Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a...

Characterization of a candidate Borrelia burgdorferib3-chain integrin ligand identified using aphage display library

Jenifer Coburn, 1* Wambui Chege, 1 LoranneMagoun, 2 Sarah C. Bodary 3 and John M. Leong 2

1Division of Rheumatology and Immunology, Tufts-NewEngland Medical Center, Box 406, 750 Washington St.,Boston, MA 02111, USA.2Department of Molecular Genetics and Microbiology,University of Massachusetts Medical Center,55 Lake Avenue North, Worcester, MA 01655, USA.3Department of Immunology, Genentech Inc.,1 DNA Way, S. San Francisco, CA 94080, USA.

Summary

The spirochaetal agents of Lyme disease, Borreliaburgdorferi (sensu lato ) bind to integrins aIIbb3,avb3 and a5b1 in purified form and on the surfacesof human cells. Using a phage display library of B.burgdorferi (sensu stricto ) DNA, a candidate ligandfor b3-chain integrins was identified. The native B.burgdorferi protein, termed p66, is known to berecognized by human Lyme disease patient sera andto be expressed on the surface of the spirochaete.We show here that recombinant p66 binds specificallyto b3-chain integrins and inhibits attachment of intactB. burgdorferi to the same integrins. When expressedon the surface of Escherichia coli , this proteinincreases the attachment of E. coli to a transfectedcell line that expresses avb3, but not to the parentalcell line, which expresses no b3-chain integrins. Loca-lization of p66 on the surface of B. burgdorferi , theability of recombinant forms of the protein to bind tob3-chain integrins and the fact that p66 and B. burg-dorferi bind to b3-chain integrins in a mutually exclu-sive manner make p66 an attractive candidatebacterial ligand for integrins aIIbb3 and avb3.

Introduction

Borrelia burgdorferi is the causative agent of Lyme dis-ease, currently the most common arthropod-borne illnessin the United States (Steere, 1989; 1998; Barbour andFish, 1993). It is also widely distributed across Europe

and parts of Asia. The agents of Lyme disease includeB. burgdorferi, B. garinii and B. afzelii, which are oftengrouped together as B. burgdorferi (sensu lato). Thethree spirochaetal species have been linked to somewhatdifferent clinical manifestations at late stages of infection(Canica et al., 1993; van Dam et al., 1993; Anthonissenet al., 1994), but the initial course of infection and the invol-vement of multiple organ systems are common to diseasecaused by all three species. The earliest sign of Lyme dis-ease is frequently the skin rash termed erythema migrans,which expands radially from the site of the tick bite andreflects the dissemination of the spirochaete through theskin. As the infection progresses, the spirochaete infectsmultiple tissues, and patients often experience flu-likesymptoms, arthralgias and neurological problems. Cardiacinvolvement and secondary erythema migrans lesionscan also occur at this stage of the disease. Late manifes-tations of infection involve the skin, central nervous sys-tem and joints and may occur months to years after thetick bite inoculation of the organism. At all stages of infec-tion, the clinical manifestations appear to arise as a directresult of spirochaetal infection of the affected tissues andorgans (Johnston et al., 1985; Nocton et al., 1994; Steere,1998). A remarkable aspect of this infection is that, in theabsence of appropriate antibiotic therapy, the bacteria areable to establish chronic infection even in the face of anintact immune system.

The course of human Lyme disease reflects the fact thatB. burgdorferi is an obligate parasite of mammalian hosts.The natural reservoirs of B. burgdorferi are small rodents,e.g. the white-footed mouse in the north-eastern UnitedStates. A number of animal models have been developedin which at least some of the aforementioned aspects ofthe human infection by B. burgdorferi are replicated. Asis the case in humans, in animals the spirochaete is ableto disseminate widely and avoid clearance by the immunesystem, thereby establishing chronic infection. It has beenpostulated that the bacteria are able to enter host cells, aproperty that would contribute to the ability to evade clear-ance, but the evidence for invasion at the cellular level invivo is inconclusive. Spirochaetes in tissue samplestaken from infected mice (Barthold et al., 1991; 1993) andhumans (Johnston et al., 1985; Nocton and Steere, 1995)appear to be extracellular and are often associated withcollagenous connective tissue and vessel walls.

Molecular Microbiology (1999) 34(5), 926–940

Q 1999 Blackwell Science Ltd

Received 28 June, 1999; revised 30 August, 1999; accepted 7September, 1999. *For correspondence. E-mail [email protected]; Tel. (þ1) 617 636 5952; Fax (þ1) 617 636 4252.

To begin to understand how the spirochaete interactswith its mammalian host, a number of laboratories havestudied the in vitro interactions of B. burgdorferi withmammalian cells and with extracellular matrix compo-nents (Garcia-Monco et al., 1989; Thomas and Comstock,1989; Szczepanski et al., 1990; Hechemy et al., 1992;Galbe et al., 1993). As is the case for many pathogenicbacteria, B. burgdorferi expresses several adhesionmechanisms that are likely to contribute to disseminationto and chronic infection of multiple tissues. Adhesion sub-strates recognized by B. burgdorferi include fibronectin(Kopp et al., 1995; Grab et al., 1998; Probert and Johnson,1998), decorin (Guo et al., 1995; 1998), heparan and der-matan sulphate glycosaminoglycans (Isaacs, 1994; Leonget al., 1995a; 1998) and integrins (Coburn et al., 1993;1998). Among these substrates, only integrins areuniquely cell associated, as the other classes also serveas components of the extracellular matrix.

Integrins are heterodimeric, divalent, cation-dependentreceptors expressed on the surfaces of virtually allnucleated mammalian cells. Representatives of B. burg-dorferi, B. garinii and B. afzelii have been shown to recog-nize integrins aIIbb3, avb3 and a5b1 (Coburn et al., 1993;1994; 1998). aIIbb3 is the classical fibrinogen receptorand is expressed only by platelets and megakaryocytes.avb3 and a5b1, which are also known as the vitronectinand fibronectin receptors, respectively, are each expressedby a variety of cell types (Springer, 1990; Hynes, 1992).The specificity of each integrin is a function of the particu-lar combination of subunits in the a–b heterodimer but, incertain cases, these complexes can display some degreeof overlap in ligand recognition. Integrins normally mediatea variety of cell–cell and cell–extracellular matrix inter-

actions, but also serve as receptors for other bacterialpathogens (Isberg and Leong, 1990; Relman et al.,1990; Leininger et al., 1991). The in vivo significance ofintegrin recognition by B. burgdorferi has not yet beenestablished but, by analogy with other bacterial patho-gens, it is likely that multiple adhesion mechanismscontribute to the virulence of the organism. Rigorousexperiments to determine the roles of the different adhe-sion pathways in B. burgdorferi infection require the iden-tification of the critical bacterial molecules involved.

To identify candidate B. burgdorferi ligands for integrinaIIbb3, we chose to use a filamentous phage display libraryof DNA from a strain that recognizes this receptor effi-ciently. Filamentous phage display libraries represent ameans of selecting efficiently for the gene encoding a pro-tein of interest (Smith, 1985). For example, large filamen-tous phage libraries have been used to identify peptidesthat bind to integrins (Koivunen et al., 1993; 1994; Healyet al., 1995), including aIIbb3 (O’Neil et al., 1992). Usingthis approach, we have identified a strong candidate forthe B. burgdorferi protein ligand that binds to integrinsaIIbb3 and avb3.

Results

Identification and characterization of filamentousphage clones that bind integrin aIIbb3

Borrelia burgdorferi strain N40 binds to integrins aIIbb3,avb3 and a5b1 (Coburn et al., 1993; 1998). To identifythe bacterial ligand for aIIbb3, a filamentous ‘surface dis-play’ phage library of genomic DNA from the cloned, infec-tious B. burgdorferi strain N40 (Coburn et al., 1993; 1998)

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 926–940

Table 1. B. burgdorferi gene fragmentsisolated from the filamentous phage library bypanning on integrin aIIbb3.

B. burgdorferi RGD Binding toClass Homologya gene referenceb sequencec aIIbb3

d

Vector NAe NA No 01 Nucleoside BB463 Yes þþþ

diphosphate kinase2 Acetate kinase BB622 No þ

3 RecG BB581 No þ

4 p66 BB603 No þþ

a. Based on database searches for matches to the nucleotide sequences. Nucleoside dipho-sphate kinase was homologous to the enzyme from Bacillus subtilis ; acetate kinase andRecG were homologous to the enzymes from E. coli.b. Numbers correspond to those assigned in the B. burgdorferi genome sequence (Fraser etal., 1997).c. Based on the sequences from the B. burgdorferi strain N40-derived phage clones and thestrain B31 genomic sequence.d. Binding is expressed as the ratio of the ELISA signal in wells coated with 1 mg ml¹1 aIIbb3

versus the signal in wells coated with the buffer control. 0 represents a ratio of# 1; þ represents a ratio of 1.0–1.2; þþ represents a ratio of 1.5–2.5; þþþ represents aratio of 1.6–6.0 (ranges indicate the results obtained in multiple assays in which class 1phage virtually always bound more efficiently than class 4 phage, which in turn bound moreefficiently than phage of classes 2 and 3). Binding data shown are representative of at leasttwo independent phage preparations and assays.e. Not applicable.

Identification of a B. burgdorferi integrin ligand 927

was used to select for phage particles that bind to aIIbb3. Inthis library, B. burgdorferi sequences that encode an openreading frame (ORF) contiguous with that of phage geneIII are expressed on the bacteriophage surface. Thelibrary was allowed to bind to purified aIIbb3 immobilizedon polystyrene wells. To increase specificity, boundphage were eluted by the cyclic RGD peptide G4120(cRGD), which inhibits B. burgdorferi attachment to b3-chain integrins (Coburn et al., 1998) and elutes bound B.burgdorferi from platelets (data not shown). Phage clonesthat were enriched by two rounds of binding to aIIbb3 andcRGD elution were screened for the presence of overlap-ping fragments of B. burgdorferi DNA. Four classes ofclones that contained overlapping fragments and hadbeen isolated from independent enrichments were identi-fied. Individual members of each class were then testedfor binding to purified aIIbb3 plated in microtitre wells(Table 1). In addition, the sequence of the B. burgdorferiDNA adjacent to the vector–insert junction was deter-mined for each candidate, and homologies to known sequ-ences were identified (Table 1). Three of the four classeswere unlikely to encode the B. burgdorferi integrin ligand,because they showed homology to proteins of other bac-teria that are not thought to be surface exposed, whilethe attachment to integrins by live B. burgdorferi requiresthat the ligand be located on the surface of the bacterium.Phage class 1, which encoded a portion of the cytoplasmicmembrane-associated nucleoside diphosphate kinase(NDK), contained an Arg–Gly–Asp (RGD) integrin recog-nition motif. RGD sequences are present in several of theligands that bind b1- and b3-chain integrins (Hynes, 1992).

This class was therefore used as a positive control in sub-sequent assays of phage–integrin interactions. Classes 2and 3, which encoded portions of the cytoplasmic proteinsacetate kinase and RecG, were not analysed further. Thereasons that these phage clones bound to aIIbb3 areunclear, as no consensus integrin recognition motifswere obvious from the genomic sequence of B. burgdor-feri strain B31 (Fraser et al., 1997) or from the portionsof the strain N40-derived sequences that we determined.

The two overlapping clones that comprised class 4,however, encoded segments of a B. burgdorferi outermembrane protein termed p66. B. burgdorferi p66 isrecognized by sera from many human Lyme diseasepatients (Dressler et al., 1993) and has been cloned bytwo other groups on that basis (Bunikis et al., 1995; Pro-bert et al., 1995). This 66 kDa antigen appears to be sur-face localized by immunogold labelling of bacterial cells(Bunikis et al., 1995), is digested by protease treatmentof living spirochaetes (Bunikis et al., 1995; Probert et al.,1995) and is encoded as a preprotein with a bacterialsecretion signal sequence (Bunikis et al., 1995; Probertet al., 1995). It has also been reported that p66 is aporin, one of two identified to date in B. burgdorferi(Skare et al., 1997). Together, these results strongly sug-gest that the 66 kDa antigen is present on the surface ofthe spirochaete. Phage clone 34-4 encoded amino acids170–404 of the reported p66 sequence (Fig. 1), whileclone 35-10 encoded amino acids 170–364 of p66. Thesequences derived from B. burgdorferi strains B31 andN40 reveal no obvious integrin recognition motifs (e.g.RGD sequences). There was a single amino acid difference


Fig. 1. Schematic representation of the p66-derived proteins used in this study. The topportion shows the protein encoded by B.burgdorferi, with the unfilled area representingthe secretion signal and the hatched arearepresenting the portion of the proteincontained in phage clone 34-4, which boundto integrin aIIbb3. The bottom portion showsthe MBP fusion proteins constructed andtested for integrin-binding activity. Theoligonucleotides used for amplification ofB. burgdorferi sequences are detailed inTable 2.

928 J. Coburn et al.

(Ile-332 → Val) in the strain N40 sequence comparedwith that of strain B31 in the region encompassed byclone 34-4.

Phage clone 34-4 reproducibly bound to integrins aIIbb3

and avb3, but binding to a5b1 was at best inefficient(Fig. 2). Similar results were obtained with clone 35-10(data not shown). The b3-chain integrin-binding propertiesof phage clone 34-4 were compared with those deter-mined previously for the parental B. burgdorferi strainN40 (Coburn et al., 1993; 1998). The attachment ofclone 34-4 to aIIbb3 was inhibited by the cyclic RGD pep-tide (cRGD) and by EDTA, as would be expected for anintegrin ligand (Fig. 2). These results parallel thoseobtained previously in studies of B. burgdorferi attach-ment to either purified aIIbb3 (Coburn et al., 1998) or plate-lets (Coburn et al., 1993) (unpublished observations). Inaddition, phage clone 34-4 bound to 835 cells, whichexpress avb3, more efficiently than to 293 cells, the paren-tal cell line that expresses no b3-chain integrins (data notshown).

Generation and analysis of MBP fusions to thecandidate B. burgdorferi integrin ligand

To ensure that the integrin-binding activity of 34-4 is notsimply an artifact of the sequence generated by the fusionof particular phage and B. burgdorferi amino acid sequ-ences and to facilitate further analysis of the integrin-bind-ing properties of this candidate, fusions of this and other

portions of B. burgdorferi p66 to maltose-binding protein(MBP) were generated (Fig. 1). Integrin interaction activ-ities of the MBP fusion proteins were then assessed byenzyme-linked immunosorbent assay (ELISA), using apolyclonal anti-MBP serum, under essentially the sameconditions used to quantify B. burgdorferi attachment tointegrins.

The MBP fusions to full-length p66 (MBP–p66) and tothe sequences contained in the phage clone 34-4, whichcontains approximately the middle third of p66 and wastherefore designated MBP–p66M, bound efficiently tointegrin aIIbb3. In contrast, neither MBP nor a fusion ofMBP with the cell-binding domain of invasin (which bindsseveral b1-chain integrins; Isberg and Leong, 1990) dis-played significant aIIbb3-binding activity (Fig. 3). TheMBP fusion to the 34-4 region plus the C-terminal portionof p66 (MBP–p66MC) bound to aIIbb3 with the highest effi-ciency, but each of the remaining fusions to p66 sequ-ences that contained the domain encompassed in thephage clone 34-4 also bound to aIIbb3. In contrast, noreproducible binding was observed with either of thefusions that did not contain the 34-4 region of p66(MBP–p66N and MBP–p66C). Similar results wereobtained when the MBP fusion proteins were immobilizedin microtitre wells and probed with soluble aIIbb3 (notshown). MBP–p66MC also bound to purified integrinavb3 (Fig. 4), and more efficiently to 835 cells, whichexpress avb3, than to the untransfected parental 293cells (data not shown). These data suggest that p66-derived sequences can recognize avb3 when the integrin


Fig. 2. Binding of phage particles to b3-chain integrins.A. Purified integrins were immobilized in microtitre wells at1 mg ml¹1, then probed with either the vector phage (fdDOG) or thephage clone 34-4, prepared as described in Experimentalprocedures. Phage particle attachment was quantified by ELISAusing an anti-M13 antibody. Binding to uncoated wells wassubtracted to give the integrin-specific signals displayed, whichwere at least 2.5-fold more than the no receptor control for bindingto aIIbb3 in the absence of inhibitor. The data shown are the means6 standard deviations of four replicates and are representative ofat least two independent experiments.B. The experiment was performed under similar conditions, withthe exception that integrin antagonists were preincubated withaIIbb3 for 30 min at ambient temperature before the addition ofphage. The EDTA concentration was 10 mM; the cRGD peptide(G4120) concentration was 150 nm.

Fig. 3. Binding of MBP–p66 fusion proteins to integrin aIIbb3.Purified aIIbb3 was immobilized in microtitre wells, then incubatedwith the MBP fusion proteins in solution. Unbound protein wasremoved by washing; bound protein was quantified by ELISA usingan anti-MBP rabbit antiserum followed by anti-rabbit IgG conjugatedto alkaline phosphatase. Binding to uncoated wells was subtractedto give the integrin-specific signals displayed. Shown are themeans þ standard deviations of four replicates; data arerepresentative of multiple experiments.A. The integrin was coated at 1 mg ml¹1, then probed with solubleproteins at 10 mg ml¹1.B. aIIbb3 was coated at 10 mg ml¹1 and probed with the fusionproteins at 20 mg ml¹1.


is expressed on the mammalian cell surface, as well as inpurified form.

To determine whether the integrin-binding properties ofthe MBP–p66 fusion proteins were similar to those of B.burgdorferi strain N40 (Coburn et al., 1993; 1998), bindingwas assessed after incubation of the receptors with integ-rin antagonists. Binding of MBP–p66MC to purified aIIbb3

was inhibited by EDTA, the cyclic RGD peptide and a func-tion-blocking anti-aIIbb3 monoclonal antibody (Fig. 5),demonstrating the specificity of the interaction of the pro-teins. Furthermore, if p66 is a ligand for integrins aIIbb3

and avb3, and the same integrin structures are recognizedby the isolated protein and by the intact bacteria, bindingof spirochaetes to the purified receptors should be compe-titively inhibited by recombinant p66 in solution. As shownin Fig. 6, attachment of B. burgdorferi strain N40 to immo-bilized aIIbb3 and avb3 was inhibited by MBP–p66MC, butnot by MBP or MBP–invasin. Although the MBP–p66fusion proteins did display some a5b1-binding activity(not shown), they did not inhibit attachment of B. burgdor-feri to this integrin (not shown).

To demonstrate further the specificity of p66–integrininteraction, we compared the integrin-binding activities ofMBP–p66 fusions with those of MBP fusions to severalother B. burgdorferi proteins. Two of these proteins, p93and BapA, contain RGD sequences and predicted secre-tion signals at the amino-termini (Luft et al., 1992; Wallichet al., 1995). The localization of p93 in the spirochaeteappears to be periplasmic (Luft et al., 1992), but that ofBapA has not been reported. The ‘outer surface proteins’(Osps) A, B and C were also tested, because each of

these lipoproteins appears to have at least some level ofexpression on the spirochaetal surface. MBP and MBP–invasin served as negative controls; fibrinogen servedas a positive control for both aIIbb3 and avb3. The p66-derived fusion proteins were by far the most efficient B.burgdorferi-derived ligands for both b3-chain integrins(Fig. 7). The MBP fusions to the Osp proteins did notbind either of the integrins significantly better than didMBP alone. Furthermore, even the fusions to BapA andp93, which contain RGD sequences, bound each of theintegrins less efficiently than did the p66-derived proteins.The results in Fig. 7 illuminate the specific integrin-bindingproperties of p66 in direct comparison with other well-characterized proteins of B. burgdorferi.

Expression of the B. burgdorferi ligand on thesurface of E. coli

The data presented in Figs 3–5 and 7 suggest that puri-fied recombinant proteins containing the central third ofB. burgdorferi p66 (p66M) bind specifically to b3-chainintegrins. To determine whether p66 might also serve asan integrin ligand when expressed on the surface of a bac-terial cell, we expressed p66 in E. coli, because geneticmanipulations of B. burgdorferi are not straightforward


Fig. 4. Binding of MBP–p66MC to integrins aIIbb3 and avb3.Integrins were immobilized in microtitre wells at 1 mg ml¹1, thenprobed with 10 mg ml¹1 either MBP–p66MC (see Fig. 1) or MBP–invasin (see Experimental procedures) as a control for specificity.MBP alone showed no binding to either integrin. Protein bindingwas quantified by ELISA using an anti-MBP antiserum. Thereceptor-specific binding activities shown are the meansþ standarddeviations of four replicates and are representative of severalexperiments. Binding of MBP–p66MC to wells with receptor wasat least threefold more than the no receptor controls.

Fig. 5. Inhibition of MBP–p66 fusion protein binding to aIIbb3 byintegrin antagonists. aIIbb3 was immobilized in microtitre wells at1 mg ml¹1, incubated for 30 min at ambient temperature with thereagents indicated, then probed with MBP–p66MC at 10 mg ml¹1.Monoclonal antibodies directed against a5b1 and aIIbb3 were usedat 10 mg ml¹1, the EDTA concentration was 10 mM, and cRGD(cyclic RGD peptide G4120; see Experimental procedures) wasused at 1.5 mM (10 × the IC50). The means and standard deviationsof four replicates are shown. Relative binding denotes the ratio ofreceptor-specific binding in the presence of each reagent divided bythe level seen in the absence of any integrin antagonists. TheOD405 was 0.215 in the ‘no additions’ condition for the experimentshown.


at this time. The mature p66 protein coding sequence wasfused to the E. coli OmpT secretion signal peptide.Expression of the fusion protein is dependent on T7RNA polymerase, which in turn is expressed only afterinduction of the lac promoter/operator by IPTG. Two inde-pendent clones that appeared to express full-length p66(pETp66) were then compared with the plasmid vectorcontrol for the ability to bind to cell line 835, whichexpresses integrin avb3. The parental cell line 293 doesnot express b3-chain integrins and was used as a control.After induction with IPTG, both p66-derived clones dis-played increased attachment to 835 cells, while bindingto 293 cells was unchanged (Fig. 8A). IPTG inductiondid not affect binding of E. coli containing the vectoralone to either cell line. Binding of the IPTG-inducedpETp66 clones to 835 cells was inhibited by avb3

antagonists, but not by an anti-aIIb antibody (Fig. 8B).The ELISA signals in wells containing either 293 cells orno cells were not affected by any reagent tested (datanot shown).

To verify that the increased attachment could be attrib-uted to expression of p66 on the bacterial surface, parallelsamples of all E. coli cells were biotinylated with a mem-brane-impermeant reagent. After fractionation of totalbacterial protein by gel electrophoresis under denaturingconditions and transfer to membranes, biotin was detectedwith a specific antibody conjugate. Induction by IPTGresulted in an increase in p66 expression and in theappearance of a new biotinylated band that co-migratedwith p66 but not with any of the bands that appeared inuninduced pETp66-containing E. coli or in the vector con-trols (data not shown). Immunoprecipitation confirmed theidentity of the new band as p66 (Fig. 8C). No biotinylationof the native periplasmic protein MBP was observed underany condition (including after immunoprecipitation), demon-strating that the outer membranes of the E. coli cells had not


Fig. 6. Inhibition of B. burgdorferi attachment to b3-chain integrinsby MBP–p66MC. Integrins aIIbb3 and avb3 were immobilized inmicrotitre wells at 1 mg ml¹1, then incubated with MBP, MBP–p66MC (see Fig. 1) or MBP–invasin (see Experimentalprocedures) at the concentrations shown for 30 min at ambienttemperature. B. burgdorferi strain N40 was added to the wells, andthe plates were centrifuged, incubated and washed as described inExperimental procedures. Bound bacteria were quantified by ELISAusing an anti-Lyme spirochaete antiserum. Data points representthe means 6 standard deviations of four replicates and representat least two independent experiments. Relative binding efficiency isdefined as the level of attachment in the presence of each reagentdivided by the level seen in the absence of any fusion protein. TheOD405 signals in the absence of inhibitor were < 0.4 for eachreceptor and represented more than 10-fold signal over the noreceptor controls.

Fig. 7. Integrin-binding activities of B.burgdorferi-derived MBP fusion proteins.MBP, the MBP fusion proteins and fibrinogenwere immobilized on microtitre wells at5 mg ml¹1, then probed with purified integrinsin solution at 10 mg ml¹1. After washing toremove unbound integrins, the boundreceptors were quantified by ELISA using ananti-b3 monoclonal antibody. No reactivitywas observed in the absence of integrin.OspA, OspB and OspC are B. burgdorferilipoproteins that are exposed on the bacterialsurface; BapA and p93 are B. burgdorferiproteins that contain secretion signals andRGD sequences. The MBP–p66 fusions aredescribed in Fig. 1; MBP and MBP–invasinserve as negative controls; fibrinogen servesas a positive control. The means þ standarddeviations of four replicates are shown; dataare representative of several independentexperiments.


been breached during the experiment. Although surfaceexpression of p66 in E. coli did not appear to be efficient,it was sufficient to allow reproducible binding to humancells that express avb3.

Discussion

At the present time, little is known about how Borrelia burg-dorferi (sensu lato) is able to establish persistent infectionin immunocompetent hosts. In many mammals, this infec-tion results in the development of disease, and Lyme dis-ease in humans may affect the joints, heart, skin andcentral nervous system. A number of groups have investi-gated the interactions of the spirochaete with an intactmodel host (Barthold et al., 1991; 1993; Ma et al., 1998)and with mammalian cells in the laboratory setting.These studies have demonstrated that, as is the casewith other pathogenic bacteria, the interaction of B. burg-dorferi with its host is complex and likely to be mediatedby a number of bacterial and host factors.

One interaction that has been characterized in in vitrostudies is the attachment of B. burgdorferi to integrins(Coburn et al., 1993; 1994; 1998). In an effort toidentify the bacterial molecule(s) that mediate binding tointegrins, we used a filamentous phage display library of


Fig. 8. b3-chain integrin recognition by E. coli expressing p66.Construction of the pETp66 plasmids, which express p66 fused tothe OmpT leader of E. coli, and details of all additional methods aredescribed in Experimental procedures.A. Attachment of E. coli to cells expressing avb3 is dependent onthe induction of p66 expression. Expression of p66 was induced bythe addition of IPTG to 1 mM (þ IPTG); parallel E. coli cultureswere left untreated (¹IPTG). Under both conditions, the plasmidcontaining no insert (pET12 vector) was compared with twoindependent clones containing the full-length p66-encodingsequence (pETp66 clones 1 and 7). The E. coli were harvested,washed and allowed to bind to 835 cells, which express avb3 as aresult of transfection of 293 cells with the genes encoding the av

and b3 integrin subunits, or to the parental cell line 293. Cell-specific attachment of E. coli was quantified by ELISA usingantibodies directed against the E. coli proteins OmpA, b-galactosidase and MBP. Induction-dependent increases in E. coliattachment to the avb3-expressing cells were observed for thepETp66 clones in several experiments.B. Attachment of E. coli expressing p66 to 835 cells is inhibited byavb3 antagonists. Binding of clone pETp66-1 after induction withIPTG was tested after preincubation of the cells with antibodiesdirected against av, avb3 or aIIb (1 mg ml¹1 each) or with the cyclicRGD peptide G4120 at 1.5 mM (10 × the IC50).C. Induction of p66 expression results in partial surface localizationof the protein. Samples of the E. coli cells made as described forthe experiment shown in (A) were biotinylated with the membrane-impermeant reagent SO4-NHS-biotin, then solubilized inimmunoprecipitation buffer (see Experimental procedures). Equalaliquots were incubated with preimmune rabbit serum, anti-p66rabbit serum or anti-MBP rabbit serum, then with proteinA-sepharose. After fractionation by gel electrophoresis underdenaturing conditions and transfer to PVDF membranes, biotinylatedproteins were revealed with an antibiotin monoclonal antibodyconjugated to alkaline phosphatase (anti-biotin). To ensure that thelack of biotinylated protein in the lanes precipitated with anti-MBPwas not caused by a failure of the immunoprecipitation, thesamples were also probed with anti-MBP, and the bands revealedwith anti-rabbit IgG conjugated to alkaline phosphatase (anti-MBP).MBP expression remained constant under all conditions, but wasnever detected with anti-biotin. Positions of molecular weightmarkers are shown at the left of the figure (in kDa).


B. burgdorferi (sensu stricto) DNA to select for genes thatencode proteins that bind to integrin aIIbb3. One candidateligand, p66, fulfils several of the many criteria that definebacterial integrin ligands. First, work by other groupshad demonstrated previously that p66 is a highly con-served protein that is localized in the outer membrane ofB. burgdorferi (Bunikis et al., 1995; Probert et al., 1995).Secondly, we showed that p66-derived sequencesexpressed as fusions to either a filamentous phage proteinor the maltose-binding protein (MBP) of E. coli bound tob3-chain integrins. This activity was inhibited by integrinantagonists that also inhibit B. burgdorferi attachment tothe same integrins. Thirdly, MBP fusions to other B. burg-dorferi-derived proteins, or to portions of p66 outside thedomain originally isolated from the phage library, did notbind to any of the three integrins tested. Fourthly, anMBP fusion protein containing p66-derived sequencesinhibited the attachment of intact B. burgdorferi to b3-chain integrins. Finally, p66 expression on the surface ofE. coli increased bacterial attachment specifically tocells expressing a b3-chain integrin, avb3. These charac-teristics make p66 an attractive candidate for the B. burg-dorferi ligand for integrins aIIbb3 and avb3.

Our data also suggest that p66 is not an efficient ligandfor integrin a5b1. Therefore, a different ligand may med-iate attachment of the spirochaete to this integrin. Onepossibility is that fibronectin might serve as a bridgebetween the bacterial cell (Probert and Johnson, 1998)and host cell integrins, such as a5b1. Our previous results,however, suggest that host-derived proteins, such asfibronectin, are unlikely to be the only integrin ligandsavailable on the surface of the spirochaete (Coburn etal., 1998), and we were unable to find evidence to supporta role for host-derived proteins in B. burgdorferi attach-ment to aIIbb3 (Coburn et al., 1993). A more definitiveidentification of the full receptor repertoire recognizedby p66 awaits the development of function-blockingmonoclonal antibodies against the protein and the genera-tion of B. burgdorferi mutants specifically deficient in p66expression.

Little is known of the structure of mature p66 in the outermembrane of B. burgdorferi. The integrin-binding domainof p66 that we have identified does not overlap with thehypothetical surface loop proposed by other groups (Buni-kis et al., 1995; 1998), but the proposed structure does notpreclude localization of other domains on the outer surfaceof the spirochaete. If p66 is a porin in the outer membraneof B. burgdorferi (as proposed by Skare et al., 1997), themature structure may contain transmembrane domainsseparated by flexible loops, some of which must be exposedto the extracellular environment. The RGD sequences ofother integrin ligands have been found to be locatedon exposed flexible loops. It is therefore consistent withthe biology of known integrin ligands that the receptor

recognition domain of p66 may also be on a loop that isexposed on the bacterial surface.

The p66 protein of B. burgdorferi joins a list of bacterialadhesins that may have multiple functions. One exampleis OmpU of Vibrio cholerae, which is a porin that alsohas haemagglutinating activity (Chakrabarti et al., 1996).The Msp and Tromp1 proteins of Treponemal specieshave also been suggested as candidate porins with adhe-sin activities (Egli et al., 1993; Blanco et al., 1995; Fenno etal., 1996), although localization of spirochaetal membraneproteins has been a matter of some controversy (Cox et al.,1996; Caimano et al., 1999). Of course, additional adhesinsparticipate in the attachment of pathogenic bacteria to mam-malian cells and tissues. B. burgdorferi, for example,expresses the lipoprotein adhesins decorin-binding protein(Guo et al., 1995; 1998) and fibronectin-binding protein(Probert and Johnson, 1998). A candidate B. burgdorferiligand for heparan and dermatan sulphate has been iden-tified recently (N. Parveen and J. M. Leong, unpublished).

B. burgdorferi p66 does not contain any consensusintegrin recognition motifs, but the same is true of invasin,the Yersinia protein that binds with high affinity to severalb1-chain integrins (Isberg and Leong, 1990; Leong et al.,1990). It will be interesting to identify the residues of p66that are crucial for recognition by aIIbb3 and avb3, and todetermine whether p66 can interact with integrins invivo. Definitive identification of p66 and other B. burgdor-feri proteins as ligands for host cell receptors and the elu-cidation of their roles in infection and disease awaits thedevelopment of efficient methods for the manipulation ofthe Borrelia genome. In the case of p66, however, thesituation may be particularly complex, as the knock-outor gross manipulation of a candidate porin may not be tol-erated by this fastidious organism. If this turns out to bethe case, identification of the specific residues involvedin integrin recognition may allow the generation of viablemutants in which integrin-binding activity has been alteredor eliminated. The expression in E. coli of p66 that is com-petent for integrin binding should facilitate the identifica-tion of such mutants.

Experimental procedures

Reagents

Polyclonal antiserum against the human fibronectin receptor(a5b1) was purchased from Telios Pharmaceuticals. The puri-fied function-blocking monoclonal antibodies (mAbs) anti-avb3 (LM609) and anti-av (CLB-706) were from Chemicon.The anti-aIIbb3 and anti-aIIb blocking mAbs, and the anti-b3

used for ELISAs, were from Immunotech. Purified anti-a5b1

blocking mAb VD1 (Tran Van Nhieu and Isberg, 1991) wasa gift from Dr Guy Tran Van Nhieu and Dr Ralph Isberg(Tufts University, Boston, MA, USA). Anti-Lyme spirochaeterabbit antiserum was a gift from Dr Allen Steere (New England



Medical Center). Rabbit antiserum against E. coli OmpA wasa gift from Dr Carol Kumamoto (Tufts University). Antiserumdeveloped in sheep against M13 phage was purchased from58 → 38 Inc. Rabbit antiserum against M13 phage was a giftfrom Dr Ron Bowditch (Scripps Institute, La Jolla, CA,USA). Rabbit antiserum directed against maltose-bindingprotein (MBP) and purified MBP were purchased from NewEngland Biolabs. Monoclonal antibiotin conjugated to alkalinephosphatase, protein A sepharose and antibiotic G418(geneticin) were from Sigma. The cyclic RGD (cRGD) pep-tide G4120 (Barker et al., 1992; Coburn et al., 1998) wasprovided by Genentech. In ELISAs using the aforementionedantibodies, readings were taken at OD405 values within thelinear range of the assay, determined through the use ofpositive and negative controls. Means and standard devia-tions were calculated using Microsoft Excel and the two-tailed t-test.

Oligonucleotides were synthesized at the Tufts ProteinChemistry Facility. Restriction enzymes and other molecularbiological reagents were purchased from New England Bio-labs, unless otherwise noted. Clones encoding fusions of mal-tose-binding protein (MBP) to the B. burgdorferi proteinsOspA, OspB, OspC and p93, as well as some of the corre-sponding purified fusion proteins, were gifts from Dr Allen C.Steere (New England Medical Center). MBP–invasin 479(Leong et al., 1990) was a gift from Dr Ralph Isberg (TuftsUniversity).

Integrins aIIbb3 and avb3 were purified by chromatographyover RGD-sepharose, as described previously (Coburn et al.,1993; 1998; Pytela et al., 1987). Integrin a5b1 was purified byaffinity chromatography over an invasin–sepharose column(Coburn et al., 1998; Leong et al., 1995b).

The buffer used throughout this work was 25 mM HEPES,pH 7.8, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 0.25 mMCaCl2 (HBS). Octyl-b-D-glucopyranoside (ObDG) wasincluded to a final concentration of 50 mM in HBS (HBSO)for integrin purification and storage. For phage selection andB. burgdorferi binding assays, the HBS was supplementedwith BSA to 1% w/v and dextrose to 0.1% w/v (HBSBD).

Bacterial strains and growth conditions

The infectious Borrelia burgdorferi strain N40 clone D10E9,as well as culture and storage conditions, have beendescribed previously (Coburn et al., 1994; 1993; 1998). Forbacterial-binding assays, stocks were thawed, washed inphosphate-buffered saline (PBS) supplemented with bovineserum albumin (BSA) to 0.2% w/v (Coburn et al., 1993;1998) and resuspended in HBSBD at a concentration of2.5 ×107 ml¹1.

E. coli K-12 strains JM109, SR2, BL21 (DE3) pLysS,MC1061 (F¹ ) and TG1 (Fþ ) were grown in standard labora-tory media (Maniatis et al., 1982). All molecular techniqueswere performed using standard protocols (Maniatis et al.,1982). DNA was sequenced using the Sequenase kit (USBiochemicals).

Mammalian cell culture

Cell line 835 was derived from the human embryonic kidneycell line 293 (which expresses av but not b3) by transfection

of the genes encoding the av plus b3 integrin subunits(Chuntharapai et al., 1993). Both cell lines were culturedunder 9% CO2 in a mixture of equal parts of DMEM (low glu-cose) and Ham’s F12 nutrient mix, supplemented with 5%fetal bovine serum and 5% newborn calf serum, 2 mM gluta-mine, 100 mg ml¹1 penicillin and 100 mg ml¹1 streptomycin.The antibiotic G418 (geneticin) was added to 835 cell culturesto 400 mg ml¹1.

Quantification of Borrelia binding to purified integrinsand to mammalian cells

B. burgdorferi binding to cultured mammalian cells and topurified integrins has been described previously (Coburn etal., 1998). Purified integrins were diluted in HBS, dispensedinto prechilled Linbro 96-well plates (ICN) and incubated over-night at 48C. The plates were washed once with HBS, thenblocked by incubation for 1 h at ambient temperature with200 ml per well HBSBD. The blocking buffer was thenreplaced with 35 ml per well HBSBD or with the same buffercontaining the reagent to be tested for inhibition of binding.After incubation for 30 min at room temperature, 15 ml perwell of a suspension of Borrelia at 2.5 × 107 ml¹1 in HBSBDwas added. The plates were then centrifuged at 1200 ×g for10 min and incubated for 30 min at room temperature.Unbound bacteria were removed by washing three timeswith 200 ml per well HBS. Bound spirochaetes were quantifiedby ELISA using a rabbit anti-Lyme spirochaete antiserum asdescribed previously (Coburn et al., 1998). None of thereagents tested affected either the motility of the bacteria orbinding to uncoated wells.

Mammalian cell lines 293 and 835 were cultured on sterileFalcon 96-well tissue culture-treated plates. Cells were platedat density of 0.25 cm2 per well (estimated from growth inflasks of defined area), and incubated under standard growthconditions for 2 days resulting in < 2 ×104 cells per well. Themedium was then replaced with 35 ml per well HBSBD, with orwithout the reagent to be tested. The remainder of the assayprotocol was exactly as described for the purified integrins(above). Integrity of the cell monolayers was verified micro-scopically at the start and end of each assay. None of thereagents used had any apparent effect on the morphologyof the cultured cells.

Construction of the filamentous phage library ofB. burgdorferi DNA

In the filamentous phage system used here, an in frame inser-tion of foreign DNA into phage gene III, which encodes a coatprotein, allows expression of the fusion protein on the phagesurface. Our filamentous phage library was constructedusing a vector derived from fdTET (Smith, 1985). This deriva-tive, fdDOG (McCafferty et al., 1990; Clackson et al., 1991;Hoogenboom et al., 1991), was a gift from Drs David Chiswelland John McCafferty (Cambridge Antibody Technology,Cambridge, UK) and was modified as follows. fdDOG replica-tive form (RF) DNA was purified by caesium chloride gradientcentrifugation and digested with Not I, which cuts at a singlesite near the 58 end of gene III. A stuffer fragment consistingof two synthetic oligonucleotides (oJLC11 and oJLC12;



Table 2) that had been preannealed to each other was ligatedinto the Not I-digested vector, at a molar ratio of < 100 stufferfragments per vector. The stuffer was designed to adapt theNot I site of fdDOG to two Sfi I sites, with stop codons in allthree reading frames between the Sfi I sites, generating thevector termed fdBUG, which cannot express the gene III pro-duct. The stop codons in gene III of fdBUG result in a reduc-tion in infectious phage particle production of at least 5000-fold (data not shown), as the corresponding protein is requiredfor infection of naive Fþ E. coli cells. To avoid selection ofphage rendered more infectious through random mutation,fdBUG was continuously maintained in the F¹ E. coli strainMC1061, which is not susceptible to fd phage infection. Diges-tion of fdBUG with Sfi I and removal of the small stuffer regionresults in 38-GTG overhangs that will not self-anneal. Thissequence allows efficient ligation to DNA fragments thathave been adapted to leave 38-CAC overhangs, therebyselecting for insertion of adapted foreign DNA, as opposedto recircularization of the vector alone. In addition, the repla-cement of the stuffer selects for inserts that result in a contin-uous gene III ORF.

Total genomic DNA (plasmid and chromosomal) from B.burgdorferi strain N40 was prepared essentially as describedpreviously (Sadziene et al., 1991). The DNA was fragmentedby partial digestion with both Csp61 and MseI, each of whichleaves a 58-TA overhang, then fractionated by agarose gelelectrophoresis. Fragments of between 0.2 and 1.0 kbpwere extracted from the gel and ligated to an equimolarmixture of three adaptors. The adaptors were formed bypreannealing synthetic oligonucleotides oJLC13þ oJLC14,oJLC15þ oJLC16 and oJLC17þ oJLC18 (Table 2). Thethree adaptors each have 58-TA overhangs at one end and38-CAC overhangs at the other. The 38-CAC overhangs arecohesive with the ends left by Sfi I digestion of fdBUG, andthe 58-TA overhangs are cohesive with those left by digestionof N40 DNA with either MseI or Csp61. Excess adaptors wereremoved by PEG precipitation of the B. burgdorferi DNA

fragments, which were then ligated into the Sfi I-digestedfdBUG vector. Ligation mixtures were then electroporatedinto E. coli MC1061 and selected on plates containing20 mg ml¹1 tetracycline. In the resulting library, termedfdLYM, the adapted N40 DNA replaces the sequences infdBUG between the Sfi I sites. Inserts containing an intactORF contiguous with phage gene III would then restore theproduction of intact, infectious phage particles. Based on anaverage B. burgdorferi DNA fragment size of 500 bp and agenome size of 1.2 Mbp, and the requirement that the codingsequence is in frame with phage coding sequence, the num-ber of clones required to represent the genome is < 99 300.The number of transformants in the library pool was< 1 946 000, or enough to represent the genome with almost20-fold redundancy.

Preparation of phage particles

Phage particles were prepared from culture supernatants offdLYM grown in E. coli strain MC1061. The library was grownovernight at 378C in L broth supplemented with 15 mg ml¹1 tet-racycline, 10¹2 trypsin-inhibitory units (TIU) ml¹1 aprotinin and1 mM benzamidine-HCl. Bacteria were removed by centrifuga-tion at ambient temperature for 10 min at 15000× g. The super-natant was filtered through a 0.45 mm low-protein-bindingcellulose acetate filter and cooled to 48C. Phage particleswere precipitated by the addition of 0.2 volumes of 20%(w/v) PEG-8000, 2.5 M NaCl, followed by gentle mixing andincubation at 48C. Phage were pelleted by centrifugation at48C for 30 min at 15 000 ×g. The pellets were gently resus-pended in the minimal possible volume of HBS supplementedwith 10¹2 TIU ml¹1 aprotinin and 1 mM benzamidine-HCl. Thetotal protein concentration was determined by measuring theabsorbance at 280 nm (OD280) of a sample of the suspension,and phage were diluted to a concentration of 2 ×109 ml¹1,based on the empirically determined formula: 1 OD280 ¼


Table 2. Synthetic oligonucleotides used in thisstudy. Construction of fdBUG

oJLC11: 58-GGCCAGTGGGGCCGTAATCAGTGACTAGGCCTCACToJLC12: 58-GGCCAGTGAGGCCTAGTCACTGATTACGGCCCCAT

Construction of fd library of B. burgdorferi DNAoJLC13: 58-GCTGGTTGGCCAAAGoJLC14: 58-TACTTTGGCCAACCAGCCACoJLC15: 58-GCTGGTTGGCCAAAAGoJLC16: 58-TACTTTTGGCCAACCAGCCACoJLC17: 58-GCTTGGTTGGCCAAAAGoJLC18: 58-TACTTTTGGCCAACCAAGCCAC

fd vector sequences used for PCR amplification of insert DNAoDOG1: 58-GTCGTCCTTTCCAGACGTTAGToDOG5: 58-CCTTTCTATTCTCACAGTGCACAGGTCCpMalC2 sequences for PCR amplification of insert DNAoMal01: 58-CGCTTTCTGGTATGCCGTGCGTAoMal02: 58-TCTCATCCGCCAAAACAGCCAAG

Cloning of p66 sequences to generate fusions to MBPoN66I: 58-AGAGGATCCATATTGGCAGCAGACGCATTGAAGGAAAoC66I: 58-ACGCGTCGACCTTCTTTTGCTATTAGCTTCCGCTGTAoJLC42: 58-GGAGGATCCGCACCTATGACTGGATTTAAAAGCACTTACoJLC43: 58-GGACTGCAGTTAAAAACCTATGCTTGCTCCTGTTGAAAATGoJLC48: 58-GCGAGTCGACCAGCTAAATTGTTCATATGTTCTTAGAAGToJLC49: 58-AGAGGATCCTGGGCGATTAAGGGTTCTGATTC

Construction of pET12p66 (other oligo used was oN66I)oJLC51: 58-GAGAGGATCCCTTTTGCTATTAGCTTCCGCTGTA


1012 phage ml¹1 (data not shown). Dilutions of phage weremade into HBSBD plus aprotinin and benzamidine to mini-mize non-specific protein interactions and proteolytic degra-dation of the library phage. In all experiments, vector controlphage were prepared from fdDOG, as fdBUG could not beused for this purpose.

Selection of phage particles that bind to integrinaIIbb3

For selection of phage that bind to aIIbb3, conditions weresimilar to those used to assay B. burgdorferi binding to integ-rins (Coburn et al., 1998), with the exception that 24-wellplates were used. Integrin aIIbb3 was coated onto non-tissueculture-treated 24-well plates (Costar) by incubation with500 ml per well of a 1 mg ml¹1 solution diluted in HBS. Afterovernight coating at 48C, the wells were washed twice with1 ml of HBS, then blocked for 1 h at ambient temperaturewith 2 ml per well HBSBD. The blocking buffer was removed,and 0.5 ml of the library or vector control phage suspensionsat 2 × 109 ml¹1 was then added to each well. After overnightincubation at 48C, the wells were washed 15 times, for 5 mineach, with 2 ml per well HBS at ambient temperature.Phage that were specifically bound to aIIbb3 were then elutedby the sequential addition of increasing concentrations of thecyclic RGD peptide G4120. This peptide was chosen becauseit efficiently inhibits B. burgdorferi strain N40 attachment toplatelets and to purified b3-chain integrins (Coburn et al.,1998), and because it elutes bound B. burgdorferi from plate-lets (unpublished observations). The concentrations used forelution ranged from 10× the IC50 (1.5 mM) to 1000× the IC50

(150 mM) (Barker et al., 1992; Coburn et al., 1998). Each elu-tion step was carried out at ambient temperature for 30 min.The eluted phage from each step were then used to infectE. coli strain TG1. Infected clones were selected by overnightgrowth at 378C on L plates containing 20 mg ml¹1 tetracycline.All colonies that arose from each elution step were pooled andused to make new phage preparations, which were individu-ally reselected for binding to integrin aIIbb3, as describedabove. The reselected phage were used to infect E. colistrain TG1, and the pooled colonies were frozen beforefurther use.

Analysis of selected phage clones

After two rounds of selection, individual colonies of phageclones in E. coli strain TG1 were purified by restreaking onL plates containing tetracycline. The B. burgdorferi DNAinserts were amplified by polymerase chain reaction (PCR)using oligonucleotides oDOG1 and oDOG5 (Table 2), whichhybridize to phage DNA sequences flanking the insertionsite. The template DNA was prepared by heating a suspen-sion of E. coli cells picked from a single colony into 20 ml ofdistilled water at 1008C for 10 min. One microlitre of this lysatewas then added to thin-walled PCR reaction tubes on ice con-taining final concentrations of 0.2 mM each primer, 0.2 mMeach dNTP (dATP, dCTP, dGTP, dTTP), 1.5 mM MgCl2,0.5 U Tfl polymerase (Epicentre Technologies) and 1 × Tflbuffer (supplied by the manufacturer) to give a final volumeof 50 ml. The tubes were transferred to the thermocycler

preheated to 928C, and amplification proceeded for 30 cycles,each consisting of 928C for 30 s, 498C for 60 s and 728C for30 s. After a final elongation step of 728C for 7 min, thetubes were held at 48C.

The amplified DNA was then digested with the restrictionenzymes Csp61, MseI, Sau 3AI and Tsp509I. The fragmentswere separated on 5% polyacrylamide gels and revealed bystaining with ethidium bromide under standard conditions(Maniatis et al., 1982). If a specific B. burgdorferi ligand foraIIbb3 was isolated in the selection protocol, clones containingidentical or overlapping inserts would be isolated from multi-ple independent wells. Clones that generated similar restric-tion fragment patterns were therefore chosen for furthercharacterization. Phage clones that did not appear to haveany similarity to any others were not analysed further. In sev-eral independent panning experiments, over 500 clones wereanalysed, but only four classes were reproducibly isolatedfrom wells that contained integrin aIIbb3 and eluted using thecyclic RGD peptide (Table 1). Phage class 1 representedbetween 30% and 50% of the clones isolated (dependingon the individual experiment), class 4 < 10%, while classes2 and 3 were isolated less frequently.

At least one individual clone from each class of phage thatgenerated similar restriction patterns was tested for binding topurified aIIbb3 according to a protocol similar to that describedfor B. burgdorferi binding assays (Coburn et al., 1998). Phagesuspension (50 ml) at 1010 ml¹1 in HBSBD plus aprotinin andbenzamidine was added to quadruplicate microtitre wellscoated with 1 mg ml¹1 aIIbb3 or the buffer control. After incuba-tion overnight at 48C, the wells were washed four times with200 ml per well HBS, then fixed with 50 ml per well 3% para-formaldehyde in PBS. Bound phage were revealed byELISA using an anti-M13 antibody, followed by the appropri-ate secondary antibody conjugated to alkaline phosphatase.In experiments performed to determine whether phage bind-ing was inhibited by specific integrin antagonists, aIIbb3-coated wells were incubated at ambient temperature for30 min with each antagonist before the addition of phage.RF (replicative form, i.e. plasmid) DNA isolated from repre-sentatives of each class of phage clone that displayed integ-rin-binding activity was sequenced using oligonucleotidesoDOG1 and oDOG5 (Table 2).

Generation and characterization of maltose-bindingprotein fusions

In frame fusions of portions of the p66 protein of B. burgdor-feri, to maltose-binding protein (MBP) were generated inpMalC2 (New England Biolabs). For most constructs, the vec-tor was cleaved with BamHI and Sal I to generate non-compa-tible cohesive overhangs that facilitate directional cloning.Insert DNA fragments were generated by PCR amplificationof B. burgdorferi DNA using primers containing the restrictionsites near the 58 ends (Table 2 and Fig. 1). Amplified DNAwas then digested with the appropriate enzymes and ligatedinto the vector. E. coli strain JM109 was transformed with theligation mixes by electroporation. Transformants were selectedon plates containing 100 mg ml¹1 ampicillin. In certain cases,the plates were also supplemented with 0.2% dextrose.After purification by restreaking, colonies were screened forthe presence of insert DNA using either PCR with primers



oMal01 and oMal02 (Table 2) or by digestion of miniprep DNApurified using a commercially available kit (Qiagen).

Plasmids containing the appropriate inserts were trans-formed into E. coli SR2 (Rankin et al., 1992). Six candidateclones from each construct were screened for inducibleexpression of the full-length fusion protein. Induction withIPTG and lysis of E. coli cells were performed as describedpreviously (Leong et al., 1990). Of those that correctlyexpressed the desired product, one was chosen for largerscale expression and purification by affinity chromatographyaccording to the manufacturer’s instructions. Purified proteinswere stored in small aliquots at ¹708C. Protein concentrationswere determined using the BCA reagent (Pierce Chemical).

Interactions between the MBP fusion proteins and purifiedintegrins were assayed using two protocols. The first methodwas adapted from that used to quantify B. burgdorferi attach-ment to integrins (Coburn et al., 1998). Briefly, integrins werecoated onto 96-well plates at 1–3 mg ml¹1 overnight at 48C.After washing with HBS and blocking with HBSBD, the wellswere probed in quadruplicate with 35 ml of the MBP fusion pro-tein diluted in HBSBD to 1–10 mg ml¹1. After incubation for 3 hat ambient temperature, the wells were washed with HBS andfixed. Fusion protein binding was quantified by ELISA usinganti-MBP rabbit antiserum.

In the second assay format, the purified MBP fusion pro-teins were immobilized in 96-well plates. Proteins were dilutedto 1–10 mg ml¹1 in HBS and dispensed at 50 ml per well. Afterovernight incubation at 48C, the wells were washed twice withHBS and blocked at ambient temperature for 45 min withHBSBD, then for an additional 15 min with HBS supplementedwith 1% BSA and 20 mM octylglucoside (HBSBO). Purifiedintegrins were diluted to 10 mg ml¹1 in HBSBO and dispensedat 35 ml per well. After incubation for 3 h at ambient tempera-ture, the wells were washed three times with 200 ml per wellHBS plus 20 mM octylglucoside. The plates were fixed in3% paraformaldehyde in PBS. Bound integrins were revealedby ELISA using a monoclonal anti-b3 antibody or a rabbit poly-clonal antiserum against a5b1.

Construction of E. coli clones that express thecandidate B. burgdorferi integrin ligand on the cellsurface

DNA encoding the full-length, mature p66 polypeptide (minusthe secretion signal sequence) was amplified from B. burgdor-feri strain N40 by PCR using primers oN66I and oJLC51essentially as described above. The amplified DNA wasdigested with the restriction enzyme BamHI, then ligatedinto the pET12a vector DNA (Novagen) that had beendigested with the same enzyme. The ligation mixes weretransformed by electroporation into E. coli strain JM109,and the transformants were selected by growth on L platescontaining 100 mg ml¹1 ampicillin and 0.2% dextrose. Restric-tion enzyme digestion of miniprep DNA prepared from trans-formants was performed to determine the orientation of theinserts. Those in the correct orientation were termedpETp66 and encoded an in frame fusion of the E. coliOmpT secretion signal and signal peptidase site to codon19 of the p66 sequence, near the native cleavage site(Probert et al., 1995). The pETp66 miniprep DNAs were

retransformed into E. coli strain BL21 (DE3) pLysS (Novagen)and selected by growth on plates containing ampicillin anddextrose. Production of p66 was tested after growth in liquidculture. Overexpression after induction for 2 h with 1 mMIPTG (Leong et al., 1990) resulted in two protein bands ofthe mobilities expected for the unprocessed and the matureforms of p66 (not shown). Both proteins were recognized byantisera directed against p66 (not shown).

For experiments in which the surface expression of p66 andthe binding of pETp66 clones were determined, the bacteriawere grown overnight at 308C in L broth supplemented with100 mg ml¹1 ampicillin, 0.2% dextrose, 1 mM benzamidine-HCl and 10¹2 TIU ml¹1 aprotinin, then diluted 1:100 intofresh broth containing the same supplements. After 2 h at308C, IPTG was added to half of each culture to 1.0 mMfinal concentration, and growth was continued for 1 h. Thecells were harvested by centrifugation at 1500 × g for 10 minat 48C and resuspended in HBS supplemented with 1 mMbenzamidine-HCl and 10¹2 TIU ml¹1 aprotinin. The opticaldensities of the samples were then determined at 600 nm,and a one-tenth volume sample was taken to ice for surfacebiotinylation. The remaining bacteria were then centrifugedas above and resuspended in HBSBD plus 1 mM benzami-dine-HCl and 10¹2 TIU ml¹1 aprotinin. Approximately 5 × 107

bacteria per well were added to 96-well plates containingnear-confluent layers of 293 or 835 cells. The plates werecentrifuged, incubated, washed and fixed as described for B.burgdorferi binding assays (above). The layers were permeabi-lized by the addition of 50 ml per well methanol and incubationfor 10 min at ¹208C. Bound E. coli were then detectedby ELISA using a mixture of rabbit antisera directed againstb-galactosidase, MBP and OmpA, each diluted 1:10 000.

Surface biotinylation of the E. coli samples was performedby the addition of a one-twentieth volume of 10 mg ml¹1 SO4-NHS-biotin (Pierce Chemical) in DMSO. After immediate mix-ing, the samples were incubated for 3 min at ambient tem-perature, after which a one-fifth volume of 1 M Tris-HCl,pH 8.0, was added. After a further 3 min incubation, the E.coli were pelleted by centrifugation for 8 min at 4300 × g.The cells were resuspended in one-twentieth the originalvolume of HBS, then lysed by the addition of gel sample buffercontaining SDS and b-mercaptoethanol (Laemmli, 1970) andheating to 958C for 5 min. Proteins were separated by electro-phoresis through a 12.5% polyacrylamide gel under denatur-ing conditions, then transferred to Immobilon polyvinylidenedifluoride (PVDF) membranes (Millipore). Blots were probedwith rabbit antisera against MBP or p66 (see below), or witha mouse monoclonal antibody against biotin. Reactivebands were revealed by incubation with the appropriate sec-ondary antibody conjugated to alkaline phosphatase and col-orimetric development. For immunoprecipitations, the E. colicells were lysed in RIPA buffer (Harlow and Lane, 1988). Theextracts were centrifuged to remove insoluble material, pre-cleared with protein A sepharose, then incubated with the rab-bit antiserum indicated at 1:60 final dilution. After 2 h at 48C,insoluble material was again removed by centrifugation; anti-bodies and complexes were precipitated by the addition ofprotein A sepharose and incubation at 48C for 1 h. After wash-ing four times in RIPA buffer, the beads were heated inLaemmli sample buffer and the proteins were fractionated,transferred and detected as described above.



Generation of rabbit antisera against theintegrin-binding domain of p66

Three rabbits were each immunized with a different prepara-tion of the integrin-binding domain of p66 (the ‘34-4’ region,Fig. 1) by AnimalPharm, according to their standard protocol.One animal was immunized with the peptide after it wascleaved from the MBP–34 fusion protein and purified by gelelectrophoresis. Two animals were immunized with an inframe fusion of the same domain to the hexa-His tag con-structed in the pET30 vector (Novagen). The resultingHis6–34 protein was insoluble in the absence of denaturantand so was purified in the presence of guanidine-HCl accord-ing to the manufacturer’s recommendations. One rabbit wasimmunized with the protein in 6 M guanidine-HCl; anotherwas immunized with the same preparation in which the guani-dine-HCl had been replaced with 4 M urea by dialysis. Allthree rabbits mounted a response that was detectable inimmunoblots against B. burgdorferi strain N40 at dilutionsgreater than 1:10 000 (unpublished observations). Preim-mune sera from the same animals showed no reactivityagainst B. burgdorferi.

Acknowledgements

This work was supported by Biomedical Science Grants fromthe Arthritis Foundation to J.C. and J.M.L., by PHS grant AI-40938 to J.C., PHS grant AI-37601 to J.M.L., and the Centerfor Gastroenterology Research on Absorptive and SecretoryProcesses at New England Medical Center, PHS grant 1P30DK39428 awarded by NIDDK. J.C. was a Genentech Fel-low of the Life Sciences Research Foundation and was sup-ported by the Lincoln National Foundation and by theEnglish, Bonter, Mitchell Foundation. J.M.L. was a PewScholar in the Biomedical Sciences. We thank Allen Steere,Carol Kumamoto, Ron Bowditch, John McCafferty andDavid Chiswell for sharing reagents, Boris Belitsky for helpfulsuggestions, Hui Liu and Daniel Caffrey for technical assis-tance, Ralph Isberg, Linc Sonenshein and Eric Rubin forreview of the manuscript, and especially Ralph Isberg forreagents and helpful discussions.

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Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a...

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