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Borrelia burgdorferi sensu lato is the spirochetal agent of Lyme borreliosis, a chronic, multisystemic illness (12, 24). The bacterium is acquired from an infected Ixodes tick, and after infecting the skin at the site of the bite, it can disseminate throughout the mammalian host. The ability of the spirochete to bind to extracellular matrix or to the surface of host cells is likely to promote tissue colonization, and the spirochete has been shown to recognize several classes of host molecules (3, 4, 7, 8), including the glycosaminoglycan (GAG) heparin (11, 16).

GAGs consist of long, linear, highly sulfated disaccharide repeats and are usually found covalently linked to a protein core as a component of proteoglycans (13, 23). Based on the extent and location of sulfation and on the composition of the disaccharide unit, GAGs can be separated into different classes, such as heparin, heparan sulfate, keratan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, or dermatan sulfate. Even within a given class of GAG there is extensive heterogeneity due to epimerization of the sugar backbone, differences in chain length, and variations in the location of N-acetyl, N-sulfate, and O-sulfate groups. The complexity and heterogeneity of GAGs complicate the analysis of protein-GAG chain interactions.

B. burgdorferi has been shown to bind to heparin, heparan sulfate, and dermatan sulfate, as well as to decorin, a dermatan sulfate/chondroitin sulfate proteoglycan (8, 11, 16). B. burgdorferi strains that bind heparin have also been shown to agglutinate rabbit erythrocytes in a heparin-inhibitable manner, suggesting that the two activities may be linked (16). It is not clear whether the recognition of different proteoglycans reflects multiple binding pathways or a single, somewhat promiscuous pathway. All GAG chains share the feature of high negative charge, and charge has been demonstrated to be critical for B. burgdorferi binding (11, 16). Nevertheless, charge is not the sole determinant of binding, because not all GAG chains are recognized by the spirochete, and the spectrum of GAGs that are efficiently bound varies among different B. burgdorferi strains (18). Thus, GAG binding by this bacterium displays an element of specificity.

Characterization of the structural requirements for heparin recognition by B. burgdorferi would promote a better understanding of the nature of this binding specificity as well as the mechanisms of cell attachment and tissue colonization by this pathogen. In the present study, we have identified some of the features of GAG chain structure that are critical for cell attachment by B. burgdorferi. We have further shown that these features are also critical for inhibition of hemagglutination, suggesting that B. burgdorferi GAG binding and hemagglutination are closely linked.

GAG chain charge and chain length are critical for recognition by B. burgdorferi. Mammalian cell attachment by strain N40, clone D10/E9 (an infectious B. burgdorferi sensu stricto isolate [3]), is inhibited by the presence of exogenous dextran sulfate, heparin, heparan sulfate, and dermatan sulfate (16, 17). This inhibition is due to an interaction between spirochetes and GAG, because treatment of bacteria with GAG inhibited cell binding even after excess GAG was removed by washing (data not shown). In contrast, treatment of cell monolayers with GAG followed by washing resulted in no inhibition of bacterial binding. Dextran and desulfated heparin failed to inhibit attachment of B. burgdorferi, suggesting that a negative charge is required for inhibitory activity (11, 16). To further explore the relationship between charge and binding inhibition, we analyzed several GAG preparations that differed in charge for the ability to inhibit B. burgdorferi N40 binding of Vero (monkey kidney) cells, an interaction that was previously shown to be mediated by proteoglycans (16). Attachment of radiolabeled B. burgdorferi N40 to Vero cells was determined in the presence of various concentrations of GAG, as described elsewhere (16, 17). As previously shown, completely desulfated, N-acetylated heparin (Seikagaku, Inc., Tokyo, Japan) had no detectable inhibitory activity, even when tested at 1 mg/ml (Table 1). N-desulfated, N-acetylated heparin, which retains O sulfation, demonstrated weak inhibitory activity.

A minimum chain length of 16 residues is required for heparin to inhibit cell binding by B. burgdorferi. The correlation between sugar chain length and inhibition prompted us to test for a minimum size requirement for GAG recognition by B. burgdorferi. Heparin fragments containing 10, 12, 16, 20, or 22 monosaccharide units (gifts of Ulf Lindahl [Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala, Sweden] and Jean Choay [Institut Choay, Paris, France] to the late Isidore Danishefsky [15, 19]) were tested for the ability to inhibit bacterial attachment to Vero cells. The 22-mer almost completely blocked attachment, while the 10- and 12-mers had no effect (Fig. 1). The 16-mer (P < 0.05 versus no-inhibitor control) and 20-mer (P < 0.005) inhibited at intermediate levels.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Identification of the minimum heparin fragment required for inhibition of attachment of B. burgdorferi to mammalian cells. Radiolabeled B. burgdorferi N40 was preincubated with 500 µg of heparin fragment per ml prior to infection of Vero cells, and bound bacteria were quantitated. Each bar represents the average ± the standard deviation of four determinations. Statistically significant differences were determined by using Microsoft Excel to calculate two-tailed t values between the no-inhibitor control (No Inhib.) and heparin fractions; the asterisks indicate P values of <0.05.

Inhibition of cell binding closely correlates with inhibition of hemagglutination. It has been postulated that the GAG-binding activity of B. burgdorferi is reflected by the agglutination of rabbit erythrocytes, because hemagglutination is inhibited by heparin and those strains that exhibit low-level hemagglutination activity also exhibit low-level GAG-binding activity (16). A further prediction of this hypothesis is that the ability of a given GAG preparation to inhibit cell attachment should correlate with its ability to inhibit hemagglutination. Used in these assays was a detergent extract of B. burgdorferi N40, prepared in bulk and frozen in aliquots at 70°C, that was found to give a more predictable hemagglutination titer than intact spirochetes (the aggregation of which introduced variability in the hemagglutination titer [data not shown]). To generate this extract, a suspension containing 10¹¹ B. burgdorferi N40 cells per ml was lysed by sonication, and the insoluble fraction was extracted in a solution containing 2% deoxycholate, 20 mM HEPES, 4 mM EDTA, and 0.01 trypsin-inhibitory units of aprotinin (Sigma Chemical Co.) per ml for 40 min at 4°C. The hemagglutination activity of the detergent-soluble fraction and the effect of each of the GAG preparations on this activity were determined as described previously (16). When we tested different classes of GAGs for the ability to inhibit hemagglutination, keratan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate had no effect whereas heparin and dermatan sulfate were inhibitory, the former at a much lower concentration than the latter (Table 1). Completely desulfated, N-acetylated heparin was devoid of hemagglutination blocking activity, and N-desulfated, N-acetylated heparin retained weak inhibitory activity. Fractions containing longer, more highly charged heparin chains were better inhibitors than fractions containing shorter, less highly charged fractions (IE and CF series fractions, Table 1). All of these results paralleled inhibition of Vero cell binding by the same GAG preparations, and within the CF heparin fractionation series, the minimum concentration for inhibition of hemagglutination correlated with the IC₅₀ for cell attachment (Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   The relative ability of heparin fractions to inhibit cell binding by B. burgdorferi correlates with the ability to inhibit hemagglutination. The IC50 for Vero cell binding and the minimum inhibitory concentration (MIC) for hemagglutination of each of the CF heparin fractions were determined as described in Table 1. The nonparametric (Spearman) correlation coefficient r and the two-tailed P value were calculated by using InStat version 2.01.

Discussion. We undertook this study to better define the requirements for GAG chain recognition by B. burgdorferi, and we found that both the charge and the chain length of GAGs are critical factors for bacterial binding. The length and/or charge of the GAG chain correlated with inhibition of cell binding by B. burgdorferi, and heparin chains with less than 16 residues lacked detectable inhibitory activity. Given the intimate relationship between the size and total charge of GAGs, it is difficult to assess the relative importance of these two features for bacterial recognition.