Inhibition of onset of overt multiplication of Chlamydia psittaci in persistently infected mouse fibroblasts (L cells).

When monolayers of mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci (strain 6BC) were dispersed in medium 199 and plated out in new flasks, the monolayers that grew out consisted almost exclusively of inclusion-free host cells that retained full resistance to superinfection with C. psittaci (covert infection). After a delay that was inversely proportional to the initial density of the newly transferred L cell population, the percentage of host cells containing visible chlamydial inclusions increased rapidly (overt infection), and most of the L cells were destroyed by extensive chlamydial multiplication (wipeout), leaving only a few survivors to start new persistently infected monolayers. When persistently infected L cell populations grown in medium 199 were transferred to Eagle minimal essential medium, the onset of overt multiplication was strongly suppressed although covert multiplication of C. psittaci continued unabated, as shown by host cell retention of resistance to superinfection and the prompt resumption of overt multiplication after transfer back into medium 199. The difference(s) between the two media responsible for the different expression of the persistently infected state was not determined. A single dose of 100 U of penicillin G per ml of medium 199 given at the time persistently infected monolayers were divided almost completely suppressed the appearance of visible signs of chlamydial infection for several weeks, although resistance to superinfection was retained at all times. The same amount of penicillin given 7 days after replating did not prevent the occurrence of the first expected wipeout, but there was a long period of inclusion-free L cell growth between the first wipeout and the second. It was concluded that covert multiplication of C. psittaci in persistently infected L cells may continue indefinitely without the appearance of visible signs of infection. The transition between covert and overt chlamydial multiplication appears to be a penicillin-sensitive, multistep process that is regulated, at least in part, by the host cell density and the composition of the growth medium.     

Association between resistance to superinfection and patterns of surface protein labeling in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.

When mouse fibroblasts (L cells) were persistently infected with Chlamydia psittaci strain 6BC, they became immune to superinfection because they no longer associated with exogenous C. psittaci in a way that led to ingestion and intracellular multiplication. At the same time, the persistently infected L cells also exhibited changes in surface structure as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiographic visualization of the surface-exposed plasma membrane proteins that had been labeled with 125I by lactoperoxidase-catalyzed iodination. The most prominent changes were the appearance of a highly labeled band with an apparent molecular weight of 35,000 and the generalized reduction in intensity of labeling of proteins migrating in the apparent molecular weight range of 60,000 to 100,000. Neither resistance to superinfection nor alteration in cell surface structure depended on the presence of visible chlamydial inclusions. When L cells were cured of persistent infection, either spontaneously or by treatment with chlortetracycline or rifampin, immunity to superinfection disappeared, and the patterns of surface-labeled proteins of the cured cells once again resembled the patterns of wild L cells. It was suggested that resistance to superinfection is the result of reversible changes in the structure of the putative host cell receptor for chlamydiae that are produced in some unknown way by the persistent chlamydial infection.     

Requirements for ingestion of Chlamydia psittaci by mouse fibroblasts (L cells).

Ingestion of 14C-amino acid-labeled Chlamydia psittaci (6BC) by mouse fibroblasts (L cells) was inhibited when the host cells were incubated for 30 min at 37 degrees C in Earle salts containing 10 mug of crystalline trypsin per ml. Tryptic digestion also inhibited the ingestion of 1-mum polystrene latex beads. Trypsin-treated L cells almost completely recovered their ability to ingest chlamydiae after 4 h at 37 degrees C in medium 199 with 5% fetal calf serum. Cycloheximide (10 mug/ml) blocked this recovery. Heating 14C-amino acid-labeled C. psittaci for 3 min at 60 degrees C inhibited its ingestion by L cells, whereas inactivating it with ultraviolet light was without effect on the ingestion rate. These results show that efficient ingestion of C. psittaci by L cells involves trypsin-labile sites on the host and heat-sensitive sites on the parasite. The failure of excess unlabeled infectious C. psittaci to promote the ingestion of 14C-labeled heat-inactivated chlamydiae suggests that direct interaction between these two sites must occur for uptake to proceed normally.     

Cytochalasin B does not inhibit ingestion of Chlamydia psittaci by mouse fibroblasts (L cells) and mouse peritoneal macrophages.

Cytochalasin B did not inhibit ingestion of Chlamydia psittaci by either mouse fibroblasts (L cells) or mouse peritoneal macrophages in concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads and Escherichia coli K-12.     

Immediate toxicity of high multiplicities of Chlamydia psittaci for mouse fibroblasts (L cells).

One hour after suspensions of mouse fibroblasts (L cells) were exposed to 500 to 1,000 L-cell 50% infectious doses of Chlamydia psittaci (6BC), the L cells failed to attach to and spread out on solid substrates, phagocytosed polystyrene latex spheres at reduced rates, incorporated less [14C]isoleucine into protein, and had smaller soluble pools of nucleoside triphosphates. The infected L cells began to die at 8 h and were all dead by 20 h. Lower multiplicities of infection took correspondingly longer to kill the L cells. These effects of high multiplicities of C. psittaci on L cells will be referred to collectively as immediate toxicity. Similar effects were obtained with other strains of C. psittaci and C. trachomatis and with other cell lines. Ultraviolet-inactivated C. psittaci retained the ability to cause immediate toxicity, but heat-inactivated chlamydiae did not. C.psittaci cells had to be ingested by L cells before they were immediately toxic but, once they were phagocytosed, they did not need to multiply or to synthesize macromolecules in order to cause immediate injury to their hosts. Immediate toxicity was not the result of depression of energy metabolism, changes in the levels of intracellular cyclic nucleotides, or release of hydrolases from lysosomes. It was suggested that a lesion is produced in the plasma membrane of the L cell every time it ingests a chlamydial cell, that each act of ingestion produces an independent lesion, and that their injurious effects are additive. Thus, the more ingestion lesions there are, the faster the host cell dies. It was also suggested that induced phagocytosis, inhibition of lysosomal fusion, and death of mice and of cells in culture may all depend on a single activity of C. psittaci.     

Kinetics of phagocytosis of Chlamydia psittaci by mouse fibroblasts (L cells): separation of the attachment and ingestion stages.

The kinetics of phagocytosis of Chlamydia psittaci (6BC) by monolayers of mouse fibroblasts (L cells) was studied with an assay that distinguished between the attachment and ingestion phases of phagocytosis. At multiplicities of 10 and 100 50% infectious doses (ID50) per L cell, virtually all of the inoculated C. psittaci had been attached and ingested after 60 min at 37 degrees C. At multiplicities of 500 to 5,000 ID50 per L cell, the initial rates of attachment and ingestion of C. psittaci to L cells increased with the multiplicity of infection, but phagocytosis stopped even though many chlamydial cells remained free in suspension and readily available for attachment to the host-cell monolayers. Phagocytosis probably ceased because the L cells were injured when they took up large numbers of chlamydial cells. This injury prevented direct determination of the number of potential binding sites for C. psittaci on each L cell. However, this number is large enough to make the rates of chlamydial attachment and ingestion predominantly dependent on the multiplicity of infection.     

Attachment of Cell Walls of Chlamydia psittaci to Mouse Fibroblasts (L Cells)

¹⁴C-labeled cell walls of the 6BC strain of Chlamydia psittaci, prepared from intrinsically labeled chlamydial cells by digestion with deoxycholate and trypsin, associated with mouse fibroblasts (L cells) in a manner comparable to that of intact C. psittaci. Almost half of the host cell-associated cell walls were not dissociated by trypsin, suggesting that they had been attached and then ingested. The attachment of cell walls to L cells was inhibited by a number of treatments known to block association of intact C. psittaci with L cells: heating the cell walls for 3 min or reacting them with antiserum against intact C. psittaci, or pretreating the L cells with trypsin or wheat germ agglutinin. Unlike intact cells of C. psittaci, cell walls were not immediately toxic for L cells, and they did not measurably adsorb neutralizing antibody. As revealed by making cell walls from intact C. psittaci labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination, cell walls contained a much smaller number of surface-labeled proteins than did whole chlamydial cells. The most abundant surface-labeled protein was one with an apparent molecular weight of 43,000. In the final step of cell wall preparation, tryptic digestion of deoxycholate-extracted cells, this major surface protein was partially cleaved to a 40,000-dalton product. When the major surface protein (both the 43,000- and 40,000-dalton moieties) was electrophoretically separated from the other cell wall proteins and used to immunize a rabbit, antibodies that neutralized the infectivity of intact C. psittaci were elicited. It was concluded that cell walls retain the ability to associate with L cells in much the same way as do intact cells of C. psittaci, but, despite the simpler structure of cell walls, the element that binds C. psittaci to host cells cannot yet be identified.     

Loss of inorganic ions from host cells infected with Chlamydia psittaci

Mouse fibroblasts (L cells) infected with the 6BC strain of Chlamydia psittaci released potassium ion (K⁺) into the extracellular milieu in a way that depended on size of inoculum and time after infection. When the multiplicity of infection was 500 to 1,000 50% infectious units (ID₅₀) per L cell, loss of intracellular K⁺ was first apparent 4 to 10 h after infection and was nearly complete at 6 to 20 h. Magnesium ion and inorganic phosphate (P_i) were also released. Similar multiplicities of ultraviolet-inactivated C. psittaci also caused release of K⁺. Leakage of inorganic ions probably resulted from immediate damage to the host-cell plasma membrane during ingestion of large numbers of chlamydiae. With multiplicities of 1 to 50 ID₅₀ per L cell, ingestion of C. psittaci was not by itself enough to cause release of K⁺ and P_i from infected L cells. There was a delay of 36 to 72 h between infection and massive leakage of intracellular ions during which time the chlamydiae multiplied extensively. Fifty ID₅₀ of ultraviolet-inactivated C. psittaci per L cell did not bring about significant leakage of K⁺, even after 72 h. The mechanism whereby these multiplicities of infection destroy the ability of host cells to retain intracellular molecules is not known. HeLa 229 cells also released K⁺ and P_i after infection, but these losses occurred more slowly than in comparably infected L cells, possibly because C. psittaci did not multiply as extensively in HeLa cells as it did in L cells. The significance of the inability of chlamydiae-infected cells to regulate the flow of molecules through their plasma membranes is discussed.     

Reduced expression of surface glycoproteins in mouse fibroblasts persistently infected with human respiratory syncytial virus (HRSV)

Summary.  BCH4 cells, persistently infected with Human Respiratory Syncytial Virus (HRSV), were obtained by Fernie et al. [12] after infection of a BALB/c mouse embryo cell line with the Long strain of HRSV. To understand the basis of HRSV persistence, the expression of HRSV RNAs and proteins was evaluated in BCH4 cells and infected parental BALB/c and fully permissive HEp-2 cells. Production of viral mRNAs was severely impaired in BCH4 cells. In addition, the expression level of the surface glycoproteins F and G was markedly reduced relative to internal viral proteins. However, virus recovered from BCH4 cells could lytically infect HEp-2 cells and expressed normal levels of surface glycoproteins. No evidence of defective genomes or interfering particles was found in BCH4 cells. Taken together, these data indicate that reduction of both viral mRNA accumulation and surface glycoprotein biosynthesis are at the basis of HRSV persistence in BCH4 cells. Accepted November 3, 2000     

Interaction of Chlamydia psittaci with mouse peritoneal macrophages.

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 the lactic dehydrogenase, beginning 2 h postinfection. This immediated macrophage cytotoxicity as abolished by pretreatment of EB with heat (5 min at 56 degrees C) and was reduced about 50% by coating EB with homologous antibody. Pretreatment of the chlamydia 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.     

Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells.

Phagocytosis of the 6BC strain of Chlamydia psittaci and the lymphogranuloma venereum 440L strain of Chlamydia trachomatis by L cells and HeLa 229 cells occurred at rates and to extents that were 10 to 100 times greater than those observed for the phagocytosis of Escherichia coli and polystyrene latex spheres. Both species of Chlamydia were efficiently taken up by host cells of a type they had not previously encountered. Phagocytosis of chlamydiae was brought about by the interaction of parasite surface ligands with elements of the host cell surface. The chlamydial ligands were readily denatured by heat, were masked by antibody, and were resistant to proteases and detergents. The host cell components were reversibly removed by proteases. Chlamydial phagocytosis was inhibited when host cells were incubated for many hours with cycloheximide. It was suggested that the presence on the chlamydial cell surface of ligands with high affinity for normal, ubiquitously occurring structures on the surface of host cells is an evolutionary adaptation to intracellular existence. The term parasite-specified phagocytosis was used to describe the efficient phagocytosis of chlamydiae by nonprofessional phagocytes and to distinguish it from the host-specified immunological and non-immunological phagocytosis carried out by professional phagocytes.     

Persistent infection of L cells with an ovine abortion strain of Chlamydia psittaci.

L cells inoculated at multiplicities of infection greater than or equal to 1 inclusion-forming unit of the abortigenic chlamydial strain B577 were destroyed within 10 to 15 days. Upon continued incubation in fresh medium, a few surviving cells repopulated the flasks, and the reemerging cultures remained persistently infected. The persistent state was characterized by cycles of repopulation with a low ratio of infected cells and cycles of extensive cytopathic changes in which greater than 90% of the cells had chlamydial inclusions and which could be delayed or even terminated by penicillin treatment. Immunofluorescence and superinfection during the period of repopulation revealed that the persistently infected cells could adsorb chlamydiae but their multiplication was arrested. This nonpermissive state could be terminated by the specific action of cycloheximide. L cells spontaneously cured from a persistent infection exhibited no change in susceptibility to chlamydiae when compared with normal L cells. However, chlamydiae derived from L cells after 7.5 months of persistence destroyed L-cell monolayers more rapidly and at lower multiplicities of infection than the wild type. This state of chlamydia-host cell interaction could not be established with the arthropathogenic strain LW613 because chlamydial infectivity was lost after the first cytolytic burst of infection in the cell cultures. The persistence described for the strain B577-L-cell system appears to differ from previously described models involving other chlamydial strains.     

Isolation and characterization of phagosomes containing Chlamydia psittaci from L cells.

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.     

Persistent infection of mouse fibroblasts (McCoy cells) with a trachoma strain of Chlamydia trachomatis.

An in vitro model of persistent infection of mouse fibroblasts (McCoy cells) with a trachoma strain (G17) of Chlamydia trachomatis has been developed. Persistently infected cultures were established by infecting McCoy cells with high multiplicities of chlamydiae. After the first cycle of chlamydial replication, the host cells multiplied more rapidly than the parasites, so that the fraction of inclusion-bearing cells declined to less than 1%. However, after 100 days, the proportion of inclusion-bearing cells rose dramatically, and the cultures alternated between periods of massive host cell destruction by chlamydiae and periods of host cell proliferation. This cycle continued indefinitely as host cell and parasite densities fluctuated periodically. The chlamydiae in the cycling populations were reidentified as the original serotype. No changes in either host cell susceptibility or chlamydial invasiveness were observed in hosts and parasites recovered from persistently infected populations. All evidence suggests that the parasite maintained itself in McCoy cell populations by cell-to-cell transfer and that an equilibrium between host and parasite multiplication was achieved when the persistently infected cultures fluctuated between periods of host cell destruction and proliferation.     

Cycloheximide-Resistant Glycosylation in L Cells Infected with Chlamydia psittaci

L cells (mouse fibroblasts), uninfected and infected with the meningopneumonitis strain of Chlamydia psittaci, were labeled with [(14)C]glucosamine, and their membranous organelles were separated by isopycnic equilibrium centrifugation of whole cell homogenates on discontinuous sucrose gradients. Glycosylation of host membranes continued throughout the infection. Cycloheximide almost completely inhibited glycosylation in uninfected L cells, but it only partially inhibited the process in infected host cells. Cycloheximide-resistant glycosylation of membrane fractions with [(14)C]glucosamine increased as the infection proceeded and was probably due to the action of chlamydial enzymes. Modification of host membranes by glycosylation may play a role in the natural development of chlamydial infections.     

Effect of interferon on the growth of Chlamydia trachomatis in mouse fibroblasts (L cells).

The effect of murine interferon on the growth of the lymphogranuloma venereum biotype of Chlamydia trachomatis (strain 440L) in murine fibroblasts (L cells) was examined. Treatment of infected cell cultures with interferon caused a reduction in the number of inclusion-bearing cells as seen by light and electron microscopy and a decrease in yields of chlamydiae as determined by infectivity assays. Interferon also inhibited cycloheximide-resistant (chlamydia-specific) protein synthesis in infected cells. The interferon effect was dose dependent, with 80 to 90% inhibition occurring at concentrations of greater than 200 IU/ml. The inhibitory effect was neutralized by anti-murine interferon globulin. Interferon did not inactivate extracellular chlamydiae, and both host cell RNA and protein synthesis were required for the development of the interferon-induced antichlamydial state. Inhibition of chlamydial growth by interferon was demonstrable in cells treated 18 h before infection or up to 4 h after infection. Cells infected after interferon was removed exhibited an antichlamydial activity decline which was complete by 30 h after interferon removal. We show that interferon treatment did not affect either entry of chlamydiae into host cells or chlamydial conversion to reticulate bodies but rather caused a reduction in the rate of reticulate body replication.     

Interaction of Chlamydia psittaci reticulate bodies with mouse peritoneal macrophages.

Noninfectious reticulate bodies of Chlamydia psittaci are readily phagocytized by thioglycolate-elicited mouse peritoneal macrophages in monolayer culture. The internalized reticulate bodies are rapidly destroyed as indicated by a 60 to 70% decrease in trichloroacetic acid-precipitable radioisotopic counts in the macrophage pellet by 10 h and a concomitant increase of the trichloroacetic acid-soluble radiolabeled chlamydial nucleic acid in the cytoplasm. This intracellular destruction of reticulate bodies in macrophages is independent of the multiplicity of infection. Reticulate bodies at a high multiplicity of infection, up to 1,000:1, are also incapable of inducing immediate cytotoxicity in macrophages as evidenced by the lack of early release of the host cell-soluble cytoplasmic enzyme lactic dehydrogenase. Thus, it appears that the virulence factors for (i) initiation or maintenance of intracellular survival via circumvention of phagolysosome formation and (ii) host cell damage are either missing or not expressed by the RB form of this bacterium.     

Localization of chlamydial group Antigen in McCoy cell monolayers infected with Chlamydia trachomatis or Chlamydia psittaci.

Chlamydial inclusions were demonstrated by indirect immunofluorescence (IF) with antiserum to the chlamydial group antigen when McCoy cell monolayers infected with either Chlamydia trachomatis or Chlamydia psittaci were fixed in formaldehyde or paraformaldehyde, provided the monolayer was not allowed to dry. If these monolayers were then air dried and restained by IF with the same antiserum but with a different fluorescence conjugate, group antigen associated with inclusion-containing McCoy cells but independent of the inclusions was revealed. This antigen was not restricted to infected cells but appeared to radiate out from them, suggesting that group antigen was released from infected cells. Similar host cell-associated antigen could be shown by IF of glutaraldehyde-fixed, air-dried monolayers, but inclusions could not be stained by IF before these preparations were dried, presumably because antibody could not penetrate glutaraldehyde-fixed cells. Electron microscopic immunoperoxidase studies of paraformaldehyde-fixed, wet monolayers located group antigen within inclusions on the outer membrane of chlamydial organisms and on single-membrane vesicles. However, when dried monolayers were labeled with the same immunoperoxidase technique, no intracellular labeling occurred, but dense staining was seen at the surface of infected cells and on adjacent membranous material. These observations are compatible with the postulate that replicating chlamydiae produce outer membrane blebs containing group antigen, which are excreted by the host cells during the chlamydial developmental cycle.