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

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.

Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin.

Infection of epithelial cells by two biovars of Chlamydia trachomatis results in the tyrosine phosphorylation of several host proteins. The most prominent change in host protein tyrosine phosphorylation involves a complex of proteins with molecular masses of 75 to 85 kDa (pp75/85) and 100 kDa (pp100). The C. trachomatis-induced tyrosine phosphorylation of pp75/85 and pp100 is observed in several cell lines, including epithelial cells, fibroblasts, and macrophages. Subcellular fractionation and detergent solubility properties of pp75/85 are consistent with its association with the cytoskeleton. Phosphoamino acid analysis demonstrates that the pp75/85 complex is phosphorylated on both tyrosine and serine residues. Immunofluorescence studies of chlamydia-infected cells by using fluorescein isothiocyanate-phalloidin and antibodies to phosphotyrosine and cortactin demonstrate that tyrosine-phosphorylated proteins, as well as cortactin, are localized to the chlamydial vacuole and that this process is facilitated by actin.

Activation of phenoxazinone synthase expression in Streptomyces lividans: characterization of the activator fragment from Streptomyces antibioticus.

We have isolated an active 719 bp fragment from the 4.3 kb region of the genome of Streptomyces antibioticus that activates a silent phenoxazinone synthase (PHS) gene in Streptomyces lividans. Sequencing of the 719 bp fragment revealed several potential open reading frames (ORFs); however, the distribution of G + C in these putative ORFs was uncharacteristic of streptomycete genes. No RNA products transcribed from the active sequence were detected by dot-blot hybridization and no proteins corresponding in size to the predicted products from the ORFs were observed when appropriate plasmids were used as templates in a streptomycete coupled transcription-translation system. Fragments of 249 and 243 bp, respectively, were obtained from the 719 bp fragment from S. antibioticus and from the S. lividans genome by PCR cloning. Both fragments activated phs in S. lividans when cloned on a high copy number plasmid.