Eukaryotic cell uptake of heparin-coated microspheres: a model of host cell invasion by Chlamydia trachomatis

Abstract

Using polystyrene microspheres coated with heparin or heparan sulfate, it was shown that coated microspheres specifically bound eukaryotic cells and were endocytosed by nonprofessional phagocytic cells. Coated microspheres displayed properties of binding to eukaryotic cells that were similar to those of chlamydiae, and the microspheres were competitively inhibited by chlamydial organisms. Endocytosis of heparin-coated beads resulted in the tyrosine phosphorylation of a similar set of host proteins as did endocytosis of chlamydiae; however, unlike viable chlamydial organisms, which prevent phagolysosomal fusion, endocytosed beads were trafficked to a lysosomal compartment. These findings suggest that heparin-coated beads and Chlamydia trachomatis enter eukaryotic cells by similar pathways.

FIG. 1

Inhibition of heparin-coated bead binding to HeLa 229 cells in the presence of exogenous heparin, heparan sulfate, chondroitin sulfate, or chlamydial organisms. (A) Microspheres coated with [³H]heparin were incubated with HeLa 229 cell monolayers for 2 h in the absence of glycosaminoglycan competitors or in the presence of 1 mg of heparin, heparan sulfate, or chondroitin sulfate per ml. (B) Dose-dependent competitive binding inhibition of [³H]heparin-coated microspheres to HeLa cells by chlamydial organisms. Coated microspheres were incubated with HeLa cell monolayers for 2 h in the presence of increasing concentrations of chlamydiae. Error bars indicate standard deviations.

FIG. 2

HeLa 229 cell attachment and uptake of [³H]heparin-coated microspheres determined by measuring the amounts of cell surface-bound (trypsin-sensitive) and cell-associated (trypsin-resistant) radioactivity at different times following inoculation and incubation at 37°C. Error bars indicate standard deviations.

FIG. 3

Transmission electron microscopy of HeLa 229 cells following incubation at 37°C for 4 h with heparan sulfate-coated polystyrene microspheres. The inset shows a higher magnification of the area immediately below the inset. Bar, 1 μm.

FIG. 4

Binding of ³⁵S-labeled chlamydial heparan sulfate-like ligand-coated microspheres to HeLa cells. (A) Dose-dependent competitive inhibition of coated microspheres and chlamydial organisms. Coated microspheres were incubated with HeLa cell monolayers for 2 h in the presence of increasing concentrations of chlamydiae. (B) HeLa cell attachment and uptake of ³⁵S-labeled chlamydial heparan sulfate-like ligand-coated microspheres. The amounts of cell surface-bound (trypsin-sensitive) and cell-associated (trypsin-resistant) microspheres were determined by scintillation counting at different times following inoculation and incubation at 37°C. ³⁵S-ligand was obtained from chlamydia-infected CHO 761 cells and purified by ion-exchange chromatography as described previously (42). Error bars indicate standard deviations.

FIG. 5

Chlamydiae and heparin-coated beads induce similar changes in host protein tyrosine phosphorylation. Immunoblot analysis of lysates from mock-infected cells (lane 1), cells infected with C. trachomatis (MoPn) for 2 h (lane 2), cells pretreated with heparin (1 mg/ml) for 1 h and then infected with chlamydial organisms for 2 h in the presence of exogenous heparin (lane 3), cells to which heparin-coated beads were added and left for 2 h (lane 4), cells to which 10-fold more heparin beads were added and left for 2 h (lane 5), or cells pretreated with heparin and then incubated with heparin-coated beads for 2 h in the presence of exogenous heparin (lane 6) that were reacted with a mouse monoclonal antibody to phosphotyrosine (4G10) as previously described (13). Molecular masses in kilodaltons are indicated on the left. Part of this figure has been reproduced from a previous publication (13).