Survival and growth of L-cell-cultivated Chlamydiapsittaci occurred in mouse macrophages in vitro. Two major factors governing the intracellular fate of chlamydiae in macrophages are: (i) the multiplicity of infection (MOI), i.e., the elementary body (EB)-to-macrophage ratio, and (ii) the state of the EB. At a low MOI (1:1), survival and growth of live, untreated chlamydiae were optimal. The chlamydiae were internalized in macrophages within 30 to 40 min. EB proceeded to differentiate into reticulate bodies, which underwent multiplication and further matured into infectious EB in the professional phagocytic cells. In contrast, at a high MOI (100:1), survival of untreated chlamydiae was greatly reduced as a result of immediate damage to the macrophages. EB that were pretreated with heat (560C for 10 to 30 min) or coated with homologous antibody were rapidly destroyed in macrophage phagolysosomes. Fusion of ferritin-labeled lysosomes with heat-treated or opsonized EB-laden phagosomes occurred in 2 to 4 h, resulting in transfer of the ferritin marker into phagolysosomes. Chlamydiae are obligate intracellular parasites. Under appropriate conditions, these patn

The entry of Chlamydia psittaci into L-929 cells was studied morphologically by transmission electron microscopy and quantitatively by a method that discriminates between attachment and uptake. Upon adsorption of 3H-labeled elementary bodies (EBs) to host cells at 4 degrees C, the EBs bound efficiently to the L-cell surface. Binding reached an equilibrium level of 55% in 3 h. Ultrastructural analysis revealed that EBs were bound preferentially to the tips and sides of microvilli at this temperature. The EBs were also observed in coated pits located at the bases of microvilli and along smooth surfaces of the host cell. No internalization was observed at 4 degrees C. When cells with prebound 3H-labeled EBs were warmed to 37 degrees C, the EBs rapidly became resistant to proteinase K removal (half time = 5 min), indicating ingested chlamydiae. At 37 degrees C, the EBs were internalized within tightly bound vesicles surrounded by an electron-dense coat of fibrillar material. EBs were also present in smooth-surfaced pits and vesicles of the host cell. Using alpha 2-macroglobulin coupled to colloidal gold (a known marker for receptor-mediated endocytosis), we observed that the entry of EBs into cells via coated pits was identical in appearance to the internalization of alpha 2-macroglobulin. Also, when the two ligands were mixed together, they could be seen within the same coated pits and were cointernalized within endocytic vesicles of the host cell. These results suggest that C. psittaci can enter nonprofessional phagocytic cells by a pathway which is similar to that of receptor-mediated endocytosis of many physiologically important macromolecules, bacterial toxins, and viruses.

L-cell-grown Chlamydia psittaci elementary bodies (EB) were rapidly phagocytized by mouse peritoneal macrophages in vitro. However, the intracellular fate of chlamydiae in macrophages appeared to be dependent on the multiplicity of infection (MOI), i.e., the EB-to-macrophage ratio, and the treatment of the EB. At an MOI of 1:1 or less, survival is maximal, and growth and multiplication of live, untreated chlamydiae did occur. In contrast, at a high MOI (100:1), survival of chlamydiae is reduced, as confirmed by release of 3H-labeled nucleic acid into the supernatant. At the high MOI, macrophage damage occurred that resulted in significant release of lactic dehydrogenase, beginning 2 h postinfection. This immediate macrophage cytotoxicity was abolished by pretreatment of EB with heat (5 min at 56°0) and was reduced about 50% by coating EB with homologous antibody. Pretreatment of the chlamydiae with heat or opsonizing antibody provides increased uptake of EB by macrophages but-may contribute to increased destruction of these obligate intracellular pathogens in professional phagocytic cells.

envelopes to initiate these events. Simultaneously with these studies, Levy and Moulder (17) published data indicating that isolated EB cell walls of C. psittaci associated with L cells in essentially the same manner as did intact whole cells. The findings reported here substantiate those of Levy and Moulder (17). In addition, EB envelopes are shown to persist in host phagosomes in the absence of P-LF. After what appears to be some type of breakdown, envelope antigens are released and emerge on the surface of the infected host cell. MATERIALS AND METHODSGrowth and purification of chlamydiae. The Cal 10 meningopneumonitis strain of Chlamydia psittaci was routinely grown in 929-L cell suspension cultures. The

Intracellular survival of Chlamydia psittaci is in part dependent on the ability of the organism to thwart phagolysosome formation. Circumvention of phagolysosome fusion could be either localized to chlamydia-laden vacuoles or generalized to all phagosomes in the host cell. To determine which of these modes is in operation the ability of chlamydia elementary and reticulate bodies to protect Saccharomyces cerevisiae from degradation in macrophage phagolysosomes was examined via acridine orange and Giemsa staining. No statistically significant difference was evident between the amount of fusion observed in coinfected macrophages and those infected with yeast cells alone. This was ot dependent on some unique interaction between the chlamydia and the yeast cells since viable count studies to determine the protection of a second organism, Escherichia coli, also failed to show significantly different amounts of inactivation of the bacteria by macrophages in the presence of C. psittaci. Therefore, the inhibition of phagolysosome fusion is localized to chlamydia-laden phagosomes.