Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Inhibition of initial adhesion of oral bacteria through a lectin from Bauhinia variegata L. var. variegata expressed in Escherichia coli G.B. Klafke1,2, S. Borsuk1, R.A. Goncßales1, F.V.S. Arruda3, V.A. Carneiro3, E.H. Teixeira3, A.L. Coelho da Silva4, B.S. Cavada5, O.A. Dellagostin1 and L.S. Pinto1 1 2 3 4 5


gico, Universidade Federal de Pelotas, Pelotas, Brazil
cleo Biotecnologia, Centro de Desenvolvimento Tecnolo Nu
rio de Micologia, Universidade Federal de Rio Grande, Rio Grande, Brazil Faculdade de Medicina, Laborato Faculdade de Medicina de Sobral, Universidade Federal do Cear
a, Sobral, Brazil
rio de Biotecnologia Molecular (LabBMol), Universidade Federal do Cear
Laborato a, Fortaleza, Brazil
rio de Mol
eculas Biologicamente Ativas, Departamento de Bioqu
ımica e Biologia Molecular, Universidade Federal do Cear
Laborato a, Fortaleza, Brazil

Keywords acquired pellicle, rBVL-I, Streptococcus mutans, Streptococcus sanguis, Streptococcus sobrinus. Correspondence
cleo Biotecnologia, Luciano S. Pinto, Nu
gico, Centro de Desenvolvimento Tecnolo Universidade Federal de Pelotas, Campus Universit
ario, s/nº 96010-900. Pelotas, RS, Brazil. E-mail: [email protected] 2013/0799: received 24 April 2013, revised 24 June 2013 and accepted 27 July 2013 doi:10.1111/jam.12318

Abstract Aims: The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. Methods and Results: Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a salivacoated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. Conclusion: We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. Significance and Impact of the Study: Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation.

Introduction Lectins are carbohydrate-binding proteins that specifically and reversibly interact with different types of carbohydrates and glycoproteins (Sharon and Lis 2002). The interaction between lectins and sugars plays an important role in several biological processes and mediates a broad range of biological activities, including inhibition of bacterial and fungal growth (Ngai and Ng 2007; Sattayasai et al. 2009; Alizadeh et al. 2011; Nunes et al. 2011). Plant lectins isolated from the seeds of Araucaria angustifolia (Santi-Gadelha et al. 2006), Eugenia uniflora (Oliveira 1222

et al. 2008) and Myracrodruon urundeuva (Sa et al. 2009), as well as from the leaves of Phthirusa pyrifolia (Costa et al. 2010) show remarkable antibacterial activity. In recent years, many studies have focused on strategies to control oral biofilm formation using lectins (Oliveira et al. 2007; Teixeira et al. 2007; Islam et al. 2009; Cavalcante et al. 2011). Long-term survival of oral cavity bacteria requires surface tissue adherence and colonization of a suitable niche in the multi-species, complex biofilm that exists in the human oral environment (Oliveira et al. 2007). Lectins can act as a tool to block microbial adhesion through the recognition of bacterial

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surface glycoconjugates and adsorbed biofilm components. This strategy might be successful in halting the development of caries at early stages (Teixeira et al. 2007). Plant lectins have been used for other biotechnological purposes (da Silva et al. 2005; Gemeiner et al. 2009), and the use of lectins in anti-adhesion therapy for oral disease has been proposed (Oliveira et al. 2007; Teixeira et al. 2007; Cavalcante et al. 2011). However, many problems, including limitations in the availability of plant resources, and a complex purification process complicate the use of native lectins. Lectin production using recombinant DNA techniques is, consequently, of great interest. The aim of the present study was to study the in vitro effects of native and recombinant forms of the B. variegata lectin (BVL) in inhibiting early adhesion of Streptococcus mutans, Strep. sanguis and Step. sobrinus to an experimentally acquired pellicle. Materials and methods Native lectin Bauhinia variegata lectin (BVL) was purified by affinity chromatography as described previously (Pinto et al. 2008). Briefly, the seeds of B. variegata were ground and extracted with Tris–HCl 0
05 mol l 1, pH 7
6 buffer, containing 0
15 mol l 1 NaCl. Each homogenate was centrifuged (10 000 g, 30 min, 4°C) and filtered. The resultant supernatants were used in haemagglutination assays and protein quantification. Construction of the expression vector The bvlI coding region was PCR-amplified from a previously constructed pCR 2
1–bvlI vector (Pinto et al. 2008). The primers used were F5′CGG GAT CCA CAA GCT CAA CCT TAA C3′ and R5′CGG GGT ACC TTA CAT ACTG GAA TAA GAG3′ (Eurofins MWG Operon, Huntsville, AL, USA). The PCR product was cleaved with BamHI and KpnI, and the resultant fragment was ligated into the BamHI- and KpnI-digested pAE vector (Ramos et al. 2004). The products of the ligation reaction were transformed into Escherichia coli TOP10 competent cells. The recombinant clone, named pAE-bvlI, was used for expression studies. Escherichia coli TOP10 cells grown on 100 lg ml 1 ampicillin-containing Luria–Bertani agar were used for plasmid maintenance. DNA sequence analysis Plasmid DNA was prepared using a GFX microplasmid purification kit (GE Healthcare, Piscataway, NJ, USA). The

Inhibition of adhesion of oral bacteria by lectins

DNA sequence was determined using the DYEnamic ET terminators sequencing kit and a MegaBACE 500 automatic DNA sequencer (GE Healthcare). The quality of the DNA sequence was verified, and the overlapping fragments were assembled with high quality and were aligned using ClustalX (Thompson et al. 1997) with default gap penalties. Homology analyses were performed using the GenBank database and BLAST (Altschul et al. 1997). Culture growth conditions and purification Escherichia coli strain BL21 (DE3) cells (EMD Millipore, Billerica, MA, USA) were transformed with pAE-bvlI and grown in LB medium containing ampicillin (100 lg ml 1) at 37°C for 16 h. A 2-ml aliquot of this enriched medium was used to inoculate 500 ml of the same medium. The resultant culture was maintained at the same culture conditions until the optical density (OD) at (550 nm) reached 0
6–0
7. Expression of the recombinant BVL-I protein was induced by the addition of IPTG at a final concentration of 0
3 mol l 1. The culture was then incubated for 16 h at 20, 30 or 37°C. Cell aliquots were collected by centrifugation and analysed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The pellet was washed with phosphate-buffered saline (PBS) and suspended in 30 ml of buffer I (0
2 mol l 1 NaH2PO4, 0
5 mol l 1 NaCl and 0
005 mol l 1 imidazole, pH 8
0). The suspension was sonicated 3 times (10 s each) at 4°C. After centrifugation, the pellet was washed with buffer I. The inclusion bodies were dissolved in 30 ml of buffer II (8 mol l 1 urea, 0
2 mol l 1 NaH2PO4, 0
006 mol l 1 NaCl and 0
005 mol l 1 imidazole, pH 8
0), and rBVL-I isolation was performed by affinity chromatography using a HiTrap chelating column (GE Healthcare) charged with Ni2+ ions. After washing with buffer II, rBVL-I was eluted with buffer III (8 mol l 1 urea, 20 mmol l 1 of NaH2PO4, 0
006 mol l 1 NaCl, 0
5 mol l 1 imidazole– HCl, pH 8
0). The purified protein was dialysed against a buffer containing 0
05 mol l 1 Tris–HCl, 0
05 mol l 1 CaCl2 and 0
05 mol l 1 MnCl2, pH 8
0. The concentration and purity of rBVL-I were determined using a BCA Protein Assay Reagent (Thermo Scientific, Rockford, IL, USA) and 12% SDS-PAGE, respectively. Antisera production and western blotting To raise BVL-specific antiserum, rabbits were immunized with purified native BVL, according to the protocol described by Gerlach and coworkers (Gerlach et al. 2002). For Western blotting, samples were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. The membrane was blocked with

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5% (w/v) nonfat dry milk in 1 9 PBS-T buffer (PBS, 0
05% Tween 20) and incubated with the rabbit-raised antiserum (1 : 200, diluted in PBS-T) and developed with peroxidase-conjugated anti-rabbit IgG (1 : 1000) or an alkaline phosphatase-conjugated anti-6 9 His antibody (1 : 2000) (Sigma-Aldrich, St Louis, MO, USA). Antibody-reactive protein bands were revealed following development with 3,3′–diaminobenzidine tetrahydrochloride (DAB) and H2O2. Haemagglutination assay To confirm lectin activity, haemagglutination assays were performed as described by Pinto and coworkers (Pinto et al. 2008). The lowest concentration required to completely agglutinate the red blood cells was determined visually. Circular dichroic (CD) measurements and secondary structure estimation Circular dichroic spectra were recorded on a JASCO J-815 spectropolarimeter (JASCO Corporation, Tokyo, Japan) equipped with a Peltier temperature control unit. The entire instrument, including the sample chamber, was constantly flushed with N2 gas during the operation. Measurements were performed using a rectangular Spectrosil (path length, 1 mm) quartz cuvette. Buffer (blank) spectra were recorded under the same conditions and subtracted from the protein spectra before further analysis. CD spectra were the average of 16 measurements at 25°C, using a scanning speed of 50 nm min 1. To obtain BVL and rBVL-I spectra, protein concentrations were adjusted to 0
26 mg ml 1 in 20 mmol l 1 Tris–HCl buffer, pH 7
6, containing 10 mmol l 1 NaCl. CD spectra, obtained in millidegrees, were converted to molar ellipticity. The secondary structure analysis was performed by CD spectrum deconvolution using the SELCON 2 program. For temperature stability studies, a protein sample was heated gradually in 2°C increments, from 25 to 80°C. At each temperature, the protein was incubated for 1 min, and the spectrum was recorded over a wavelength range of 200–250 nm. Fluorescence emission spectroscopy Fluorescence measurements were performed on an ISS K2 (ISS, Champaing, IL, USA) spectrofluorometer in the steady state mode, using quartz cuvettes with a path length of 1 cm and volume of 1
0 ml. Samples were excited at 280 nm, with the emission being monitored in the range of 395–450 nm.

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Inhibition of oral bacteria Culture conditions Streptococcus mutans UA159 American Type Culture Collection (ATCC) 25175, Strep. sanguis ATCC10556 and Step. sobrinus ATCC6715 were cultured in Todd–Hewitt broth (THB) for 24 h at 37°C and 10% CO2. One isolated colony was then picked and inoculated into brain– heart infusion broth (BHI) and cultured under the same conditions described above. The bacterial concentration, extrapolated from the bacterial growth curve, was adjusted to 1 9 108 cells ml 1. Effect of lectins on bacterial growth The antimicrobial activity of the lectins was assessed using the method described by Franzblau and coworkers (Franzblau et al. 1998). The method is based on microdilution testing in 96-well polystyrene plates (Polysorp Nunc, Roskilde, DK, USA) according to the National Committee for Clinical Laboratorial Standards (NCCLS) guidelines. The bacterial growth was spectrophotometrically monitored at 620 nm (Biotrak II Plate Reader – GE Healthcare). The lectins were tested at a concentration range of 500– 7
8 lg ml 1. The minimal inhibitory concentration (MIC) was defined as the lowest concentration that decreased bacterial growth to an OD below 0
05, at 620 nm (Islam et al. 2009). Effect of lectins on early adhesion in saliva-coated surface Collection of saliva. Aliquots of saliva from three healthy individuals were collected under masticatory stimulation, at least 2 h after eating, drinking or teethbrushing. To ensure a more uniform formation and to increase the number of receptors on the film, the aliquots were cooled in ice immediately after harvesting and mixed to generate a ‘pool’ of saliva (Gibbons et al. 1986), which was clarified by centrifugation at 10 000 g for 20 min at 4°C and sterilized by filtration using a 0
22-lm filter. A consent form was obtained from all individuals who donated saliva.

Early adhesion on saliva-coated wells. The assay of biofilm formation in microtitre plates was performed following the method described by O’Toole and Kolter (O’Toole and Kolter 1998), with some modifications. The lectins were tested at concentrations of 10, 100 and 200 lg ml 1. First, 100 ll of clarified saliva was distributed in triplicate wells and incubated at 37°C for 2 h. The excess was then removed, and the plate was washed twice with PBS, pH 7
2. Lectins (100 ll) were then added, and the samples

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were incubated for 2 h at 37°C. After repeating the washing procedure, 100 ll of the different bacterial cultures (1 9 108 cells ml 1) or PBS (negative control) was added to evaluate the early adhesion achieved during 2 h of incubation at 37°C. Finally, crystal violet dye was added to the wells, and adherence was quantified at 575 nm (Peeters et al. 2008).

(data not shown). The 6 9 His-tagged rBVL-I protein was purified by Ni-NTA affinity chromatography under denaturing conditions. The recombinant protein was dialysed to remove the denaturing agent and then concentrated by lyophilization, yielding about 40 mg l 1 of E. coli culture suspension. Biochemical characteristics of rBVL-I

Statistical analyses The data are presented as mean  SEM and were analysed using ANOVA followed by the Tukey’s post hoc test, with significance being denoted by a P-value of ≤0
05. Results Expression of recombinant BVL-I in E. coli The expression of rBVL-I was induced with IPTG for 16 h. The recombinant protein, having a molecular weight of c. 28 kDa, was detected by Western blot (Fig. 1). To induce the expression of a soluble form of the protein, we studied the effects of changing the temperature during induction. rBVL-I induced at 20°C was partially soluble; the recombinant protein was found in both pellet and supernatant. However, rBVL-I produced at 30 or 37°C was insoluble and remained in the pellet

(a) 181·3

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5

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119·5 82·2

48·8 37·1

25·9 19·4 14·8

Figure 1 (a) Expression of recombinant BVL-I in Escherichia coli BL21 (DE3). M, molecular mass markers; lane 1, E. coli BL21(DE3)/pAE-bvlI not induced by IPTG; lane 2, E. coli BL21(DE3)/pAE-bvlI induced by IPTG; lane 3, E. coli BL21(DE3)/pAE not induced by IPTG; lane 4, E. coli BL21(DE3)/pAE induced by IPTG. (b) Western blot of rBVL-I purified by one-step affinity chromatography. Lane 5, purified rBVL-I not reduced; lane 6, purified rBVL-I reduced.

The mass value of rBVL-1 determined by SDS-PAGE correlated with the predicted molecular mass of 28 kDa (Fig. 1a). When assessed under nonreducing conditions by SDS-PAGE and Western blotting (Fig. 1b), the molecular mass of rBVL-I correlated with the predicted value of the dimer form, indicating that rBVL-I forms a homodimer in solution. Western blotting showed that the recombinant protein was recognized by BVL-specific antisera in both reducing and nonreducing conditions (Fig. 1b). Haemagglutination activity Lectin activity was analysed by rabbit erythrocyte haemagglutination. No differences were observed between the recombinant and native forms. The minimal concentration required to cause haemagglutination was 0
096 lg ml 1 for rBVL-I and 0
052 lg ml 1 for BVL. The agglutination of the rabbit erythrocytes occurred immediately upon addition of the cells to the lectin-containing medium. Estimation of secondary structure Figure 2 displays the CD spectra of BVL and rBVL-I in the native state. The far UV CD analysis of the lectins shows a large negative peak at 221 nm, characteristic of proteins with a predominance of b-sheets in the secondary structure. The secondary structure was studied using the SELCON 2 program, and the results indicate that the b-sheet secondary structures are predominant on both lectins, being c. 57%. The predicted secondary structure for BVL includes a-helices (6%), turns (28%) and other contributions (9%). rBVL-I contained a-helices (7%), turns (28%) and other contributions (8%). The thermal denaturation of the lectins was characterized by measuring the ellipticity changes at 221 nm induced by a temperature increase from 25 to 80°C and exhibited a cooperative sigmoidal behaviour (Fig. 3). Both BVL and rBVL-I were shown to be thermostable up to 40°C. The thermal unfolding showed a transition midpoint at 55°C and followed the observation of two-state mechanism (native and denatured). BVL and rBVL-I displayed an intrinsic fluorescence typical of folded globular proteins

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2

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[θ](103.cm2.deg.dmol–1)

[θ](103.cm2.deg.dmol–1)

0 –2 –4 –6 –8 –10

–4 –6 –8 –10 –12

–12 –14 200

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Figure 2 Circular dichroic (CD) spectra of native BVL and rBVL-I (0
26 mg ml 1) in 20 mmol l 1 Tris–HCl, pH 7
6, containing 10 mmol l 1 NaCl, at 25°C. ( ) Native BVL; ( ) rBVL.

with an kcm of c. 336 nm, which is characteristic of tryptophan side chains in a nonpolar environment (Fig. 4).

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70

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Figure 3 Changes in ellipticity of BVL and rBVL-I as a function of temperature. Insets give temperature dependence of the circular dichroic (CD) signal at 221 nm. Lectin sample (0
26 mg ml 1 in 20 mmol l 1 Tris–HCl, pH 7
6 containing 10 mmol l 1 NaCl) was heated gradually in 5°C steps, from 25 to 80°C. At each temperature, the protein was incubated for 1 min and the spectrum was recorded over a wavelength range of 200–250 nm. Measurements were performed using a rectangular 1 mm path quartz cuvette. (■) Native BVL; (●) rBVL.

Inhibition of early adhesion 18 16 Fluorescence, arbitary units

Assays were performed to test the potential of both the native and recombinant forms of the lectin in inhibiting adhesion and growth of the oral micro-organisms. The antibacterial activity was determined by the MIC assay, using 108 cells ml 1 of Strep. sanguis, Step. mutans and Step. sobrinus. Both native (BVL) and recombinant (rBVL-I) forms were found unable to reduce growth of these bacterial strains (data not shown). In contrast, at all concentrations tested, rBVL-I and BVL significantly reduced early adhesion of Step. mutans and Strep. sanguis to a saliva-coated surface (Fig. 5). However, when used at 200 lg ml 1, BVL was more effective than rBVL-I in inhibiting adhesion of Step. mutans, probably because it has more protein dimer form.

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Discussion In this study, we constructed a recombinant pAE-bvlI expression plasmid to produce the active lectin from B. variegata in E. coli. Cells grown at 37°C expressed a large amount of recombinant protein. Bauhinia lectins have been extensively purified and characterized (Young et al. 1985; Coelho and Silva 2000; Silva et al. 2001, 2007, 2011; Farias et al. 2004; Lin and Ng 2008; Pinto et al. 2008). Bauhinia variegata lectin shares characteristics similar to those from the Caesalpiniaceae family. However, there are many problems inherent to the use of native lectins, 1226

Figure 4 Fluorescence emission spectra of BVL and rBVL-I. For fluorescence measurements, the protein concentration was 0
18 mg ml 1 in 20 mmol l 1 Tris–HCl, pH 7
6 containing 10 mmol l 1 NaCl. The sam) Native BVL; ( ) rBVL. ples were excited at 280 nm, at 25°C. (

such as the limitations in the availability of plant resources and complex purification procedures. For example, Kusui et al. (1991) constructed a cDNA library for the BPA lectin, using RNA isolated from germinated Bauhinia purpurea seeds. A number of isoforms of this lectin were identified using genetic cloning and bioinformatic techniques.

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Initial adherence inhibition test for S. mutans

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Figure 5 Initial adherence inhibition testing for oral bacteria. The lectins were resuspended in PBS. The different letters on top of the bars indicate statistically significant differences (P ≤ 0
05). Error bars equal standard error of the mean (SEM). ( ) Negative control; ( ) rBVL; ( ) rBVL.

Purifying each isoform using conventional techniques allows definition of the functional properties and biological activities of the components of protein mixtures.

However, different isoforms can have diverse activities (Eck et al. 1999; Raemaekers et al. 1999). Moreover, difficulties in purifying the components of these heterogeneous mixtures preclude detailed study of their multiple functions and properties. As an alternative, the expression of plants’ lectins in bacteria has been demonstrated to be an efficient method for the study of each isoform (Raemaekers et al. 1999). Despite the success in the expression of lectin rBVL-I, most of the protein was deposited in insoluble inclusion bodies. A similar result has also been observed following the recombinant expression of the lectins of Abrus pulchellus (Goto et al. 2003), Erythrina cristagalli (Stancombe et al. 2003), Phlebodium aureum (Tateno et al. 2003), Oryza sativa (Kong et al. 2004), Zingiber officinale (Chen et al. 2005) and B. purpurea (Kusui et al. 1991). The recombinant protein derived from B. purpurea was recovered from inclusion bodies, and no haemagglutinating activity was observed. The haemagglutinating activity of purified rBVL-I demonstrated that the denaturing conditions used during protein solubilization did not affect this activity. As glycosylation cannot efficiently occur in E. coli, this posttranslational modification may not be required for this property. In addition, CD (Fig. 2) and fluorescence (Fig. 4) measurements confirmed that the recombinant form of this lectin is structurally and functionally similar to the native form. These results corroborate with those obtained from CD studies of B. variegata var. Candida seeds (Silva et al. 2007). Protein structural analysis indicates that the b-sheet is a predominant feature (c. 57%) of the secondary structure of both BVL and rBVL-I. Comparatively, the CD spectra analysis of B. variegata var. Candida seeds indicates that 65% of the secondary structure consists of b-sheets. These results suggest that BVL, rBVL-I and B. variegata var. Candida lectins are probably structurally similar. Using thermal denaturation studies of BVL and rBVL-I, the lectin was shown to be thermostable up to 40°C (Fig. 3). The thermal unfolding showed a transition midpoint at 55°C and followed the observation of two-state mechanism (native and denatured). Earlier studies indicate that the lectins from Bauhinia pentandra and Bauhinia bauhinioides seeds are more thermostable and retain their haemagglutinating activity after incubation at 70 and 60°C, respectively (Silva et al. 2001, 2011). These results suggest that care should be taken during storage to avoid thermal denaturation of BVL. The haemagglutinating activity of rBVL-I was maintained during production. The lowest protein concentration required for rabbit erythrocyte haemagglutination (0
096 lg mL 1) was lower than that observed for several other plant lectins, including the recombinant rice (Oryza

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sativa L.)-derived SALT lectin (Branco et al. 2004). Additionally, the divalent cations, Ca2+ and Mn2+, are required for the sugar-binding activity of several lectins, but the incorporation of these into the buffer during rBVL-I dialysis did not increase the haemagglutinating activity. These results were corroborated by experiments with native lectins from B. variegata var. Candida seeds (Silva et al. 2007) and with those from B. bauhinioides seeds (Silva et al. 2011), wherein lectin activity was unchanged following sequential dialysis against EDTAand NaCl-containing buffers. To study the biological activity of rBVL-I, we compared early adhesion inhibition of oral bacteria by the recombinant and native forms of the lectin. Many lectins have antibacterial activity, and this effect (on both grampositive and gram-negative bacteria) occurs through interactions with components of the bacterial cell wall (Paiva et al. 2010). These components include teichoic and teichuronic acid, peptidoglycans and lipopolysaccharides. A study revealed that isolectin I (from Lathyrus ochrus seeds) binds to muramic acid and muramyl dipeptides through hydrogen bonds. These bonds occur between ring hydroxyl oxygen atoms of sugars and the lectin carbohydrate-recognition domains. Hydrophobic interactions between the Tyr100 and Trp128 lectin residue side chains (Ayouba et al. 1994; Bourne et al. 1994) also play a role. Besides being able to recognize certain bacterial cell wall components, lectins may also inhibit bacterial biofilm formation in the oral cavity (Black et al. 2004). The cariogenic potential of Streptococcus spp. is well known; Step. mutans is strongly implicated in the development of caries (Holt et al. 2000). Our results indicate a relevant rBVL-I-induced inhibition of early adhesion of Strep. sanguis, Step. mutans and Step. sobrinus. Although the involvement of a galactose-specific lectin has been described earlier in the accession process for Step. mutans (Gibbons and Qureshi 1979), another galactose-specific lectin, VML, inhibited Strep. sanguis adhesion alone (Teixeira et al. 2007). The presence of GalNAc-binding adhesins on surface of Strep. sanguis (Ruhl et al. 2004) may provide an explanation for this specific inhibitory activity. In a previous study, Lee and coworkers (Lee et al. 1998) examined interactions between lectins of various specificities and several species of Streptococcus spp. and observed that different lectins interacted with only specific species, corroborating the results of this study. Significant bacterial biofilm growth inhibition by plant lectins was reported by Islam and colleagues (Islam et al. 2009). In addition, our group recently demonstrated inhibitory effects of seed lectins from plants of the Diocleinae subtribe, in Step. mutans, and S. oralis biofilm formation (Cavalcante et al. 2011). However, no studies have explored the use of recombinant lectins or have 1228

compared the properties of recombinant and native forms. In summary, we report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. This study establishes a successful protocol to express and purify the functional recombinant BVL-I protein, in quantities large enough for the analysis of its biological activities, which include erythrocyte haemagglutination and inhibition of early adhesion by oral bacteria. The lectin may be a promising anti-adhesive agent, but further investigation is necessary to determine whether it might be effective in preventing or reducing biofilm formation in the oral cavity. Also, the present study was conducted using single-species cultures. Further studies using multispecies cultures will further elucidate effectiveness of BVL as antibiofilm agents on saliva-coated hydroxyapatite. Acknowledgements Financial support was provided by CAPES Foundation (Grant 19120/2009-3 and 02841/09-6) and by Fundacß~ao de Amparo a Pesquisa do Rio Grande do Sul (FAPERGS, Grant 11/1842-5). Gabriel Baracy Klafke received a scholarship from CAPES Foundation. Conflict of interest No conflict of interest declared. References Alizadeh, H., Leung, D.W. and Cole, A.L. (2011) Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata. Phytochemistry 72, 94–99. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402. Ayouba, A., Causse, H., Van Damme, E.J.M., Peumans, W.J., Bourne, Y., Cambillau, C. and Roug
e, P. (1994) Interactions of plant lectins with the components of the bacterial cell wall peptidoglycan. Biochem Syst Ecol 22, 153–159. Black, C., Allan, I., Ford, S.K., Wilson, M. and McNab, R. (2004) Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis. Arch Oral Biol 49, 295–304. Bourne, Y., Ayouba, A., Rouge, P. and Cambillau, C. (1994) Interaction of a legume lectin with two components of the bacterial cell wall. A crystallographic study. J Biol Chem 269, 9429–9435.

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Journal of Applied Microbiology 115, 1222--1230 © 2013 The Society for Applied Microbiology