L. casei Capable of Binding to Fibronectin 563

Lactobacillus casei Acquires the Binding Activityto Fibronectin by the Expression of the FibronectinBinding Domain of Streptococcus pyogeneson the Cell Surface

*For correspondence. Email akira-kushiro@yakult.co.jp;Tel. +81-42-577-8962 ext. 2403; Fax. +81-42-577-3020.

J. Mol. Microbiol. Biotechnol. (2001) 3(4): 563-571.

© 2001 Horizon Scientific Press

JMMB Research Article

Akira Kushiro*, Takuya Takahashi, Takashi Asahara,Hirokazu Tsuji, Koji Nomoto, and Masami Morotomi

Yakult Central Institute for Microbiological Research,1796 Yaho, Kunitachi, Tokyo 186-8650, Japan

Abstract

Fibronectin binding domain was expressed on the cellsurface of Lactobacillus casei strain Shirota whichhardly adheres to fibronectin. DNA for the fibronectinbinding domain of the sfbI gene, which encodes afibronectin binding protein of Streptococcus pyogenesATCC 21059, was amplified with polymerase chainreaction, cloned into a surface display vector pSAK332,and introduced into L. casei. The fibronectin bindingdomain was expressed as a fusion protein consistingof staphylokinase of Staphylococcus aureus and theanchor sequence of cell wall-associated 763 proteinaseof Lactococcus lactis NCDO 763. The fibronectinbinding ability of the resulting L. casei was confirmedwith Western blot analysis, immunoelectronmicroscopic analysis, and adherence to fibroblastcells. These results indicate that L. casei has acquireda new phenotype to bind fibronectin upon theexpression of the fibronectin binding domain on thecell surface. This L. casei also shows binding affinityto fibrinogen, indicating that fibronectin bindingdomain is involved in the binding to fibrinogen as well.

Introduction

Lactobacilli have been used for fermentation of dairyproducts. They have recently drawn much attention asprobiotic bacteria for their beneficial effects on humanhealth (Havenaar and Huis in’t Veld, 1992) and also beenrecognized as GRAS (generally recognized as safe)(Salminen et al., 1998). It has been reported thatLactobacillus casei strain Shirota has antitumor activity(Matsuzaki et al., 1985) and protective effect againstbacterial infections (Nomoto et al., 1985) in preclinicalstudies. The administration of heat-killed L. casei strainShirota to the model mouse of superficial bladder cancershowed high antitumor effect (T. Takahashi, unpublished).

Morales et al. (1976) first reported the use ofintravesical Mycobacterium bovis bacillus Calmette-Guérin(BCG) in the treatment of superficial bladder cancer, and

prospective randomized trials have shown intravesical BCGto be a new treatment of superficial bladder cancer (Lammet al., 1991). However, instillation of live organisms of BCGis occasionally associated with serious systemic sideeffects such as fever, serum-like syndromes,granulomatous toxicity, sepsis and even death (Lamm etal., 1992). Ratliff et al. (1988) have demonstrated thatantitumor effects of BCG are due to activation of immuneresponses which are mediated by attachment of BCG tothe bladder wall through fibronectin. L. casei strain Shirota,in contrast, exhibits no detectable binding to fibronectin.We could, therefore, expect higher antitumor activity of L.casei strain Shirota which is genetically engineered toacquire binding capacity to fibronectin.

Fibronectin binding proteins have been wellcharacterized and several genes were cloned andsequenced for Staphylococcus aureus (Signäs et al., 1989,Jönsson et al., 1991), Streptococcus dysgalactiae(Lindgren et al., 1993), and Streptococcus pyogenes (Selaet al., 1993, Talay et al., 1994, Kreikemeyer et al., 1995).In the case of S. pyogenes, a gene coding for the fibronectinbinding protein was designated as sfbI (Talay et al., 1992),sfbII (Kreikemeyer et al., 1995) or prtF (Hanski andCaparon, 1992). The amino acid sequences of SfbI andPrtF are 70% homologous. Fibronectin binding proteins ofstaphylococci and streptococci have a common feature thatthe repetitive sequences, which are abundant in acidicamino acid residues, play a major role for fibronectinbinding.

We show here that fibronectin binding domain includingfour repeats of a unit of 37 amino acid residues is expressedon the cell surface of L. casei strain Shirota, and that theresulting L. casei acquires a new phenotype for binding toboth fibronectin and fibrinogen. The increased adhesionof the recombinant L. casei to fibroblast cells is alsodiscussed.

Results

Construction of the Plasmid pSAKSfb14A new surface display vector called pSAK332 (Figure 1)has a coding sequence for the preform (a signal sequenceplus a mature form consisting of 163 amino acid residues)of staphylokinase protein (SAK) of S. aureus (Sako andTsuchida, 1983) which is juxtaposed to the cell wall anchorsequence of the C-terminal 332 amino acid residues ofcell wall-associated 763 proteinase of Lactococcus lactisNCDO 763 (Kiwaki et al., 1989). The anchor sequencecontains sorting signals such as an LPXTG motif,hydrophobic domain, and a positively charged tail, whichare commonly observed in gram-positive bacterial cell wall-

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564 Kushiro et al.

associated proteins (Schneewind et al., 1992). L. caseiharboring pSAK332 successfully expressed SAK on its cellsurface and showed fibrinolysis activity (M. Kiwaki, personalcommunication). These results indicate that both the signaland anchor sequences function properly in L. casei, andthat a functional domain can be expressed on the cellsurface as a fusion protein with SAK and anchor sequence.

Signäs et al. (1989) reported that synthetic peptides

mimicking the repetitive sequence of fibronectin bindingprotein of S. aureus interact with fibronectin. This suggeststhat no rigid three-dimensional structure is required forfibronectin binding. We have picked up the sfbI gene of S.pyogenes ATCC 21059 to be cloned into a surface displayvector, because S. pyogenes ATCC 21059 is available oncommercial source.

The plasmid pSAK332 has a SalI site between SAKand the anchor sequence, and this SalI site is a cloningsite for this surface display vector. DNA for the fibronectinbinding domain (Asn-364 through Thr-526 of SfbI proteinas shown Figure 1) was amplified with polymerase chainreaction (PCR) using Sp01 and Sp02 primers which aredesigned to include SalI and XhoI site at the 5' region,respectively. The PCR product was digested with SalI andXhoI, and the digested product was then cloned into a SalIsite of pSAK332. The resulting plasmid pSAKSfb14 wassequenced to verify that the fibronectin binding domain isinserted in the same frame as SAK and the anchorsequence (Figure 1). The plasmid pSAKSfb14 was thenintroduced into L. casei strain Shirota by electroporation.

Detection of Fibronectin Bound on the Cell Surface ofL. casei Strain Shirota/pSAKSfb14Fibronectin was incubated with L. casei strain Shirota(parental strain), L. casei strain Shirota/pSAK332 (vectorplasmid not carrying the sfbI) and L. casei strain Shirota/pSAKSfb14, and then bactrerial cells were washed toremove nonspecifically bound fibronectin, and were boiledto release fibronectin from the cells to the supernatant.The supernatants were separated with SDS-PAGE andfibronectin was detected by Western blot analysis. A large

Figure 1. Construction of pSAKSfb14. Emr represents the erythromycin resistant gene, pUC ori and pAMb1 ori represents replication origin of pUC19 and ofpAMb1, respectively. Amino acid residues of fibronectin binding domain used in this study are indicated in one letter. SAK represents the signal and maturesequence of staphylokinase of S. aureus, and anchor represents C-terminal 332 amino acid residues of 763 proteinase of L. lactis.

Figure 2. Fibronectin binding analysis of the cell surface of L. casei strainShirota, L. casei strain Shirota/pSAK332 and L. casei strain Shirota/pSAKSfbI14. Bacterial cells were incubated with fibronectin and boiled torelease the captured fibronectin to the supernatant. Fibronectin in thesupernatant was detected by Western blotting. Lane 1, Molecular weightstandard; lane 2, L. casei strain Shirota/pSAKSfb14; lane 3, L. casei strainShirota; lane 4, L. casei strain Shirota/pSAK332; lane 5, Fibronectin (200ng). The molecular masses of standard proteins are denoted in kilodalton(kDa).

L. casei Capable of Binding to Fibronectin 565

amount of fibronectin was recovered from L. casei strainShirota/pSAKSfb14 (Figure 2, lane 2), whereas fibronectinwas not observed from either L. casei strain Shirota or L.casei strain Shirota/pSAK332 (Figure 2, lane 3, 4). Theelectrophoretic mobility of fibronectin released from L. caseistrain Shirota/pSAKSfb14 was similar to that of fibronectinadded to the bacteria, indicating that the fibronectinmolecules are bound to L. casei strain Shirota/pSAKSfb14(Figure 2, lane 2, 5). L. casei strain Shirota/pSAK332, whichexpresses SAK on its cell surface, did not trap fibronectin(Figure 2, lane 4), implying that SAK and C-terminal anchorsequence are not involved in binding to fibronectin.

The supernatant of cell culture of L. casei strain Shirota/pSAKSfb14 was precipitated by trichloroacetic acid andthe binding capacity of precipitated proteins was analysedby Western blotting. Several proteins showed fibronectinbinding ability. This is probably due to excretion of the fusionprotein from the cell surface of L. casei strain Shirota/pSAKSfb14. However, any corresponding fragments werenot detectable by Coomassie staining (data not shown).The amount of the fusion protein excreted from cell surfacewould be a low level.

Assays of the Binding Activity to Fibronectin orFibrinogen of BacteriaTo compare the binding activity of L. casei strain Shirota/pSAKSfb14, fibronectin binding activity of variousLactobacillus strains were examined. As shown in Table 1,the fibronectin binding activity of L. casei strain Shirota/pSAKSfb14 was the highest among Lactobacillus strains,whereas L. casei strain Shirota and L. casei strain Shirota/pSAK332 had no significant activity. This result is consistentwith that of Western blot analysis described above (Figure2). The binding activity of S. pyogenes ATCC 21059, fromwhich the fibronectin binding domain was originated, was

about 1.7 times as much as that of L. casei strain Shirota/pSAKSfb14 (Table 1).

We also examined the binding activity of L. casei strainShirota/pSAKSfb14 to fibrinogen (Table 1). L. casei strainShirota/pSAKSfb14 showed the fibriongen binding activity,whereas L. casei strain Shirota and L. casei strain Shirota/pSAK332 had no significant activity. This result indicatesthat the repetitive sequence expressed on the cell surfaceshows binding capacity not only to fibronectin, but also tofibrinogen. Fibrinogen binding activity of L. casei strainShirota/pSAKSfb14 is, however, much lower than that ofS. pyogenes ATCC 21059 (Table 1).

Immunoelectron Microscopic AnalysisImmunoelectron microscopic analysis was carried out toconfirm that the fibronectin and fibrinogen molecules arecaptured on the cell surface of L. casei strain Shirota/pSAKSfb14. Bacterial cells incubated with fibronectin orfibrinogen were further treated with primary antibody andgold colloidal particle-labeled secondary antibody to detectfibronectin or fibrinogen molecules trapped on the cellsurface. As shown in Figure 3A and 3B, gold colloidalparticles were observed outside the peptidoglycan layerof L. casei strain Shirota/pSAKSfb14. This result indicatesthat L. casei strain Shirota/pSAKSfb14 expressesfibronectin binding domain and traps fibronectin moleculeson the cell surface. On the other hand, gold colloidalparticles were not detected either on the cell surface of L.casei strain Shirota (Figure 3C) or L. casei strain Shirota/pSAK332 (Figure 3D).

Fibrinogen was also detected on the cell surface of L.casei strain Shirota/pSAKSfb14 (Figure 3E), whereas nofibrinogen was observed on the parental strain (Figure 3F).

Table 1. Binding activity of Lactobacillus strains and S. pyogenes

566 Kushiro et al.

Figure 3. Immunoelectron microscopic detection of fibronectin or fibrinogen bound on the surface of L. casei cells. (A), (B) Binding capacity of L. casei strainShirota/pSAKSfb14 to fibronectin. Gold colloidal particles were detected outside the cell wall peptidoglycan. (C) Binding capacity of L. casei strain Shirota tofibronectin. (D) Binding capacity of L. casei strain Shirota/pSAK332 to fibronectin. (E) Binding capacity of L. casei strain Shirota/pSAKSfb14 to fibrinogen.Gold colloidal particles were observed on the cell surface. (F) Binding capacity of L. casei strain Shirota to fibrinogen.

L. casei Capable of Binding to Fibronectin 567

Adhesion to FibroblastBecause fibronectin is secreted in large amounts in culturesof fibroblast cells (Hedman et al., 1978), we examined theadhesion of L. casei strain Shirota/pSAKSfb14, L. caseistrain Shirota and S. pyogenes ATCC 21059 to murinefibroblast STO cells (Martin and Evans, 1975).Approximately 1x104 of bacterial strains were added tomonolayers of STO cells and incubated for three hours.The wells were washed with PBS, and the STO cells weretreated with 0.3% Triton X-100 in PBS to dissolve the STOcells along with the adherent bacteria. The well contentswere plated to determine the number of adherent bacteria.Percent adherence was calculated by dividing the numberof adherent CFU (colony forming units) by the number ofinoculated CFU and multiplying by 100. As shown in Figure4, L. casei strain Shirota/pSAKSfb14 bound to monolayersof STO cells about three times as much as its parentalstrain did (p<0.001). L. casei strain Shirota/pSAKSfb14bound more than 5% of inoculum, whereas L. casei strainShirota bound less than 2%. S. pyogenes ATCC 21059was not recovered from monolayers of STO cells in thisexperiment.

We further performed inhibition experiments in thepresence of anti-fibronectin antibody, i.e., monolayers ofSTO cells were pre-treated with anti-fibronectin antibody.The attachment of L. casei strain Shirota/pSAKSfb14 toSTO cells was significantly inhibited by anti-fibronectinantibody (p<0.01), implying that L. casei strain Shirota/pSAKSfb14 adhered to fibroblast through fibronectin(Figure 4).

The adhesion of L. casei strain Shirota/pSAKSfb14 toSTO cells was also confirmed by light microscopicobservation after Giemsa staining. A large number ofbacterial clusters of L. casei strain Shirota/pSAKSfb14 wereobserved on the surface of STO cells (Figure 5A), whereasonly a small number of bacteria were visible on the control(Figure 5B). These results indicate that the fibronectinbinding domain expressed on the L. casei cell surfacedirectly promotes the adherence to STO cells.

Discussion

For the sake of biological response modifier and safety,genetically-engineered lactic acid bacteria have beendeveloped for antigen delivery vehicle for oral immunization(Pouwels et al., 1998, Slos et al., 1998), and intranasalimmunization of mouse with Staphylococcus carnosusexpressing fibronectin binding domain on the cell surfaceresulted in improved antibody response (Liljeqvist et al.,1999). S. carnosus is used as starter cultures in meatfermentation applications, but not used in dairy products.On the other hand, L. casei has been used in fermentedmilk and generally considered as safe (GRAS). Consideringthe simplicity of oral ingestion of fermented products, L.casei which acquires fibronectin binding capacity may havean advantage to be an effective tool for live vaccinedevelopment. There are several reports for the expressionof fibronectin binding protein or fibronectin binding domainin bacteria like Escherichia coli (Flock et al., 1987, Talay etal., 1991, Lindberg et al., 1992, Sela et al., 1993), S.

Figure 4. Quantitative analysis of adherent bacteria to STO cells. The results were presented as averages from three independent experiments, which wereperformed in quadruplicate, and the bars indicate standard deviations from the means. For inhibition studies, monolayers of STO cells were pre-treated withanti-fibronectin antibody. The paired t-test was used in order to evaluate the statistical significance of the differences.

568 Kushiro et al.

pyogenes (Hanski et al., 1992), Enterococcus faecalis(Hanski et al., 1992) and S. carnosus (Liljeqvist et al., 1999).In contrast to these bacteria, L. casei is a GRAS andprobiotic bacterium. We present here the first evidence forthat fibronectin binding domain is expressed on the cellsurface of probiotic bacteria.

The fibronectin binding capacity of L. casei strainShirota/pSAKSfb14 is lower than that of S. pyogenes ATCC21059 (Table 1). We consider, however, that increasing ofthe plasmid copy number or the stability of the fusion proteincould enhance the fibronectin binding capacity of L. caseistrain Shirota/pSAKSfb14. The adherence of S. pyogenesATCC 21059 to fibroblast cells (STO cells) is not detectablein this study. Hanski and Caparon (1992) have shown thatthe adherence of S. pyogenes JRS4 to respiratory epithelialcells (HTE cells) is a high level and the fibronectin bindingprotein plays an important role in adherence. We currentlydo not know the reason why S. pyogenes ATCC 21059 didnot adhere to fibroblast cells. The adherence of S.pyogenes cells to fibroblast cells has not been reportedyet.

L. casei strain Shirota/pSAKSfb14 shows fibrinogenbinding capacity. Katerov et al. (1998) have reported thatfibronectin binding protein, PrtF of S. pyogenes type M15,binds also fibrinogen, and its fibrinogen binding domain islocated in the N-terminal region, whereas the fibronectinbinding domains are in the C-terminal region. Anotherfibrinogen binding motif of FBP54 of S. pyogenes type M5was revealed to be N-ternimal 89 amino acid residuescontaining repeat sequence (Courtney et al., 1994). Thoseamino acid sequences involved in fibrinogen binding arenot homologous to the fibronectin binding domain used inthis work. We present here that the repetitive sequenceexpressed on the cell surface showed not only fibronectinbinding but also fibrinogen binding activity (Table 1, Figure3), though it is not a high level.

Bacterial attachment to host tissue is usually the firststep leading to colonization and development of infectiousdisease. Natanson et al. (1995) have studied 109 strainsof group A streptococci including clinical isolates andsuggested a correlation between fibronectin binding andthe pathogenic potential. To exclude the possibility of theundesirable effect by expressing fibronectin binding protein,L. casei strain Shirota/pSAKSfb14 was injectedintravenously into a rabbit model of catheter-inducedinfective endocarditis (Durack and Beeson, 1972). L. caseistrain Shirota/pSAKSfb14 was rapidly killed andundetectable in the tissues of liver, kidney, spleen, andvegetation, and did not cause infective endocarditis (datanot shown). These results imply that other factors incombination with fibronectin binding ability may beresponsible for infective endocarditis. We have detectedthat several proteins excreted from the cell surface of L.casei strain Shirota/pSAKSfb14 showed fibronectin bindingcapacity, and the possibility that those proteins aredeleterious could not be denied. Excreted proteins,however, did not lead rabbit to death in the case ofintravenous injection of L. casei strain Shirota/pSAKSfb14.

Bacterial attachment to host tissue is also an importantstep to modulate immune system. Attachment of BCG tobladder wall was thought to be a requisite first step in theinitiation of the antitumor response and fibronectin was

shown to be an important mediator for attachment in vitro(Ratliff et al., 1988). The distribution of fibronectin wasexamined in normal human bladder mucosa (Pode et al.,1986). Fibronectin was absent from the apical surface ofepithelial cells, but was observed at the basementmembrane and in the submucosa. However, in areas ofurothelial damage, BCG attached to the bladder wall viafibronectin and activated immune response (Ratliff et al.,1988). Recently, Zhao et al. (2000) demonstrated thatfibronectin attachment protein of BCG plays an importantrole in the in vivo attachment of BCG to the bladder walland in the induction of BCG-treated antitumor activity. Onthe other hand, a double-blinded placebo-controlled trialby oral administration of L. casei strain Shirota showed astatistically significant decrease in the recurrence ofsuperficial bladder cancer after transurethral resection (Asoet al., 1995), and the habitual intake of L. casei strainShirota was suggested to reduce the risk of bladder cancer(Ohashi et al., 2000). From the view of prophylaxis, long-term ingestion of lactic acid bacteria would be a preventivemeasure for the occurrence of bladder cancer. We expect,therefore, that probiotic bacteria with both antitumor activityand fibronectin binding capacity have a potential for bladdercancer therapy.

We are currently studying the antitumor activity of L.casei strain Shirota/pSAKSfb14 to the model mouse ofsuperficial bladder cancer.

Experimental Procedures

Bacterial Strains and Growth MediumAll Lactobacillus strains were from the stock of ourlaboratory. S. pyogenes ATCC 21059 was obtained fromAmerican Type Culture Collection. E. coli JM109 was usedas a host strain for plasmid construction. MRS medium(Difco) was used for cultivation of L. casei, Brain HeartInfusion (Difco) for S. pyogenes, and LB (Difco) for E. coli.Relevant antibiotic concentrations were 20 µg/ml oferythromycin for L. casei and 500 µg/ml for E. coli, and100 µg/ml of ampicillin for E. coli.

Isolation of Chromosomal DNA from S. pyogenesChromosomal DNA was isolated from overnight culture ofS. pyogenes ATCC 21059. Washed bacteria weresuspended in lysis buffer (0.1% lysozyme, 0.01% N-Acetylmuramidase SG (Seikagaku Co, Japan), 10%sucrose, 50 mM Tris-HCl pH 8.0, 1 mM EDTA). Followinga 90 min incubation at 37°C, SDS solution was added atthe final concentration of 1%. The lysate was heated at65°C for 90 min to inactivate nuclease, and proteinase K(Promega) was added at 200 µg/ml and incubated forfurther three hours at 37°C. After phenol extraction andethanol precipitation, ribonuclease A was added to the DNAsolution. Chromosomal DNA was again extracted withphenol/chloroform and recovered by ethanol precipitation.

Cloning of the Fibronectin Binding Domain of the sfbIGene into the Surface Display VectorThe surface display plasmid pSAK332 was a generousgift from M. Kiwaki (Yakult Institute for MicrobiologicalResearch). It is a shuttle vector between E. coli and L.casei, and recombination of plasmid was completed in E.

L. casei Capable of Binding to Fibronectin 569

Figure 5. Microscopic analysis of the adherence of L. casei cells to STO cells. (A) L. casei strain Shirota/pSAKSfb14. (B) L. casei strain Shirota. L. casei cellswere incubated with fibroblast STO cell monolayers for three hours, washed and stained with Giemsa stain. L. casei cells are the small, rod shape, darklystained objects on the surface of STO cells.

coli. Primers used for amplification of the sfbI gene wereas follows:Sp01, 5'-CGCGGTCGACAATGAAACAGGTTTTTCAGGAAATATGGTT-3'(1360-1389)Sp02, 5'-GCGCCTCGAGGGTATCTTCAACAATGGTCACTGTTTCACT-3'(1848-1819).Numbers in parentheses correspond to the nucleotidenumber of the sfbI gene. Sp01 and Sp02 has Sal I andXho I site at the 5' region, respectively. Coding sequenceof sfbI gene is from 11th nucleotide of each primer.

The fibronectin binding domain of the sfbI wasamplified with PCR using the primers described above.Reactions were carried out in a total volume of 50 µl in acocktail containing 1.6 mM MgCl2, 200 mM dNTPs, 200ng of chromosomal DNA of S. pyogenes, 10 pmol of eachprimer, and 2.5 units of elongase (Gibco). The cycle was94°C for 30 sec, 63°C for 30 sec, 68°C for 2 min, andrepeated 35 times. The PCR product was digested withSal I and Xho I, and the 1.2 kb fragment was extractedfrom agarose gel. The plasmid pSAK332 was digested withSal I and dephosphorylated. The 1.2 kb fragment wascloned into the Sal I-digested pSAK332 to yield pSAKSfb14.The plasmid pSAKSfb14 was sequenced to verify that thefibronectin binding domain is inserted in the same readingframe as SAK and the anchor sequence.

ElectroporationL. casei strain Shirota grown exponentially were chilled onice for 15 min. The cells were collected and washed with10% glycerol several times to remove salt ions. PlasmidDNA was added to 40 µl of the cell suspension in a 0.2 cmcuvette (BioRad) and the mixture was pulsed using GenePulser (Bio-Rad) at 1.5 kV, 200 Ω, 25 µF. 0.9 ml of MRSbroth was immediately added and competent cells wereincubated at 37°C for 60min. Cells were plated on MRSagar containing erythromycin at the concentration of 20µg/ml.

Western Blot AnalysisWestern blot analysis was performed according to vanPutten et al. (1998) with minor modifications. Approximately1x108 bacteria in the stationary phase were collected bycentrifugation at 10,000 x g at 4°C for 5 min, and washedwith 500 µl of PBST buffer (0.05% Triton X-100 in PBS).For blocking, they were incubated with 250µl of 1% BSA inPBST buffer at 37°C for 60 min. Then, cells were incubatedwith 1 µg of fibronectin from human plasma (BoehringerMannheim) in 100 µl of PBST buffer at 37°C for 60 min.Bacteria were collected by centrifugation, washed with500µl of PBST buffer for three times to exclude thefibronectin non-specifically attached to bacteria. They weresuspended in 100 µl of sample solution and boiled for 5min to release the captured fibronectin to the supernatant,and then aliquots of supernatant were subjected to SDS-PAGE (7.5% gel). The proteins were electrophoreticallytransferred to an Immobilon-PSQ membrane (Millipore) byusing semidry blotter (Sartoblot II-S, Sartorius). Fibronectinwas detected by incubation with the rabbit anti-humanplasma fibronectin antibody (primary antibody, BectonDickinson, dilution 1:10,000 in PBST buffer) for 60 min atroom temperature, followed by incubation with alkalinephosphatase-conjugated goat anti-rabbit IgG antibody(secondary antibody, Promega, dilution 1:7,500 in PBSTbuffer) for 60 min, and then developed with nitro bluetetrazolium (NBT, Promega) and 5-bromo-4-chloro-3-indolyl -phosphate (BCIP, Promega) in alkalinephosphatase buffer (100 mM Tris-HCl buffer pH 9.5, 100mM NaCl, 5 mM MgCl2).

Assays of Binding Activity to Fibronectin or Fibrinogenof BacteriaBinding activity to fibronectin or fibrinogen of bacteria wasexamined as follows. Approximately 1x108 bacteria in thestationary phase were collected by centrifugation at 10,000x g at 4°C for 5 min, and washed with PBST buffer. They

570 Kushiro et al.

were incubated with 1% BSA in PBST buffer at 37°C for60 min for blocking. Cells were then incubated with 1 µg offibronectin or fibrinogen (human plasma, Sigma) in 100 µlof PBST buffer at 37°C for 60min. After washing with PBSTbuffer, cells were suspended with anti-fibronectin antibodyor anti-fibrinogen antibody (primary antibody, BecktonDickinson, dilution 1:10,000 in PBST buffer), respectivelyat 37°C for 60 min. Cells were washed with PBST bufferand resuspended with the secondary antibody (dilution1:7,500 in PBST buffer) at 37°C for 60 min. After washingwith PBST buffer, 1x107 of cells were incubated with p-nitrophenyl phosphate (Sigma) as substrate in 1ml ofalkaline phosphatase buffer at 37°C for 5 min, and thenthe absorbance at 405 nm was measured. One unit ofalkaline phosphatase activity is defined as the amount ofalkaline phosphatase to release 1 nmol of p-nitrophenolper 1 min.

Immunoelectron Microscopic AnalysisPre-embedding method was used for immunoelectronmicroscopic analysis. Bacteria were treated as describedabove in Assays of binding activity to fibronectin orfibrinogen of bacteria except for the use of goat anti-rabbitIgG antibody-gold conjugate (particle size 15 nm, EYLaboratories) as a secondary antibody. After incubationwith the secondary antibody and washing with PBS, theywere fixed with 2.5% glutaraldehyde in 0.1 M phosphatebuffer (pH 7.4) for 90 min. The cells were then embeddedin 3% warm agar. The agar block was solidified, washedwith 0.1 M phosphate buffer, fixed with 1% osmiumtetraoxide in 0.1 M phosphate buffer for two hours, andthen dehydrated stepwise in ethanol (50 to 100%) at roomtemperature. The agar block was cut into ultrathin sectionswith a Reichert ultramicrotome. The sections were stainedwith 5% aqueous solution of uranyl acetate for 10 min andobserved under a transmission electron microscope (ModelJEM1200Ex, JEOL) at 80kV.

Adhesion AssayMurine fibroblast STO cells were grown to form monolayersin D-MEM medium (Difco) supplemented with 10% fetalbovine serum (Difco) at 37°C in the presence of 5% CO2in eight-well chamber slides (Labteck II chamber, Nunc).Bacteria in the stationary phase were collected, washedthree times with PBS and suspended with D-MEM mediumwithout fetal bovine serum. The medium of STO cells wasalso replaced with D-MEM medium without serum.Approximately 1x104 of bacterial strains were added to theSTO cells and incubated at 37°C in 5% CO2 for three hours.The wells were gently washed four times with PBS not todisrupt the monolayers. After rinsing, the STO cells weretreated with 0.3% Triton X-100 in PBS to dissolve the STOcells along with the adherent bacteria. The well contentswere agitated and the dilutions were plated on MRS agarto determine the number of adherent bacteria per well. Theactual number of bacteria added to each well wasdetermined by colony counting. Percent adherence wascalculated by dividing the number of adherent CFU (colonyforming units) by the number of inoculated CFU andmultiplying by 100. For inhibition studies, the anti-fibronectinantibody was added to the monolayers (dilution 1:100)before adding bacteria. For the light microscopic analysis,

the STO cells with adherent bacteria were stained withGiemsa stain and examined.

Acknowledgments

We gratefully thank to M. Kiwaki for providing the plasmidpSAK332, and S. Iwata and M. Ando for theimmunoelectron microscopic analysis. We also wish tothank to T. Sato and A. Hirashima for critically reading ofmanuscript, and S. Nishikawa for supporting ourexperiments.

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