Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment - PubMed (original) (raw)

Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment

V B Carruthers et al. Infect Immun. 2000 Jul.

Abstract

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition by Toxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.

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Figures

FIG. 1

FIG. 1

IF detection of trails produced by gliding Toxoplasma on protein-coated glass. Addition of heparin (Hep), heparan sulfate (HepS), CSA, and CSC at 25 mg/ml significantly inhibited both substrate attachment and the formation of trails. The trails were detected by staining for the surface antigen SAG1 as described in Materials and Methods. CTL, gliding in medium in the absence of additions. Bars, 5 μm.

FIG. 2

FIG. 2

GAGs inhibit gliding motility. Incubation of parasites with increasing concentrations (6.2, 12.5, and 25 mg/ml) of heparin (Hep), heparan sulfate (HepS), CSA, CSC, and dextran sulfate (DS), but not dextran (D), resulted in a significant inhibition of attachment to serum-coated glass (A) and relative gliding as monitored by the formation of trails (B). Control represents the extent of attachment or gliding in medium lacking additives. The data are reported as average percentages of control plus SE; n = 3.

FIG. 3

FIG. 3

Soluble GAGs disrupt Toxoplasma attachment to human fibroblasts in a biphasic, dose-dependent manner. (A) CSC, CSA, or the synthetic polyanion dextran sulfate (DS) slightly enhanced attachment at concentrations of <1 mg/ml but then markedly inhibited attachment at higher concentrations, whereas nonsulfated dextran (D) had little effect. (B) Heparin (Hep) also showed a biphasic response, enhancing binding at low doses and inhibiting it at higher concentrations. Heparan sulfate (HS) as well as de-N-sulfated heparin (dNS) competed for binding of parasites to host cell monolayers only at higher concentrations, while fucoidin (Fuc) (a polymer of sulfated

l

-fucose) had little affect. HFF monolayers in 96-well plates were preincubated with 0 to 20 mg of GAGs/ml, and the attachment of parasites was quantified using a colorimetric assay for β-Gal activity. The data are reported as mean percent inhibition, where untreated controls represent 100%; n = 3.

FIG. 4

FIG. 4

GAG-deficient host cells (pgsA-745) were ∼40% less susceptible to infection by parasites than wild-type CHO-K1 host cells (Ctl), and their residual capacity for attachment was unaffected by soluble GAGs. Wild-type and mutant host cells were pretreated with medium alone or 5 mg of heparin (H), dextran sulfate (DS), or dextran (D) per ml, and host-cell-associated parasites were quantified by IF microscopy. The data are expressed as the mean number of parasites per host cell plus SE; n = 3. ∗∗, statistically significant at a P value of ≤0.01 (Student's t test).

FIG. 5

FIG. 5

Inhibition of parasite attachment to HFFs following enzymatic treatments to remove cell surface GAGs. Treatment with heparinase I (HepI) at 1.7 × 10−3 IU/ml or 1.7 × 10−4 IU/ml significantly reduced parasite attachment (∗∗, P ≤ 0.01). Treatment with heparinase III (HepIII) reduced parasite attachment at 1.7 × 10−3 IU/ml and to a lesser extent at 1.7 × 10−4 IU/ml (∗, P ≤ 0.05). Treatment with chondroitinase ABC (Case-ABC) at 1 IU/ml reduced parasite attachment only slightly. The data are expressed as the mean plus SE; n = 2.

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