Decorin-binding adhesins from Borrelia burgdorferi

Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi . Ticks deposit B. burgdorferi into the dermis of the host, where they eventually become associated with collagen fibres. We demonstrated previously that B. burgdorferi is unable to bind collagen, but can bind the collagen-associated proteoglycan decorin and expresses decorin-binding proteins (Dbps). We have now cloned and sequenced two genes encoding the proteins, DbpA and DbpB, which have a similar structure, as revealed by circular dichroism (CD) spectroscopy of recombinant proteins. Competition experiments revealed a difference in binding specificity between DbpA and DbpB. Western blot analysis of proteinase K-treated intact B. burgdorferi and transmission electron microscopy studies using antibodies raised against recombinant Dbps demonstrated that these proteins are surface exposed. DbpA effectively inhibits the attachment of B. burgdorferi to a decorin substrate, whereas DbpB had a marginal effect, suggesting a difference in substrate specificity between the two Dbps. Polystyrene beads coated with DbpA adhered to a decorin-containing extracellular matrix produced by cultured skin fibroblasts, whereas beads coated with OspC did not. Taken together, these data suggest that Dbps are adhesins of the MSCRAMM (microbial surface component-recognizing adhesive matrix molecule) family, which mediate B. burgdorferi attachment to the extracellular matrix of the host.     

Variable small protein (Vsp)-dependent and Vsp-independent pathways for glycosaminoglycan recognition by relapsing fever spirochaetes

Tick-borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp-dependent and Vsp-independent GAG-binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism.     

Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi

Binding of glycosaminoglycans (GAGs) by Borrelia burgdorferi, the Lyme disease spirochete, has the potential to promote the colonization of diverse tissues. GAG binding by B. burgdorferi is associated with haemagglutination and we have identified a 26 kDa protein, which we have termed Bgp (Borrelia GAG-binding protein), on the basis of its ability to bind to heparin and erythrocytes. Bgp was found in outer membrane fractions of B. burgdorferi and on the surface of intact bacteria, as assayed by labelling with a membrane-impermeable biotinylating agent or anti-Bgp antibodies. Purified recombinant Bgp agglutinated erythrocytes, binds to the same spectrum of GAGs as the B. burgdorferi strain from which the cloned bgp sequence was obtained, and inhibited B. burgdorferi binding to purified GAGs and to cultured mammalian cells. Thus, Bgp is a strong candidate for a GAG-binding adhesin of B. burgdorferi.     

Common and unique contributions of decorin-binding proteins A and B to the overall virulence of Borrelia burgdorferi

As an extracellular bacterium, the Lyme disease spirochete Borrelia burgdorferi resides primarily in the extracellular matrix and connective tissues and between host cells during mammalian infection, where decorin and glycosaminoglycans are abundantly found, so its interactions with these host ligands potentially affect various aspects of infection. Decorin-binding proteins (Dbps) A and B, encoded by a 2-gene operon, are outer surface lipoproteins with similar molecular weights and share approximately 40% identity, and both bind decorin and glycosaminoglycans. To investigate how DbpA and DbpB contribute differently to the overall virulence of B. burgdorferi, a dbpAB mutant was modified to overproduce the adhesins. Overproduction of either DbpA or DbpB resulted in restoration of the infectivity of the mutant to the control level, measured by 50% infectious dose (ID(50)), indicating that the two virulence factors are interchangeable in this regard. Overproduction of DbpA also allowed the mutant to disseminate to some but not all distal tissues slightly slower than the control, but the mutant with DbpB overproduction showed severely impaired dissemination to all tissues that were analyzed. The mutant with DbpA overproduction colonized all tissues, albeit generating bacterial loads significantly lower than the control in heart and joint, while the mutant overproducing DbpB remained severely defective in heart colonization and registered bacterial loads substantially lower than the control in joint. Taken together, the study indicated that DbpA and DbpB play a similar role in contribution to infectivity as measured by ID(50) value but contribute differently to dissemination and tissue colonization.     

Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach

Lyme disease is caused by the spirochete Borrelia burgdorferi following transmission from infected Ixodes ticks to human hosts. Following colonization of the skin, spirochetes can disseminate throughout the body, resulting in complications that can include ocular, cardiac, neural, and skeletal disease. We have previously shown that B. burgdorferi expresses two closely related decorin-binding adhesins (DbpA and DbpB) of the MSCRAMM (microbial surface component recognizing adhesive matrix molecule) type that can mediate bacterial attachment to extracellular matrices in the host. Furthermore, three Lys residues in DbpA appear to be critical for the binding of DbpA to decorin. We have now characterized the interaction of DbpA and decorin further by using a synthetic peptide approach. We synthesized a panel of peptides that spanned the DbpA sequence and examined their ability to inhibit the binding of intact DbpA to decorin. From these studies, we identified a decorin-binding peptide that lost this activity if the sequence was either scrambled or if a critical Lys residue was chemically modified. A minimal decorin-binding peptide was identified by examining a set of truncated peptides. One peptide is proposed to contain the primary decorin-binding site in DbpA. By comparing the amino acid sequences of 29 different DbpA homologs from different B. burgdorferi sensu lato isolates, we discovered that the identified decorin-binding sequence was quite variable. Therefore, we synthesized a new panel of peptides containing the putative decorin-binding sequence of the different DbpA homologs. All of these peptides were active in our decorin-binding assay, and consensus decorin binding motifs are discussed.     

Evidence of Dbps (decorin binding proteins) among European strains of Borrelia burgdorferi sensu lato and in the immune response of LB patient sera

Decorin binding proteins DbpA and DbpB act as Borrelia burgdorferi (B. burgdorferi) adhesins to decorin, and are able to elicit a persistent antibody response in the mouse; accordingly DbpA protein would seem to be promising in immunoprofilaxis of Lyme borreliosis (LB). This study examines the distribution of Dbp epitopes in European strains of B. burgdorferi, of different genospecies and the presence of antibodies to Dbps in human sera from patients suffering from early and late LB, as revealed by immunoblotting. Different levels of expression of Dbp epitopes were found both among and within genospecies; data from human sera indicate that Dbps are expressed during infection though not as strongly as in the mouse infection.     

Mast cell glycosaminoglycans

Mast cells contain granules packed with a mixture of proteins that are released on degranulation. The proteoglycan serglycin carries an array of glycosaminoglycan (GAG) side chains, sometimes heparin, sometimes chondroitin or dermatan sulphate. Tight packing of granule proteins is dependent on the presence of serglycin carrying these GAGs. The GAGs of mast cells were most intensively studied in the 1970s and 1980s, and though something is known about the fine structure of chondroitin sulphate and dermatan sulphate in mast cells, little is understood about the composition of the heparin/heparan sulphate chains. Recent emphasis on the analysis of mast cell heparin from different species and tissues, arising from the use of this GAG in medicine, lead to the question of whether variations within heparin structures between mast cell populations are as significant as variations in the mix of chondroitins and heparins.     

Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin-binding protein BBK32 is required for optimal infectivity

The aetiological agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.     

Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.     

Mechanism of C. trachomatis attachment to eukaryotic host cells.

A novel trimolecular mechanism of microbial attachment to mammalian host cells was characterized for the obligate intracellular pathogen Chlamydia trachomatis. Using purified glycosaminoglycans (GAGs) and specific GAG lyases, we demonstrated that a heparan sulfate-like GAG present on the surface of chlamydia organisms is required for attachment to host cells. These observations were supported by inhibition of attachment following binding of heparan sulfate receptor analogs to chlamydiae and by demonstrating that chlamydiae synthesize a unique heparan sulfate-like GAG. Furthermore, exogenous heparan sulfate, as an adhesin analog, restored attachment and infectivity to organisms that had lost these attributes following treatment with heparan sulfate lyase. These data suggest that a GAG adhesin ligand mediates attachment by bridging mutual GAG receptors on the host cell surface and on the chlamydial outer membrane surface.     

Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells.

Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface-expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2-terminal part as well as in the C-repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC-chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein-mediated adhesion of S. pyogenes.     

The influence of high ambient glucose level on the production of pericellular glycosaminoglycans by cultured endothelial cells

The 14C-acetate metabolic labeling of glycosaminoglycans (GAGs) was used to investigate the effect of high glucose level on the production of hyaluronic acid (HA), heparan sulphate (HS), chondroitin sulphate (CS) and dermatan sulphate (DS) by human immortalized umbilical vein endothelial cells. It is demonstrated that 30 mM glucose decreased the accumulation of HS and increased the accumulation of CS and DS in the cell layer, pericellular matrix and conditioned medium in 48 h of incubation. The modulation of the overall metabolism of sulphated GAGs by high glucose is in contrast to the observed redistribution of HA from the conditioned medium to the pericellular matrix of endothelial cells. The preincubation at 30 mM glucose increased also the attachment of hyaluronidase-treated endothelial cells to HA-coated surface and had no effect on the cell attachment to poly-D-lysine, indicating the alterations of CD44 binding to immobilized HA. The treatment of endothelial cells with p-nitrophenyl-beta-D-xylopyranoside, which inhibits the coupling of CS to the core protein, attenuated high glucose-induced pericellular HA accumulation and decreased cell attachment to HA-coated surface. It is supposed the implication of CD44-related CS in the accumulation of pericellular HA by endothelial cells exposed to high glucose level.     

Borrelia burgdorferi erpT expression in the arthropod vector and murine host

The expression of a Borrelia burgdorferi gene, erpT was investigated throughout the spirochaete life cycle in the arthropod vector and the murine host. Three phage clones from a B. burgdorferi DNA expression library synthesized a 30 kDa antigen that was recognized by antibodies in the sera of B. burgdorferi -infected mice but not mice hyperimmunized with B. burgdorferi lysates. Differential antibody binding suggested that this protein was preferentially expressed in vivo . This antigen was designated ErpT, based upon 99.6% homology with the BBF01 sequence in the B. burgdorferi genome. ErpT was not detected on spirochaetes cultured in BSK II medium by indirect immunofluorescence or in B. burgdorferi lysates by immunoblotting, implying that ErpT is not readily produced in vitro. erpT mRNA was not discernible by Northern blot but was identified by RNA polymerase chain reaction in vitro , indicating that erpT is expressed at low levels by cultured spirochaetes. erpT expression was then investigated in the vector and mice because B. burgdorferi do not normally reside in culture medium. RNA polymerase chain reaction and immunofluorescence studies demonstrated that erpT was expressed by a small minority of B. burgdorferi (11/500, 2.2%) within unfed ticks and then repressed during engorgement. erpT mRNA or ErpT antibodies were first detected in B. burgdorferi -infected mice at 4 weeks, suggesting that erpT was not expressed in the early stages of murine infection. Then, during persistent infection, RNA polymerase chain reaction showed that erpT was expressed by B. burgdorferi within the joints, heart and spleen, but not by spirochaetes in the skin. Immunization of mice with ErpT was antigenic but was not protective. These studies demonstrate that B. burgdorferi erpT is differentially expressed throughout the B. burgdorferi life cycle, in both the vector and the mammalian host, and is primarily expressed in extracutaneous sites during murine infection.     

Vero cell surface proteoglycan interaction with the microneme protein NcMIC(3) mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii

Neospora caninum and Toxoplasma gondii are characterised by a very low host cell specificity, thus they are able to infect a wide range of different cells in vivo and in vitro. Infection of the host cell by tachyzoites is a process which is preceded by adhesion onto the host cell surface. The receptors on the host cell surface which would allow N. caninum to establish a physical interaction have not been investigated so far. Here we report the role of host cell surface proteoglycans as receptors for the adhesion of N. caninum tachyzoites to Vero cell monolayers. We found that N. caninum tachyzoites, similar to T. gondii tachyzoites, can bind to sulphated proteoglycans which naturally occur on the surface of mammalian cells, including heparin/heparan sulphate, chondroitin sulphates, as well as to the artificially sulphated glycosaminoglycan dextran sulphate. Although removal of heparan sulphate from the host cell surface results in decreased adhesion of T. gondii tachyzoites, binding of N. caninum tachyzoites is not affected by this treatment. Conversely, enzymatic removal of chondroitin sulphate A, B and C decreases N. caninum adhesion but does not affect T. gondii binding to Vero cells. Thus, T. gondii and N. caninum tachyzoites exhibit differential adhesive properties with regard to host cell surface glycosaminoglycans. Additional experiments employing Triton X-100 solubilised NcSRS2 and NcMIC3 showed that NcSRS2 binds to the host cell surface, but not through those sulphated glycosaminoglycans investigated in this study. In contrast, NcMIC3 binding to the host cell surface is dramatically influenced by these modifications. Further experiments showed that the NcMIC3 adhesive motif comprised of four consecutive epidermal growth factor-like domains expressed as a recombinant protein exhibits a high binding activity for sulphated glycosaminoglycans. These results suggest that host cell surface proteoglycan interaction of N. caninum differs from that observed for T. gondii, and that the epidermal growth factor-like adhesive motif in NcMIC3 could be involved in this process.     

Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans.

The secretory enzyme extracellular superoxide dismutase (EC-SOD) occurs in at least three forms, which differ with regard to heparin affinity: A lacks affinity, B has intermediate affinity, and C has relatively strong affinity. The affinity of EC-SOD C for various sulphated glycosaminoglycans (GAGs) was assessed (a) by determining the concentration of NaCl required to release the enzyme from GAG-substituted Sepharose 4B and (b) by determining the relative potencies of the GAGs to release EC-SOD C from heparan sulphate-Sepharose 4B. Both methods indicated the same order of affinity. Heparin bound EC-SOD C about 10 times as avidly as the studied heparan sulphate preparation, which in turn was 10 and 150 times as efficient as dermatan sulphate and chondroitin sulphate respectively. Chondroitin sulphate showed weak interaction with EC-SOD C at physiological ionic strength. Heparin subfractions with high or low affinity for antithrombin III were equally efficient. The binding of EC-SOD C to heparin-Sepharose was essentially independent of pH in the range 6.5-9; below pH 6.5 the affinity increased, and beyond pH 9.5 there was a precipitous fall in affinity. The inhibitory effect of NaCl on the binding of EC-SOD C to GAGs indicates that the interaction is of electrostatic nature. EC-SOD C carries a negative net charge at neutral pH, and it is suggested that the binding occurs between the negative charges of the GAG sulphate groups and a structure in the C-terminal end of the enzyme that has a cluster of positive charges. These results are compatible with the notion that heparan sulphate proteoglycans on cell surfaces or in the intercellular matrix may serve to bind EC-SOD C in tissues.     

Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540

Glycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0-30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Activation of human monocytic cells by Borrelia burgdorferi and Treponema pallidum is facilitated by CD14 and correlates with surface exposure of spirochetal lipoproteins.

Here we examined the involvement of CD14 in monocyte activation by motile Borrelia burgdorferi and Treponema pallidum. B. burgdorferi induced secretion of IL-8 by vitamin D3-matured THP-1 cells, which was inhibited by a CD14-specific mAb known to block cellular activation by LPS and the prototypic spirochetal lipoprotein, outer surface protein A. Enhanced responsiveness to B. burgdorferi also was observed when THP-1 cells were transfected with CD14. Because borreliae within the mammalian host and in vitro-cultivated organisms express different lipoproteins, experiments also were performed with "host-adapted" spirochetes grown within dialysis membrane chambers implanted into the peritoneal cavities of rabbits. Stimulation of THP-1 cells by host-adapted organisms was CD14 dependent and, interestingly, was actually greater than that observed with in vitro-cultivated organisms grown at either 34 degrees C or following temperature shift from 23 degrees C to 37 degrees C. Consistent with previous findings that transfection of Chinese hamster ovary cells with CD14 confers responsiveness to LPS but not to outer surface protein A, B. burgdorferi failed to stimulate CD14-transfected Chinese hamster ovary cells. T. pallidum also activated THP-1 cells in a CD14-dependent manner, although its stimulatory capacity was markedly less than that of B. burgdorferi. Moreover, cell activation by motile T. pallidum was considerably less than that induced by treponemal sonicates. Taken together, these findings support the notion that lipoproteins are the principle component of intact spirochetes responsible for monocyte activation, and they indicate that surface exposure of lipoproteins is an important determinant of a spirochetal pathogen's proinflammatory capacity.     

Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a phage display library

The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins alphaIIbbeta3, alphavbeta3 and alpha5beta1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for beta3-chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to beta3-chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses alphavbeta3, but not to the parental cell line, which expresses no beta3-chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to beta3-chain integrins and the fact that p66 and B. burgdorferi bind to beta3-chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins alphaIIbbeta3 and alphavbeta3.