, respectively. After washing the plates three times with PBST, 100 µl of TMB (KPL, USA) was added to each well and incubated for 5 min. The reaction was stopped by adding 100 µl of 0.5% hydrofluoric acid in each well. Each plate was read at 630 nm by an ELISA plate reader (Bioteck EL-312; BioTeck, USA). Each value represents the mean ± standard error of the mean (SEM) of three trials performed in triplicate samples. Statistically significant (p < 0.05) differences are indicated by asterisks.

Fig. 4
Isothermal titration calorimetry (ITC) profile of LigBCtv with Fn as a typical ITC profile in this studyA: heat differences obtained from 25 injections. B: Integrated curve with experimental point (◆) and the best fit (-). The thermodynamic parameters are shown in Table 1.

Binding assays by epifluorescence microscopy (EPM) and confocal laser-scanning microscopy (CLSM)

To measure the binding of Leptospira to Fn by EPM, Leptospira (108) were added to each well (eight well culture slides) coated with 1 mg Fn or BSA (negative control) in 100 µl of PBS and incubated at 37℃ for 6 h (Fig. 1D). To measure the binding inhibition of Leptospira to the MDCK cells by Fn, 108 Leptospira were pre-incubated with 10 µg of Fn or BSA (negative control) in 100 µl of PBS for 1 h at 37℃ prior to the addition of 106 MDCK cells and incubated 6 h at 37℃ (Fig. 1E). To measure the binding inhibition Leptospira to Fn by LigBCen or LigBCtv by EPM, 50 nM of GST-LigBCen, GST-LigBCtv or GST (negative control) in 100 µl PBS was added to each of the Fn or BSA (negative control and data not shown) (1 mg per 100 µl) coated wells for 1 h at 37℃. Then, the Leptospira(108) were added to each well and incubated for 6 h at 37℃ (Fig. 3C). To determine the binding inhibition of Leptospira to the MDCK cells by LigBCen or LigBCtv by CLSM, the MDCK cells (106) were preincubated with 50 nM of GST-LigBCen, GST-LigBCtv or GST (negative control) in 100 µl of PBS for 1 h at 37℃ respectively. Then, the Leptospira (108) were added to each well and incubated for 6 h at 37℃ (Fig. 4C). For the detection of Leptospira binding in Figs. 1D, E, and Fig. 3C, hamster anti-Leptospira antibodies (1:100) and Alexa 488-conjugated goat anti-hamster IgG (1:250) were used as primary and secondary antibodies, respectively. To determine the attachment of Leptospira and the binding of GST-LigBCen, GST-LigBCtv or GST, Fig. 4C, rabbit anti-GST (1:250) and hamster anti-Leptospira antibodies (1:100) served as primary antibodies, and FITC conjugated goat anti-rabbit IgG (1:250) and Alexa 594-conjugated goat anti-hamster IgG (1:250) were used as secondary antibodies. Fixation and immunofluorescence staining were performed as previously described [44] with slight modifications. Briefly, Leptopsira and the MDCK cells were fixed in 2% paraformaldehyde for 60 min at RT. For the antibody labeling, fixed bacteria were incubated in PBS containing 0.3% BSA for 10 min at RT. The primary and secondary antibodies, in the PBS containing 0.3% BSA, were incubated sequentially for 60 min at RT. After incubation with the primary and secondary antibodies, the glass slides were mounted with coverslips using Prolong Antifade (Molecular Probe, USA) and viewed with a 60 × objective by EPM (Nikon, Japan) or CLSM (Olympus, Japan). An Olympus Fluoview 500 confocal laser-scanning imaging system, equipped with krypton, argon and He-Ne lasers on an Olympus IX70 inverted microscope with a PLAPO 60 × objective, was used. The settings were identical for all captured images. Images were processed using Adobe Photoshop CS2. For counting the attachment of Leptospira to the MDCK cells or Fn, three fields were selected to count the number of binding organisms. All studies were repeated three times and the number of Leptospira attached to the MDCK cells were counted by an investigator blinded to the treatment group.

GST pulldown assay

The GST pull-down assay was performed as previously described [57]. Purified proteins or GST (negative control) were loaded onto 0.5 ml glutathione-Sepharose beads (Amersham Biosciences Piscataway, USA) at 4℃ overnight. The beads were then washed three times with the lysis buffer containing 30 mM Tris acetate, 10 mM sodium phosphate, pH 7.4, 0.1% Tween 20, 1 mM EDTA, 2 µg/ml leupeptin, 4 µg/ml aprotinin, 1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The MDCK cells (106) were lysed in the lysis buffer and used immediately after lysis. A 500 µl aliquot of cell lysate or human plasma Fn (40 µg/ml) was incubated with purified proteins immobilized on glutathione-Sepharose beads at 4℃ for 3 h. After incubation, the beads were separated by centrifugation, washed three times with the lysis buffer and boiled in Laemmli sample loading buffer consisting of 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.25 mM PMSF, and 0.1% bromophenol blue in 20% glycerol. The eluted proteins were subjected to 6% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The membranes were incubated in 5% skim milk in PBS/T overnight and then incubated with mouse anti-Fn antibody (1:1,000). The immunocomplexes were detected with an HRP-conjugated goat anti-mouse IgG antibody (1:5,000).

Small interfering RNA (siRNA) inhibition of LigB binding

The siRNA duplexes directed against the sequence 5'-gcagcacaacuuccaauua-3' of Fn and negative siRNA duplex, 5'-auucuaucacuagcgugac-3', were selected by the software, siDESIGN [43] and synthesized by Dharmacon (USA). The RNA duplexes were introduced into the MDCK cells by the method of lipofection [18], and 8 × 105 cells were transfected with 0.4 µg negative siRNA and Fn-siRNA. Adhesion assays were performed 72 h after lipofection [51]. The knockdown efficiency of endogeneous Fn expression was determined as previously described [57] with slight modification. The total protein contents of the MDCK cells (106) were analyzed using Western immunoblotting as described under 'GST pulldown assays'. The protein bands of actin derived from the MDCK cells were measured as a control using a mouse anti-actin antibody (1:5,000). The band intensity was measured by densitometry using the Image J software (National Institutes of Health, Bethesda, MD, USA) [53]. A LigB binding assay was performed 72 h after lipofection. To determine the binding of LigB fragments to Fn, each fragment (50 nM) was added to the MDCK cells (106) transfected with Fn or negative siRNA. To determine the binding of each fragment and the expression of Fn in the MDCK cells, rabbit anti-GST (1:250) and mouse anti-Fn (1:250) served as the primary antibodies, and FITC-conjugated goat anti-mouse IgG (1:250) and Texas Red-conjugated goat anti-rabbit IgG (1:250) were used as secondary antibodies. Fixation, immunofluorescence staining, image detection, and processing were carried out as described in previous sections. All experiments were performed in triplicate.

Isothermal titration calorimetry

The experiments were carried out with CSC 5300 microcalorimeter (Calorimetry Science, USA) at 25℃ as previously described [47]. In a typical experiment, the cell contained 1 ml of a solution of proteins, and the syringe contained 250 µl of a solution of Fn at a concentration that was 20 times higher than the protein concentration in the cell. Both solutions were in PBS pH 7.5. The titration was performed as follows: 15 to 25 injections of 10 µl (Table 1) with a stirring speed of 250 rpm, and the delay time between the injections was 5 min. Data were analyzed using Titration BindingWork 3.1 software (Calorimetry Science, USA) that was fit to an independent binding model. The concentration of Fn and LigB used in this study was based on our preliminary titration experiments (data not shown).

Table 1
Thermodynamic parameters for the interaction of Fn and truncated LigB

Statistical analysis

Statistically significant differences between samples were determined using the Student's t-test following logarithmic transformation of the data. Two-tailed p-values were determined for each sample, and a p < 0.05 was considered significant. Each data point represents the mean ± SE of a sample tested in triplicate. An asterisk indicates that the result was statistically significant.

Results

Attachment of Leptospira to the MDCK cells was mediated by fibronectin

The binding of leptospiral cells to various ECM components was determined by ELISA. As shown in Fig. 1A,Leptospira were strongly bound to Fn, but not to other ECM molecules (Fig. 1A). Furthermore, the binding of Leptospira to Fn was dose dependent (Fig. 1B). When Leptospira were pretreated with Fn, binding to the MDCK cells was decreased (Fig. 1C). There was an approximately 3.5-fold increase in the immobilization of Leptospira in the Fn-coated wells compared to the controls (Fig. 1D). Moreover, Fn was observed to block the attachment of Leptospira, by approximately 47%, when the Fn treated Leptospira were added to the MDCK cells (Fig. 1E). Thus, Fn appears to mediate the attachment of Leptospira to the MDCK cells.

Interaction between LigB and Fn

To determine whether LigB interacts with Fn, we truncated the LigB protein into three parts, LigBCon, LigBCen and LigBCtv, (Fig. 2A) due to the difficulty of expressing and purifying the full length LigB [33]. First, we analyzed the interaction of each LigB fragment with Fn using a GST-pull down assay. Our results showed that both human plasma Fn and Fn derived from the MDCK cell lysates could bind both LigBCen and LigBCtv, but not LigBCon (Figs. 2B and C). Since LigBCen and LigBCtv showed a positive pull down result, the interaction between LigBCen and LigBCtv with Fn was further studied by ELISA. We found that both the binding of LigBCen and LigBCtv to Fn, and the inhibition of the attachment of Leptospira to Fn by LigBCen and LigBCtv, were dose-dependent (Figs. 3A and B). Moreover, the EPM images revealed an up to 40% reduction in the attachment of Leptospira to Fn in the presence of LigBCen and LigBCtv (Fig. 3C). Finally, in order to quantitatively evaluate the binding affinity between Fn and LigB fragments, the dissociation constants (Kd) were measured by ITC (Table 1). Fig. 4 shows the data from a typical ITC experiment. The interaction appears to be exothermic with a favorable enthalpy and unfavorable entropy. The calculated Kd values for Fn binding to LigBCen and LigBCtv were 0.01 µM and 8.55 µM, respectively (Table 1). The binding of LigBCon could not be detected by ITC (data not shown). These findings are in agreement with our previous results. Altogether, these data indicate that Fn specifically interacts with LigBCen and LigBCtv fragments.

Fig. 2
The interaction between LigB and Fn by the GST-pull down assay (A) A schematic diagram showing the structure of LigB and the truncated LigB protein used in this study. (B). Human plasma Fn ( lane 2 to lane 5 ) or cell lysates of the MDCK cells (lane 7 to lane 10) was applied to the GST beads preimmobilized by GST, GST-LigBCon, GST-LigBCen, or GST-LigBCtv at 4℃ for 3 h. The pull down complex was analyzed by immunoblot analysis using Fn antibodies. Lane 1 and lane 6 contain 1 µg of human plasma Fn and the cell lysate from 1 × 106 MDCK cells, respectively, to serve as a positive reference. Lane 2 and lane 7 are GST-LigBCen, lane 3 and lane 8 are GST-LigBCtv, lane 4 and lane 9 are GST-LigBCon, and lane 5 and lane 10 are GST. The molecular mass of the human Fn and canine Fn (MDCK cells) was 261 kDa and 271 kDa, respectively, and the relative positions of the standards are given in kDa on the left.

LigBCen and LigBCtv mediate the attachment of Leptospira to the MDCK cells

To determine if LigB is used by Leptospira to adhere to the MDCK cells, various concentrations of LigBCen or LigBCtv were added to the MDCK cells, and binding was detected by ELISA and immunofluorescence staining. Our results clearly showed that LigBCen and LigBCtv were bound to the MDCK cells in a dose dependent manner (Fig. 5A). Pretreatment of the MDCK cells with LigBCen or LigBCtv reduced the attachment of Leptospira by ~32%. The reduction of Leptospira attachment was also dose-dependent (Figs. 5B and C). We further elucidated the receptor role of Fn in the MDCK cells for its possible ligand, LigB on the surface of Leptospira, by RNA interference to decrease the endogeneous Fn expression in the MDCK cells. As shown in Fig. 6A, transfection of the cells with siRNA duplex specific for canine Fn resulted in a ~36% reduction of the Fn expression, relative to the control cells. The binding of LigBCen and LigBCtv to Fn siRNA-transfected MDCK cells was significantly reduced (Figs. 6B and C). These results suggest that Fn serves as a receptor for LigB that mediates Leptospira adhesion.

Fig. 5
The binding of LigBCen or LigBCtv to the MDCK cells reduced leptospiral adhesion (A) Binding of LigBCen or LigBCtv to the MDCK cells. Various concentrations (0, 2, 4, 6, or 8 nM) of GST-LigBCen, GST-LigBCtv or GST (negative control) was added to the MDCK cells (105). The binding of each of these proteins to the MDCK cells were measured by ELISA. (B) LigBCen or LigBCtv inhibits the binding of Leptopsira to MDCK cells. The MDCK cells were incubated with various concentrations (0, 2, 4, 6, or 8 nM) of GST-LigBCen, GST-LigBCtv or GST (negative control) prior to the addition of Leptopsira (107). The adhesion of Leptospira to the MDCK cells (105) was detected by ELISA. The reduced percentage of attachment was determined relative to the attachment of Leptopsira in the untreated MDCK cells. (C). LigBCen or LigBCtv inhibited the binding of Leptopsira to the MDCK cells. The MDCK cells (106) were pre-treated with 50nM of GST-LigBCen, GST-LigBCtv and GST (negative control) prior to the addition of the Leptopsira (108). The adhesion of Leptospira or the binding of these proteins to the MDCK cells were detected by CLSM. In (A) and (B), each value represents the mean± SEM of three trials in triplicate samples. Statistically significant values (p < 0.05) are indicted by *. In (C), the CLSM settings were identical for all the captured images. Images were processed using Adobe Photoshop CS2.

Fig. 6
The binding of LigBCen or LigBCtv to Fn siRNA transfected MDCK cells was reduced (A). Detection of the expression of Fn and actin in the MDCK cells 72 h after transfected by Fn or negative siRNA. Fn and α-actin were detected by immunoblotting probed by actin antibody or Fn antibody. (B) Binding of GST-LigBCen or (C) GST-LigBCtv was reduced by the siRNA transfected cells. (D) GST served as a negative control. Fifty nM of GST-LigBCen, GST-LigBCtv or GST was added to Fn or the negative siRNA transfected MDCK cells. Expression of Fn and the binding of these proteins to the MDCK cells were detected by CLSM. The CLSM settings were identical for all the captured images. Images were processed using Adobe Photoshop CS2.

Discussion

Adhesion to host cells is pivotal for many pathogenic bacteria including Leptospira spp. Since pathogenic Leptospira spp. can infect a variety of tissues including liver, kidney and lung, study of the host-pathogen interaction is extremely important for improved understanding of leptospirosis. Recently, the leptospiral genome has been sequenced and a number of tentative virulence factors have been proposed [3, 31, 42]. However, their exact roles in leptospiral pathogenesis remain to be established. To date, several leptospiral adhesion molecules have been identified. These include a 36 kDa Fn-binding protein [30], a 24 kDa laminin-binding protein [1] and LigA, LigB and LigC proteins [25, 33, 34]. These molecules may play an important role in the pathogenesis of leptospiral infection since they are able to bind to ECMs such as collagens I and IV, laminin and fibronectin [6, 24].

Pathogenic Leptospira spp. have been previously reported to adhere to extracellular matrices [15, 16] including Fn. Fns are dimers of two similar peptides linked at their C-termini by two disulfide bonds [8] and serve as receptors for several bacteria, including spirochetes [7, 11, 12, 19, 20, 23, 28, 32, 38, 40, 46, 50, 55]. Our results showed that Fn immobilized Leptospira. In addition, Fn was observed to block the attachment of Leptospira to MDCK cells if the Leptospira were pre-treated with Fn. These results support the recent report that Fn might be an important molecule involved in the pathogenic adherence of Leptospira spp. to host cells [6, 24].

We demonstrated the interaction between LigB and Fn. It was shown that the LigBCen and LigBCtv fragments were bound to Fn, by GST-pulldown assays, ELISA and ITC measurements. The low Kd values for LigBCen indicated that the LigB-Fn interaction was specific. This evidence strongly suggests that LigB is a Fn-binding protein. A study reported by Choy et al. [6] showed that LigB U1 and LigB U2 (LigBCen equivalent) could strongly bind to Fn, while the LigB CTD (LigBCtv equivalent) binds weakly to Fn. However, the Kd values of LigBCen and LigBCtv to Fn that we obtained were slightly different than those reported by Choy et al. [6]. The differences in the obtained Kd values could be explained by (i) the protein fragments evaluated in this study (LigBCen and LigBCtv) were not exactly the same length fragments (LigBU1, LigBU2 and LigBCTD) and (ii) the method we used (ITC) to measure the Kd differed from that of Choy et al. [6].

Since pathogenic Leptospira spp. adheres to renal tubular epithelial cells and induces a severe tubulointerstitial nephritis leading to renal failure [58], it is possible that LigB is responsible for the binding of Leptospira to the renal tubular epithelium. Our results indicated that LigB binds to the MDCK cells via the LigBCen or the LigBCtv fragments. However, the LigBCen was observed to bind to both the MDCK cells and Fn with a greater affinity than the LigBCtv. The microscopic images also showed that not all of the Fn was co-localized with the LigB. This result suggests that LigB might bind to two or more receptors. Our results elucidate the process of Leptospira attachment to the MDCK cells, as noted in a previous study [52], and demonstrated how Fn can block leptospiral attachment to the MDCK cells.

Our results clearly confirm that LigB is one of the microbial surface components that recognize adhesive matrix molecules (MSCRAMM) members that bind to the ECM including Fn. The transmembrane domain of LigB is predicted to reside within the conserved region, with only the variable region exposed on the surface [33, 34]. These results support our data that Fn-binding domains of LigB are localized in the variable regions. This is not surprising since similar findings have been reported for other MSCRAMMs [13, 37, 39]. In Borrelia, the binding motifs in the decorin-binding proteins, DbpA and B, are located in the central regions, which vary among the different Borrelia strains (B. burgdorferi, B. garnii, and B. afzeli) [37]. The Fn-binding domain of the Fn-binding protein, BBK32 is also variable among the different Borrelia strains [39]. The repetitive D1, D2 and D3 elements of Staphylococcus aureus Fn-binding protein, which bind the N-terminal 29 kDa of Fn, also vary [13].

Since both LigBCen and LigBCtv bind to Fn, but with different affinities, this suggests that there is more than one potential Fn-binding domain. In Mycobacterium avium, two Fn-binding domains are located on two non-contiguous segments of 24 amino acids in the Fn attachment protein-A [45]. The FnBPA of Staphylococcus aureus contains three repetitive elements, D1, D2 and D3 and each binds the N-terminal 29 kDa fragment of Fn [13]. Seven additional Fn-binding elements are located in the N-terminal of the D repeats [48]. In Streptococcus dysgalactiae, there are five Fn-binding segments within the C-terminus of the Fn binding protein F1/(FnBB) [47, 48]. Therefore, it is likely that several binding sites might be present in the LigB variable region. However, we were unable to identify a similar Fn-binding motif in the other known Fn-binding proteins.

In conclusion, we have shown that LigBCen and LigBCtv bind to Fn and have confirmed that LigB is a member of the MSCRAMMs. Since pathogenic Leptospira spp. initially attaches to mucosal epithelial cells prior to entry into the bloodstream and subsequent dissemination to multiple organs such as the kidney, liver and lung, Lig proteins may play a pivotal role in the pathogenesis of leptospirosis. Fn is one of the most important ECMs on epithelial cells and serves as a receptor for leptospiral adherence [6, 15, 24]. Thus, further studies into the interaction of Lig proteins and ECMs are warranted.

Acknowledgments

This work was supported in part by the Harry M. Zweig Memorial Fund for Equine Research, the New York State Science and Technology Foundation (Center for Advanced Technology) and the Biotechnology Research and Development Corporation. We would also like to thank Dr. Marci Scidmore for help with the epifluorescence microscope and confocal laser florescence microscope techniques and Dr. Bhargavi Jayaraman and Charlene Mottler for their help with the isothermal titration calorimetry techniques. We also thank our laboratory members, especially Drs. Syed Faisal and Tavan Janvilisri, for their suggestions during the course of this study and to Drs. Marci Scidmore, Linda Nicholson, and Sean McDonough for the critical reading of this manuscript.

References

    1. Barbosa AS, Abreu PA, Neves FO, Atzingen MV, Watanabe MM, Vieira ML, Morais ZM, Vasconcellos SA, Nascimento AL. A newly identified leptospiral adhesin mediates attachment to laminin. Infect Immun 2006;74:6356–6364.
    1. Barocchi MA, Ko AI, Reis MG, McDonald KL, Riley LW. Rapid translocation of polarized MDCK cell monolayers by Leptospira interrogans, an invasive but nonintracellular pathogen. Infect Immun 2002;70:6926–6932.
    1. Bulach DM, Zuerner RL, Wilson P, Seemann T, McGrath A, Cullen PA, Davis J, Johnson M, Kuczek E, Alt DP, Peterson-Burch B, Coppel RL, Rood JI, Davies JK, Adler B. Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. Proc Natl Acad Sci USA 2006;103:14560–14565.
    1. Cameron CE, Brouwer NL, Tisch LM, Kuroiwa JM. Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin. Infect Immun 2005;73:7485–7494.
    1. Cameron CE, Brown EL, Kuroiwa JM, Schnapp LM, Brouwer NL. Treponema pallidum fibronectin-binding proteins. J Bacteriol 2004;186:7019–7022.
    1. Choy HA, Kelley MM, Chen TL, Moller AK, Matsunaga J, Haake DA. Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect Immun 2007;75:2441–2450.
    1. Coburn J, Fischer JR, Leong JM. Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete. Mol Microbiol 2005;57:1182–1195.
    1. Darnell J, Lodish H, Baltimore D. In: Molecular Cell Biology. 2nd ed. New York: Scientific American Books; 1990. pp. 802-824.
    1. Edwards AM, Jenkinson HF, Woodward MJ, Dymock D. Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405. Infect Immun 2005;73:2891–2898.
    1. Faine SB, Adher B, Bolin C, Perolat P. In: Leptospira and Leptospirosis. 2nd ed. Medbourne: MedSci; 1999. pp. 67-71.
    1. Fischer JR, LeBlanc KT, Leong JM. Fibronectin binding protein BBK32 of the Lyme disease spirochete promotes bacterial attachment to glycosaminoglycans. Infect Immun 2006;74:435–441.
    1. Grab DJ, Givens C, Kennedy R. Fibronectin-binding activity in Borrelia burgdorferi. Biochim Biophys ACTA 1998;1407:135–145.
    1. Ingham KC, Brew S, Vaz D, Sauder DN, McGavin MJ. Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin: affinity, stoichiometry, and modular requirements. J Biol Chem 2004;279:42945–42953.
    1. Isberg RR, Voorhis DL, Falkow S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell 1987;50:769–778.
    1. Ito T, Yanagawa R. leptospiral attachment to extracellular matrix of mouse fibroblast (L929) cells. Vet Microbiol 1987;15:89–96.
    1. Ito T, Yanagawa R. Leptospiral attachment to four structural components of extracellular matrix. Nippon juigaku zasshi 1987;49:875–882.
    1. Jerse AE, Yu J, Tall BD, Kaper JB. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA 1990;87:7839–7843.
    1. Jiang ST, Chiang HC, Cheng MH, Yang TP, Chuang WJ, Tang MJ. Role of fibronectin deposition in cystogenesis of Madin-Darby canine kidney cells. Kidney Intl 1999;56:92–103.
    1. Kim JH, Singvall J, Schwarz-Linek U, Johnson BJ, Potts JR, Hook M. BBK32, a fibronectin binding MSCRAMM from Borrelia burgdorferi, contains a disordered region that undergoes a conformational change on ligand binding. J Biol Chem 2004;279:41706–41714.
    1. Konkel ME, Christensen JE, Keech AM, Monteville MR, Klena JD, Garvis SG. Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein. Mol Microbiol 2005;57:1022–1035.
    1. Kopp PA, Schmitt M, Wellensiek HJ, Blobel H. Isolation and characterization of fibronectin-binding sites of Borrelia garinii N34. Infect Immun 1995;63:3804–3808.
    1. Levett PN. Leptospirosis. Clin Microbiol Rev 2001;14:296–326.
    1. Li X, Liu X, Beck DS, Kantor FS, Fikrig E. Borrelia burgdorferi lacking BBK32, a fibronectin-binding protein, retains full pathogenicity. Infect Immun 2006;74:3305–3313.
    1. Lin YP, Chang YF. A domain of the Leptospira LigB contributes to high affinity binding of fibronectin. Biochem Biophys Res Commun 2007;362:443–448.
    1. Matsunaga J, Barocchi MA, Croda J, Young TA, Sanchez Y, Siqueira I, Bolin CA, Reis MG, Riley LW, Haake DA, Ko AI. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol Microbiol 2003;49:929–945.
    1. Matsunaga J, Lo M, Bulach DM, Zuerner RL, Adler B, Haake DA. Response of Leptospira interrogans to Physiologic Osmolarity: Relevance in Signaling the Environment-to-Host Transition. Infect Immun 2007;75:2864–2874.
    1. Matsunaga J, Sanchez Y, Xu X, Haake DA. Osmolarity, a key environmental signal controlling expression of leptospiral proteins LigA and LigB and the extracellular release of LigA. Infect Immun 2005;73:70–78.
    1. May M, Papazisi L, Gorton TS, Geary SJ. Identification of fibronectin-binding proteins in Mycoplasma gallisepticum strain R. Infect Immun 2006;74:1777–1785.
    1. Meites E, Jay MT, Deresinski S, Shieh WJ, Zaki SR, Tompkins L, Smith DS. Reemerging leptospirosis, California. Emerg Infect Dis 2004;10:406–412.
    1. Merien F, Truccolo J, Baranton G, Perolat P. Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae. FEMS Microbiol Lett 2000;185:17–22.
    1. Nascimento AL, Ko AI, Martins EA, Monteiro-Vitorello CB, Ho PL, Haake DA, Verjovski-Almeida S, Hartskeerl RA, Marques MV, Oliveira MC, Menck CF, Leite LC, Carrer H, Coutinho LL, Degrave WM, Dellagostin OA, El-Dorry H, Ferro ES, Ferro MI, Furlan LR, Gamberini M, Giglioti EA, Goes-Neto A, Goldman GH, Goldman MH, Harakava R, Jeronimo SM, Junqueira-de-Azevedo IL, Kimura ET, Kuramae EE, Lemos EG, Lemos MV, Marino CL, Nunes LR, de Oliveira RC, Pereira GG, Reis MS, Schriefer A, Siqueira WJ, Sommer P, Tsai SM, Simpson AJ, Ferro JA, Camargo LE, Kitajima JP, Setubal JC, Van Sluys MA. Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacteriol 2004;186:2164–2172.
    1. Nyberg P, Sakai T, Cho KH, Caparon MG, Fassler R, Bjorck L. Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes. EMBO J 2004;23:2166–2174.
    1. Palaniappan RU, Chang YF, Hassan F, McDonough SP, Pough M, Barr SC, Simpson KW, Mohammed HO, Shin S, McDonough P, Zuerner RL, Qu J, Roe B. Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J Med Microbiol 2004;53:975–984.
    1. Palaniappan RU, Chang YF, Jusuf SS, Artiushin S, Timoney JF, McDonough SP, Barr SC, Divers TJ, Simpson KW, McDonough PL, Mohammed HO. Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans. Infect Immun 2002;70:5924–5930.
    1. Palaniappan RU, McDonough SP, Divers TJ, Chen CS, Pan MJ, Matsumoto M, Chang YF. Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection. Infect Immun 2006;74:1745–1750.
    1. Parma AE, Cerone SI, Sansinanea SA. Biochemical analysis by SDS-PAGE and western blotting of the antigenic relationship between Leptospira and equine ocular tissues. Vet Immunol Immunopathol 1992;33:179–185.
    1. Pikas DS, Brown EL, Gurusiddappa S, Lee LY, Xu Y, Hook M. Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach. J Biol Chem 2003;278:30920–30926.
    1. Probert WS, Johnson BJ. Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31. Mol Microbiol 1998;30:1003–1015.
    1. Probert WS, Kim JH, Hook M, Johnson BJ. Mapping the ligand-binding region of Borrelia burgdorferi fibronectin-binding protein BBK32. Infect Immun 2001;69:4129–4133.
    1. Raibaud S, Schwarz-Linek U, Kim JH, Jenkins HT, Baines ER, Gurusiddappa S, Hook M, Potts JR. Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes. J Biol Chem 2005;280:18803–18809.
    1. Rathinam SR, Rathnam S, Selvaraj S, Dean D, Nozik RA, Namperumalsamy P. Uveitis associated with an epidemic outbreak of leptospirosis. Am J Ophthalmol 1997;124:71–79.
    1. Ren SX, Fu G, Jiang XG, Zeng R, Miao YG, Xu H, Zhang YX, Xiong H, Lu G, Lu LF, Jiang HQ, Jia J, Tu YF, Jiang JX, Gu WY, Zhang YQ, Cai Z, Sheng HH, Yin HF, Zhang Y, Zhu GF, Wan M, Huang HL, Qian Z, Wang SY, Ma W, Yao ZJ, Shen Y, Qiang BQ, Xia QC, Guo XK, Danchin A, Saint Girons I, Somerville RL, Wen YM, Shi MH, Chen Z, Xu JG, Zhao GP. Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 2003;422:888–893.
    1. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nature biotechnol 2004;22:326–330.
    1. Rzomp KA, Scholtes LD, Briggs BJ, Whittaker GR, Scidmore MA. Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner. Infect Iimmun 2003;71:5855–5870.
    1. Schorey JS, Holsti MA, Ratliff TL, Allen PM, Brown EJ. Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria. Mol Microbiol 1996;21:321–329.
    1. Schroder A, Schroder B, Roppenser B, Linder S, Sinha B, Fassler R, Aepfelbacher M. Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells. Mol Biol Cell 2006;17:5198–5210.
    1. Schwarz-Linek U, Pilka ES, Pickford AR, Kim JH, Hook M, Campbell ID, Potts JR. High affinity streptococcal binding to human fibronectin requires specific recognition of sequential F1 modules. J Biol Chem 2004;279:39017–39025.
    1. Schwarz-Linek U, Werner JM, Pickford AR, Gurusiddappa S, Kim JH, Pilka ES, Briggs JA, Gough TS, Hook M, Campbell ID, Potts JR. Pathogenic bacteria attach to human fibronectin through a tandem beta-zipper. Nature 2003;423:177–181.
    1. Segura ER, Ganoza CA, Campos K, Ricaldi JN, Torres S, Silva H, Cespedes MJ, Matthias MA, Swancutt MA, Lopez Linan R, Gotuzzo E, Guerra H, Gilman RH, Vinetz JM. Clinical spectrum of pulmonary involvement in leptospirosis in a region of endemicity, with quantification of leptospiral burden. Clin Infect Dis 2005;40:343–351.
    1. Seshu J, Esteve-Gassent MD, Labandeira-Rey M, Kim JH, Trzeciakowski JP, Hook M, Skare JT. Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi. Mol Microbiol 2006;59:1591–1601.
    1. Shi J, Scita G, Casanova JE. WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium. J Biol Chem 2005;280:29849–29855.
    1. Thomas DD, Higbie LM. In vitro association of leptospires with host cells. Infect Immun 1990;58:581–585.
    1. Vendrame F, Segni M, Grassetti D, Tellone V, Augello G, Trischitta V, Torlontano M, Dotta F. Impaired caspase-3 expression by peripheral T cells in chronic autoimmune thyroiditis and in autoimmune polyendocrine syndrome-2. J Clin Endocrinol Metab 2006;91:5064–5068.
    1. Verma A, Hellwage J, Artiushin S, Zipfel PF, Kraiczy P, Timoney JF, Stevenson B. LfhA, a novel factor H-binding protein of Leptospira interrogans. Infect Immun 2006;74:2659–2666.
    1. Wann ER, Gurusiddappa S, Hook M. The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen. J Biol Chem 2000;275:13863–13871.
    1. Werts C, Tapping RI, Mathison JC, Chuang TH, Kravchenko V, Saint Girons I, Haake DA, Godowski PJ, Hayashi F, Ozinsky A, Underhill DM, Kirschning CJ, Wagner H, Aderem A, Tobias PS, Ulevitch RJ. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nature immunol 2001;2:346–352.
    1. Xu Q, Yan B, Li S, Duan C. Fibronectin binds insulin-like growth factor-binding protein 5 and abolishes Its ligand-dependent action on cell migration. J Biol Chem 2004;279:4269–4277.
    1. Yang CW, Wu MS, Pan MJ. Leptospirosis renal disease. Nephrol Dialysis Transplant 2001;16 Suppl 5:73–77.
    1. Yang CW, Wu MS, Pan MJ, Hsieh WJ, Vandewalle A, Huang CC. The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells. J Am Soc Nephrol 2002;13:2037–2045.

Metrics
Share
Figures

1 / 6

Tables

1 / 1

PERMALINK