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 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.

Characterization and intracellular trafficking pattern of vacuoles containing Chlamydia pneumoniae in human epithelial cells

“…More than two decades ago, Friis (1972) was the ®rst to conclude that phagosomes harbouring C. psittaci do not fuse with lysosomes within L cells. This notion was supported by several subsequent experiments performed mainly on macrophages infected with the same species (Wyrick and Brownridge, 1978;Zeichner, 1982;1983). Similarly, more recent studies on the traf®cking routes of C. psittaci or C. trachomatis in the target epithelial cells have pointed to a complete disconnection of chlamydial inclusions from the endocytic pathway and the lack of phagosomal acidi®cation (Heinzen et al, 1996;Schramm et al, 1996;Taraska et al, 1996).…”

The intracellular life of Chlamydia psittaci: how do the bacteria interact with the host cell?

“…Observations of fusion between chlamydial inclusions that contain microorganisms of the same species but not between inclusions containing di¡erent species [57] have led to the suggestion that the properties of the inclusion membrane of C. psittaci and C. trachomatis (and probably of C. pneumoniae and C. pecorum, too) are di¡erent, and that these properties are positively controlled by the organisms in the inclusion. Zeichner [117] demonstrated that the chlamydial vacuole is di¡erent from other`conventional' vacuoles since the membrane protein composition of C. psittaci-containing vacuoles appears to di¡er from the protein composition of the host cell plasma membrane. He performed polyacrylamide gel electrophoresis on chlamydial vacuoles isolated by dextran gradient centrifugation visualizing 10 major proteins instead of a whole set of the host cell (L cells) cytoplasmic membrane proteins.…”

“…Chlamydia can recognize and exploit speci¢c receptor sites on the host cytoplasmic membrane that trigger formation of a type of endosome that normally does not fuse with lysosomes (coated-pit or receptor-mediated endocytosis is not programmed for lysosomal fusion) [23,41,117].…”

“…EB envelope factor(s) might mask or enzymatically degrade recognition molecule(s) on the phagosome surface that normally marks it as an organelle with which the lysosome can fuse [24,117]. 6.…”

The intracellular life ofChlamydia psittaci: how do the bacteria interact with the host cell?

“…Observations of fusion between chlamydial inclusions that contain microorganisms of the same species but not between inclusions containing different species [57]have led to the suggestion that the properties of the inclusion membrane of C. psittaci and C. trachomatis (and probably of C. pneumoniae and C. pecorum , too) are different, and that these properties are positively controlled by the organisms in the inclusion. Zeichner [117]demonstrated that the chlamydial vacuole is different from other ‘conventional’ vacuoles since the membrane protein composition of C. psittaci ‐containing vacuoles appears to differ from the protein composition of the host cell plasma membrane. He performed polyacrylamide gel electrophoresis on chlamydial vacuoles isolated by dextran gradient centrifugation visualizing 10 major proteins instead of a whole set of the host cell (L cells) cytoplasmic membrane proteins.…”