Diverse Lyme Disease Spirochetes Bind Integrin oLIIbI33 on

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Burgdorfer, W., S. Hayes, and D. Corwin. 1989. Pathophysiology ... Comstock, L. E., and D. D. Thomas. 1989. .... Thomas, D. D., and L. E. Comstock. 1989.
INFECTION AND IMMUNITY, Dec. 1994, p. 5559-5567 0019-9567/94/$04.00+0 Copyright X) 1994, American Society for Microbiology

Vol. 62, No. 12

Diverse Lyme Disease Spirochetes Bind Integrin oLIIbI33 on Human Platelets JENIFER COBURN,' STEPHEN W. BARTHOLD,2 AND JOHN M. LEONG`* Division of Rheumatology and Immunology, Tufts-New England Medical Center, Boston, Massachusetts 02111,'1 and Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-80162 Received 14 June 1994/Returned for modification 19 August 1994/Accepted 12 September 1994 Lyme disease is a chronic, multisystemic infection caused by Borrelia bwgdorferi sensu lato. An infectious strain of B. burgdorferi was previously shown to bind to human platelets via the integrin "IHAj3. In this study, a diverse group of Lyme disease spirochetes was tested for platelet- and aOII,3-binding activity. This collection included representatives of each of the three species that cause Lyme disease, B. bwgdorfe,i (sensu stricto), B. garinji, and B. afzelii. Strains were characterized for infectivity in mouse models or were low-passage isolates from human patients. Each of the 11 infectious strains bound to platelets immobilized in microtiter wells and in suspension. Binding to platelets in suspension was specifically inhibited by a blocking anti-allbP3 antibody, and representatives of each species bound to purified QIIAj3. The strains that did not bind aIIbB3 or platelets were all noninfectious. No obvious relationship was observed between binding to platelets and expression of the bacterial outer surface protein OspA, OspB, or OspC, as assessed by immunoblotting. These results demonstrate that integrin ac 03-binding activity is widespread among the Bomrlia species that cause Lyme disease and are consistent with a role for %aIIA binding in the transmission and/or pathogenesis of Lyme disease.

Lyme disease is currently the most common arthropodborne illness in the United States (3, 40) and has also been widely reported in Europe and Asia. The causative agent, Borrelia burgdorferi (sensu lato), has been isolated from patients and from ticks in diverse geographic regions. Clinical manifestations of B. burgdorferi infection are complex and can be divided into three stages (21, 40). The first stage, localized infection, is characterized by a skin rash, erythema migrans, that spreads radially from the site of the tick bite. The second stage, early disseminated infection, can include flu-like symptoms, secondary erythema migrans, carditis, arthralgia, and a variety of neurological manifestations. The third stage, or chronic infection, may occur years later and can affect the skin, joints, and central nervous system. In most patients, the clinical manifestations of Lyme disease respond to antibiotic therapy, suggesting that they are at least in part the result of direct spirochetal infection of the affected tissues (21, 32). Interaction with host tissues and cells is likely to play a key role in each of the stages of Borrelia infection. Binding to eukaryotic cells might be expected to be involved in the transmission of the spirochete between arthropod and mammalian hosts, in the apparent tropism of B. burgdorferi for particular tissues, and in bacterial dissemination and persistence in the mammalian host. B. burgdorferi has been shown to bind a variety of cell types, including tick cells (7, 23), platelets (14), and cultured mammalian epithelial cells, endothelial cells, and fibroblasts (16, 42, 43). Binding of the infectious North American B. burgdorferi strain N40 to human platelets is mediated by the integrin a11b13 (10). Integrins are divalent cation-dependent, heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix interactions and communication between the extracellular environment and the cytoskeleton (for a review, see reference 19). For each receptor, the specificity of

ligand recognition is determined by the particular combination of the a and 1 chains in the complex. a11,3 is a plateletspecific integrin that binds to a number of adhesion molecules, e.g., fibrinogen and von Willebrand factor, and plays a critical role in hemostasis and thrombosis. aimP3 is fully competent for ligand binding only after platelet activation, a complex process during which platelets acquire increased adhesive properties in response to vascular damage (22). Several bacterial pathogens have been shown to bind to platelets, and this interaction is thought to facilitate the establishment of certain infections, e.g., endocarditis caused by streptococci and staphylococci (9, 17). In the case of B. burgdorferi, binding to platelets might promote dissemination to diverse tissues by concentrating spirochetes at regions of endothelial damage, where activated platelets would be most common. Alternatively, adhesion to platelets might contribute to the transmission of the spirochete, as tissue damage surrounding the site of tick attachment might result in the activation of platelets. If platelet binding is important for Borrelia infection, this activity should be consistently found among infectious Lyme spirochete isolates. In previous work, a low-passage, infectious clone of B. burgdorferi N40 bound to platelets, but a highpassage, noninfectious clone of strain HB19 did not (10). This observation raised the question of how widespread plateletbinding activity might be among Lyme disease spirochetes. Isolates are available from diverse geographic locations, and the original B. burgdorferi (sensu lato) has now been divided into three species (2, 47). B. burgdorferi (sensu stricto; group 1) now comprises most North American and some European isolates, while B. garinii (group 2) and B. afzelii (group VS461 or group 3) comprise European and Asian strains. Although there appear to be some differences in the clinical manifestations caused by the different species (8, 46), Lyme disease in diverse geographic locations is a multistage illness with cardiac, dermatologic, neurologic, and rheumatologic manifestations (21). Persistent infection can be established by each of the three species of Lyme spirochete, even in the face of the specific and expanding host immune response.

* Corresponding author. Mailing address: Division of Rheumatology and Immunology, Tufts-New England Medical Center, Box 406, 750 Washington St., Boston, MA 02111.

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TABLE 1. B. burgdorferi, B. ganinii, and B. afzelii strains used in this work Strain'

Infectious N40 LP3 LP4 LP5 LP7 CA-20 Sh-2-82 HB19 PBi PBo PKo Noninfectious HB19 N40 G39/40 297 CA-11 CA-20 CA-17 PBi

VS102 G2 PBr VS461 a

Clone

Origin

D1O/E9 Uncloned Uncloned Uncloned Uncloned 2A 1A7 Uncloned Uncloned Uncloned 25

Tick, United States Human skin, United States Human skin, United States Human skin, United States Human skin, United States Tick, United States Tick, United States Human blood, United States Human CSF,d Germany Human CSF, Germany Human skin, Germany

10, 26 A. Steere (13) A. Steere (13) A. Steere (13)

B. B. B. B.

A. Steere (13) P. Rosa (36) T. Schwan (37, 38) S. Barthold (41) V. Preac-Mursic (48) V. Preac-Mursic (48) V. Preac-Mursic (33, 48)

B. burgdorferi B. burgdorferi B. burgdorferi B. garinii B. afzelii B. afzelii

1

Human blood, United States Tick, United States Tick, United States Human CSF, United States Tick, United States Tick, United States Tick, United States Tick, United States Tick, United States Human CSF, Germany Human CSF, Germany Tick, Switzerland Human CSF, Germany Human CSF, Germany Human CSF, Germany Tick, Switzerland

10, 26 26, this study K. Georgilis (15) 26 P. Rosa (30) P. Rosa (30) P. Rosa (30) P. Rosa (30) P. Rosa (30) V. Preac-Mursic (48) V. Preac-Mursic (48) R. Marconi, (29) P. Rosa (29, 36) P. Rosa (29, 36) V. Preac-Mursic (48) R. Marconi (29)

D1O/E9 A6 All/B11 1A 4A 14A 14A 13C Uncloned Uncloned Uncloned 12 22 Uncloned Uncloned

All Borrelia strains used in this study were adapted to growth at

Reference(s) or source

Species

burgdorferi

burgdorferi

Passage

6-15 3-5

burgdorferi

3-5

burgdorferi B. burgdorferi

3-5

B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. garinii B. garinii B. garinii B. garinii B. garinii B. garinii B. afzelii

3-5 8-15 8-14 LOwC 8 5 5-7 Highc 50 Highc 15 LowC

LowC LowC Highc 8-15 148 312 8-12 Highc Highc 15 8-12

Infectivityb

A B B B B C D A A, B B, E E, F E E E E E E E E E NDe E E E E E E

34°C in MKP/HS (see Materials and Methods).

b Definition of infectivity (see Materials and Methods): A, causes disseminated infection in 3-week-old

C3H/HeN mice; B, low-passage isolate from human Lyme disease patient; C, infectious in 3-week-old C3H/HeN mice but recovered only from the inoculation site; D, infectious in white-footed mice (37); E, not recovered after intradermal inoculation of 3-week-old C3H/HeN mice; F, infectious in the gerbil model. c Exact in vitro passage number unknown. Low passage is defined as fewer than 10 passages, at 1 to 2 passages per week in culture. High passage is defined as more than 50 passages. d CSF, cerebrospinal fluid. e ND, not determined.

In this study, we evaluated platelet-binding activity among a collection of 27 strains that includes members of each of the three spirochete species that cause Lyme disease and compared binding activity with infectivity for each strain. We found that all of the infectious Borrelia strains tested bind platelets and that integrin aIIb3 mediates binding by representatives of all three species. In addition, the few isolates tested that did not bind platelets were not infectious.

MATERIALS AND METHODS Bacterial strains and growth conditions. The Borrelia strains used in this study are listed in Table 1. Strains N40, G39/40, 297, and Sh-2-82 were cloned by limiting dilution (15, 26, 38). All other cloned strains were obtained by plating the parental strain in semisolid agar (10, 30, 33, 36). In this laboratory, each passage was defined as a 1:100 dilution of a culture at approximately 108 bacteria per ml. Cultures were passaged on average approximately once per week, but this interval varied with individual strain growth rate. The cloned low-passage B. burgdorferi strains N40, CA-20 clone 4A (CA-20-4A), and CA-11-lA and the cloned high-passage strain HB19 were used to determine the culture conditions that allowed optimal platelet-binding activity (Table 2). Growth at 34°C in MKP medium (34) containing human serum (MKP/HS) (10) resulted in maximal binding by N40, CA-20-4A, and CA-11-lA. Strain N40 binding activity was maximal when the cultures

reached a bacterial concentration of 0.5 x 108 to 1 x 108/ml (not shown). Strain HB19 did not bind platelets after growth in any of the conditions tested. On the basis of these results, all bacterial strains tested in this study were adapted to growth at 34°C in MKP/HS and harvested at 0.5 x 108 to 1 X 108 spirochetes per ml in order to optimize bacterial binding to platelets. Glycerol was added to the cultures to a final concentration of 20%, and these bacterial stocks were stored in aliquots at -70°C. Platelets. Human blood was drawn into citrate anticoagulant (44) and centrifuged for 15 min at 140 x g at room temperature. Platelets were isolated from platelet-rich plasma by gel filtration over Sepharose 2B (Pharmacia) in Tyrode's buffer (TB; 2 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES], 137 mM NaCl, 12 mM NaHCO3, 1 mM MgCl2, 2.7 mM KCl, 5.6 mM dextrose [pH 7.35]) (44). Activated platelets were prepared by incubation with 0.2 U of thrombin (Sigma Chemical, St. Louis, Mo.) per ml for 20 min at room temperature. Assay of B. burgdorferi binding to platelets. Two methods were used to measure binding of bacteria to platelets, similar to the methods previously described (10). For both assays, bacterial stocks stored at -70°C were thawed and immediately diluted with 2 volumes of PBS/BSA (phosphate-buffered saline [PBS; 16.9 mM K2HPO4, 4.8 mM KH2PO4, 149.9 mM NaCl] containing 0.2% bovine serum albumin [BSA]). Bacteria were harvested by centrifugation and washed twice in >250 volumes

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TABLE 2. Culture conditions for optimization of B. binding to platelets

Platelet binding

Culture

Culture

Strain'

N40

Culture

Cutuemp temd

(OC)C

Suspensiond

Solid Phasee (%

MKP/HS

34 25 25-*34

2+ 1.5+ 2+

25.8 ± 1.43 13.3 ± 1.48 31.2 ± 2.95

MKP/RS

34 25

1.5+

NDf

1+ 1+ 1+ 0.5+ 0 0 0 0 0 2+ 0.5+ 2+

ND ND ND ND ND ND ND ND ND 34.5 ± 2.02 10.2 ± 1.38 21.4 ± 1.45

2+ 0

ND ND

mediumb

25-*34 BSK I/IHS BSK II/RS

HB19

burgdorferi

MKP/HS MKP/RS BSK II/HS

CA-11-lA

MKP/HS

CA-20-4A

MKP/HS MKP/RS

34 34 34 25 34 25 34 34 25 25--34 34 34

a The B. burgdorferi strains used in this study were N40-D1O/E9, low passage and infectious; HB19-1, high passage and noninfectious; CA-11-1A, low passage and noninfectious; and CA-20-4A, low passage and noninfectious. b Culture medium was MKP (34) or BSK II (4) supplemented with either human serum (HS) or rabbit serum (RS). All cultures were adapted to the medium indicated by subculturing a late-log-phase culture at least twice, with each subculture consisting of a 1:100 dilution at intervals of approximately 1 week. I For each temperature, bacteria were adapted as described above for culture medium conditions. 25->34 refers to bacteria that were adapted to growth at 25°C and then readapted to growth at 34°C. d Coded replicate samples were scored for binding by an independent observer using dark-field microscopy (magnification, x400). 0, no binding was observed in .20 fields; 0.5+, a single bacterium bound to a platelet was only occasionally seen; 1+, .1 bacterium was bound to 21 platelet in five fields; 1.5+, .1 platelet in every field was bound by at least 1 spirochete; 2+, .2 bacteria were bound to each of .5 platelets in every field. Data shown are representative of at least two separate assays. I Data shown are means ± standard errors of the percent inoculum bound as determined by DAPI staining in the solid-phase assay as described in Materials and Methods. Data shown are representative of three separate assays and were obtained with the same bacterial preparations used for the suspension assay. f ND, not determined.

of PBS/BSA and then resuspended in MKP without serum (MKP-S). Spirochetes were enumerated by using a PetroffHausser counting chamber and adjusted to -5 x 108 bacteria per ml. Before each assay, all strains were visually examined to ensure bacterial integrity and motility. Spirochetes were thoroughly dispersed at the start of each assay. All strains were tested on at least three separate occasions, using both assays. Suspension assay. Activated platelets and bacteria were mixed at a ratio of -10 spirochetes per platelet in a final medium consisting of 1 part TB plus 2 parts MKP-S. After 20 min at room temperature, an independent observer scored binding in randomly sorted, coded replicates, using dark-field microscopy at a magnification of X400. At least 20 fields, each containing approximately 30 platelets and 300 spirochetes, were examined for each sample. Binding was scored on a nonlinear scale of 0 to 2+ (see footnotes to Tables 1 to 4). A score of 0.5+ indicates that binding was observed but only on rare occasions. A score of 1 + required that .1 spirochete was bound to -1 platelet in five fields (roughly 0.1% of the bacteria bound to platelets), 1.5+ meant that .1 platelet was bound by 21 bacterium in every field (roughly 0.5% of the bacteria were bound), and for 2+ binding, .2 bacteria bound to each of .5

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platelets in every field (between approximately 5 and 80% of the spirochetes were attached to platelets). To determine whether binding was mediated by the platelet integrin a11b3 (10), platelets were preincubated for 10 min with a blocking anti-CtIbft3 monoclonal antibody (CD 41a; AMAC, Inc., Westbrook, Maine) at 20 ,ug/ml. Solid-phase assay. Activated platelets (107 per well) were added to 96-well black polystyrene Fluoroplates (Labsystems, Helsinki, Finland) and centrifuged at room temperature for 15 min at 425 x g. Platelet monolayers were covered (35 ,ul per well) with MKP-S diluted 1:3 with 10 mM HEPES (pH 7.8)-10 mM glucose (M/3), and then approximately 7.5 x 106 freshly dispersed spirochetes were added in 15 ,ul of MKP-S. Many Borrelia strains have a tendency to self-aggregate, especially at high bacterial concentrations, a phenomenon that could potentially artificially increase the apparent binding efficiency. We therefore used a bacterial inoculum adjusted to minimize the degree of self-aggregation. At the same time, in order to achieve levels of binding that were detectable by the fluorescent DNA stain 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, Oreg.; see below), centrifugation for 20 min at room temperature at 1,200 x g was required to facilitate bacterium-platelet contact. The plates were then incubated for 30 min at room temperature to allow stable bacterial binding. Unbound bacteria were removed by washing three times with PBS; bound spirochetes and platelets were fixed in 3% (wt/vol) paraformaldehyde in PBS (50 RI per well). After the paraformaldehyde was dried overnight at room temperature, cells were permeabilized by adding 50 p.1 of prechilled methanol per well and incubating the cells for 10 min at -20°C. The methanol was discarded, and the plates were dried at room temperature. Fifty microliters of 0.1 p.g of DAPI per ml in PBS was added to each well, and the plates were incubated for 10 min at room temperature in the dark. After two washes in PBS and addition of 50 ,ul of fresh PBS per well, bound bacteria were quantitated by reading the plate in a Titertek Fluoroskan II plate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The inoculated bacteria were quantitated by centrifuging 15 p.1 of the bacterial suspensions onto platelet monolayers covered with 50 p.1 of paraformaldehyde per well, drying overnight, and processing as described above. Percent inoculum bound for each experiment was calculated by using absolute numbers of bacteria converted from fluorescence units, using a standard curve established with two B. burgdorferi strains over a wide concentration range. Assay of binding to purified atlbII3. Integrin 0I4b13 was purified from human platelets (American Biological Technologies, Seguin, Tex.) by RGD-Sepharose chromatography as described previously (10, 35). Although other integrins present on platelets (e.g., a003) might also bind to RGD-Sepharose, in comparison with the level of x14b,43 expression, these integrins are not abundant (18). In our purified aiI3 preparation, neither at, nor PI (which would account for the presence of all known platelet integrins) was detectable by either enzymelinked immunosorbent assay or immunoblotting. Fluoroplate microtiter wells were coated with purified aIIAb13 (10 ,ug/ml in TB [see above]) overnight at 4°C. Control wells were coated with buffer alone or, to control for nonspecific protein-protein interactions, with 10 p.g of human fibronectin (Promega, Madison, Wis.) or a fusion protein containing the cell-binding domain of the Yersinia pseudotuberculosis invasin protein (MBP-Inv479) (25) per ml. Wells were washed briefly with TB and then blocked for 4 to 6 h at 4°C in TB containing 1% BSA. Binding of spirochetes to purified a1b,133 was assayed essentially as described previously (10) except that bacteria were not radiolabeled. Briefly, 50 p.1 of dispersed borreliae at 2.5 x

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108/ml was added to coated wells immediately after removal of the blocking solution. Binding and quantitation of bacteria with DAPI were then carried out as described above for platelets. Surface biotinylation and immunoblot analysis of bacterial strains. Bacteria were prepared for immunoblotting as described above for binding assays. After the second PBS/BSA wash, the membrane-impermeant reagent NHS-LC-biotin (Pierce, Rockford, Ill.) at 10 mg/ml in dimethyl sulfoxide was added to 0.1 mg/ml in order to specifically label surface expressed proteins. After 5 min at room temperature, ethanolamine (pH 8) was added to 100 mM. Bacteria were washed once in MKP-S and then suspended in 2% sodium dodecyl sulfate (SDS) loading buffer containing 2-mercaptoethanol. To determine whether each of the 27 strains expressed the wellcharacterized outer surface proteins OspA, OspB, and OspC, and to begin to assess the potential roles of these proteins in binding to platelets, immunoblot analysis of bacterial proteins was performed according to standard protocols (24, 45). Antibiotin antibody probing revealed that proteins corresponding to the expected mobilities of the Osp proteins were surface localized (not shown). Rabbit polyclonal antisera used were anti-OspA antiserum 3705G and anti-OspB antiserum 5044G, generous gifts from D. Dorward, and an anti-OspC antiserum from T. Schwan. A monoclonal antibody directed against biotin was purchased from Sigma. Reactive proteins were detected by using anti-rabbit immunoglobulin G conjugated to alkaline phosphatase. Infectivity assays. Strains isolated from human Lyme disease patients and passaged fewer than five times in vitro were considered infectious (LP3, LP4, LP5, LP7, and PBo; Table 1). One strain (Sh-2-82) was proven infectious in white-footed mice, another (PKo) was proven infectious in gerbils, and other strains (N40, CA-20-2A, low-passage HB19, and PBi) were proven infectious in the C3H/HeN mouse model (1) in this study (Table 1). Three-week-old C3H/HeNCrl mice (Charles River Laboratory, Raleigh, N.C.) were injected intradermally with 100 RI of MKP/HS containing either 104 or 106 live spirochetes grown under our standard conditions (described above). After 2 weeks, the mice were sacrificed, and the inoculation site, ear, bladder, and spleen were cultured in BSK II (4) medium for 2 weeks as described previously (1, 5). Cultures were examined by dark-field microscopy, and those that showed spirochete growth within 2 weeks were considered positive. Strains that were recovered from any of the sites tested were considered infectious. RESULTS Assessment of binding to platelet monolayers by diverse Borrelia strains. Previous work showed that a low-passage clone of B. burgdorferi N40 bound to platelets efficiently, while a high-passage clone of strain HB19 did not (10). To determine whether the ability to bind platelets is commonly found among Lyme disease spirochetes, a collection of 27 strains (Table 1), including infectious and noninfectious representatives of each Lyme disease spirochete species, was tested for plateletbinding activity after culturing under standard conditions developed in this laboratory (see Materials and Methods). The 11 infectious strains, representing the three Lyme spirochete species, were defined as infectious by one of the following criteria. Isolates from Lyme disease patients that had been passaged fewer than five times in culture were considered infectious (Table 1). In addition, most of the strains characterized for integrin-binding activity in this study were tested for the ability to establish infection after intradermal inoculation

INFECT. IMMUN.

of 3-week-old C3H/HeN mice (1, 5); those that were recovered from ear, spleen, bladder, and/or the inoculation site were also included in the infectious group. Two strains, PKo and Sh282-1A7, had been shown to be infectious in gerbils and white-footed mice, respectively (33, 37). The 16 strains defined as noninfectious were not recovered from C3H/HeN mice after intradermal inoculation (Table 1). A nonradioactive assay was developed to assess plateletbinding activities among these diverse isolates, taking advantage of the fact that platelets are anucleate cells. The fluorescent DNA-specific reagent DAPI was used to quantitate platelet binding by the bacteria (see Materials and Methods). Because this assay does not involve antibody probing, it does not depend on the expression of any particular Borrelia antigen. The inoculum used in these experiments was kept as low as possible so that bacterial self-aggregation was minimized, but this low inoculum required centrifugation of bacteria onto platelet monolayers in order to achieve binding at levels detectable by DAPI (see Materials and Methods). Each strain was tested for platelet binding multiple times to account for day-to-day variation in bacterial and platelet preparations. Each of 11 of the infectious strains, including at least one representative of each Lyme spirochete species, bound platelets efficiently (Fig. 1). Most of the noninfectious strains displayed lower or undetectable activity. No strain bound to control wells without platelets (not shown). It was possible that the apparent binding in the solid-phase assay could be artificially increased by self-aggregation of the bacteria, a property commonly found among low-passage Lyme disease spirochetes. To determine whether individual spirochetes were bound to platelet monolayers, a modification of this assay was performed with platelet monolayers on coverslips. The binding assay was performed under the conditions of our standard microtiter plate assay, and the bacteria were visualized by fluorescence microscopy. Individual bacteria of the low-passage B. burgdorferi N40 were seen bound to the platelet monolayers on coverslips (Fig. 2a). Occasional aggregates were also observed but did not account for the majority of binding. Consistent with the results obtained in the microtiter well assay, few bacteria of the high-passage strain HB19 were seen bound to platelet monolayers (Fig. 2b). Borrelia binding to platelets in suspension. A visually scored assay of binding to platelets in suspension was also used to assess the binding activity and the extent of self-aggregation for each of the 27 strains characterized in the solid-phase assay. Spirochetes were incubated with platelets in suspension, and coded replicate samples were scored for binding by an independent observer using dark-field microscopy (see Materials and Methods). In this assay, discrimination of stable binding from transient colocalization was possible because the motility of bound spirochetes caused rapid oscillatory motion of the platelets. All of the binding-proficient strains bound via the tips of individual spirochetes (not shown). The 11 infectious isolates each displayed platelet-binding activity in suspension, consistent with the results of the solidphase assay (Table 3; Fig. 1). Of the 16 noninfectious strains, 12 also bound platelets (Table 3). The four noninfectious strains that did not bind platelets in the suspension assay also bound least efficiently in the solid-phase assay. A few strains appeared to have different relative plateletbinding activities in the two assays. The centrifugation of the bacteria onto platelet monolayers in the solid-phase assay may promote stable attachment via multiple pathways. For example, in addition to binding atHbP3, many Borrelia strains express a second cell-binding pathway mediated by cell surface proteoglycans (20, 27). In fact, all of the strains that apparently bound

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aIIbP3 BINDING BY LYME SPIROCHETES

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