Isolation and characterization of phagosomes containing Chlamydia psittaci from L cells

Abstract

The obligate intracellular procaryote Chlamydia psittaci enters host cells by a mechanism similar to, but distinct from, conventional phagocytosis. To better understand chlamydial uptake, L-cell phagosomes containing a single chlamydial cell were isolated and studied. Two rounds of dextran rate-zonal gradient centrifugation of L cells homogenized 1 h after infection with C. psittaci yielded phagosomes relatively free of other membranous structures. In double-label experiments, the phagosomes were enriched over a 40-fold for radioactivity derived from chlamydiae as compared with the initial homogenate. Several lines of evidence showed that the structures isolated on dextran gradients were chlamydial phagosomes. These structures and free chlamydiae banded at different positions on discontinuous sucrose gradients. The difference was destroyed by the nonionic detergent Nonidet P-40, which disrupts plasma membranes but has no effect on C. psittaci. Material labeled on the surface of the L-cell plasma membrane cosedimented with the phagosome fractions. Electron microscopy of these fractions revealed structures having the appearance of a chlamydial elementary body surrounded by a unit membrane. Sodium dodecyl sulfate-polyacrylamide gels of the phagosome membranes displayed 10 major protein bands, less than the total number of surface-labeled proteins in the L-cell plasma membrane. Seven of the proteins of phagosome membranes had electrophoretic mobilities corresponding to those of proteins exposed on the surface of L cells. Two of them were cleaved by both trypsin and chymotrypsin, enzymes that decrease the susceptibility of L cells to infection with C. psittaci. These proteins may therefore be involved in the attachment and ingestion of C. psittaci by L cells.