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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Chlamydia pneumoniae is a bacterial obligate intracellular parasite with a developmental cycle common to all members of the genus Chlamydia. Like other chlamydiae, the developmental cycle of C. pneumoniae occurs entirely within a membrane-bound intracellular vacuole, termed an inclusion, that is non-fusogenic with endosomal or lysosomal compartments. To characterize the vesicular interactions of the C. pneumoniae inclusion, we used a fluorescent analogue of ceramide, {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-derythro-sphingosine (C6-NBD-Cer), that has previously been used to characterize the endogenous synthesis and transport of sphingolipids from the Golgi apparatus to Chlamydia trachomatis and Chlamydia psittaci inclusions. Sphingolipids are trafficked to C. pneumoniae inclusions in a time-, temperature- and energy-dependent manner with properties very similar to the delivery of sphingomyelin to C. trachomatis inclusions. These results indicate that interactions of the inclusion with a subset of sphingomyelin-containing exocytic vesicles is a property common to all species of chlamydiae.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Organisms of the genus Chlamydia are obligate intracellular parasites. Three species of Chlamydia are recognized as causative agents of human diseases. Chlamydia trachomatis is the causative agent of trachoma and a variety of sexually transmitted diseases. Chlamydia psittaci is a zoonotic disease that can be transmitted to humans to cause psittacosis. Chlamydia pneumoniae is a human respiratory pathogen that commonly causes such respiratory syndromes as pneumonia, bronchitis, pharyngitis and sinusitis. C. pneumoniae has been also associated with other acute or chronic diseases such as atherosclerosis, asthma, sarcoidosis, otitis media, erythema nodosum and Reiter's syndrome (Kuo et al., 1995). Members of the genus Chlamydia are characterized by a biphasic life cycle comprising functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication (Moulder, 1991). This developmental cycle takes place entirely within an intracellular vesicle, termed an inclusion, that is not acidified and does not fuse with lysosomes (Schramm and Wyrick, 1995; Heinzen et al., 1996; Taraska et al., 1996; Al-Younes et al., 1999). The chlamydial developmental cycle is initiated by endocytosis of an elementary body (EB) by a eukaryotic host cell. The EBs rapidly differentiate to reticulate bodies (RBs), which multiply within the inclusion. After a number of cell divisions, RBs differentiate back to EBs and these are released when the host cell lyses at ≈72 h after infection.

It has been demonstrated that the fluorescent vital stain for Golgi apparatus, {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-derythro-sphingosine (C6-NBD-Cer), is specifically delivered from the Golgi apparatus of the infected host cell to the C. trachomatis inclusion (Hackstadt et al., 1995). C6-NBD-Cer, like endogenous ceramide, is processed to sphingomyelin or glucosylceramide within the Golgi apparatus before transport to the plasma membrane via a vesicle-mediated process (Lipsky and Pagano, 1985). C. trachomatis (Hackstadt et al., 1996) or C. psittaci (Rockey et al., 1996) inclusions interrupt this exocytic pathway to intercept sphingomyelin-containing vesicles which fuse with the inclusion in a time-, temperature- and energy-dependent fashion. Interaction with this pathway is dependent upon chlamydial activities, as evidenced by the requirement for de novo chlamydial transcription and translation. Once fusion of these vesicles with the inclusion occurs, sphingomyelin is then rapidly incorporated into the chlamydial cell wall. Once incorporated by chlamydiae, the probe is no longer exchanged or transported intracellularly (Hackstadt et al., 1997).

To study vesicular interactions of the C. pneumoniae inclusion, C6-NBD-sphingomyelin trafficking was characterized. We found only slight differences between C. trachomatis and C. pneumoniae in the process of sphingomyelin uptake. We conclude that the transport of sphingomyelin from the Golgi apparatus to the chlamydial inclusion is a property common among chlamydiae. In addition, we have confirmed that the C. pneumoniae inclusion membrane, like the inclusion membrane of other chlamydial species (Heinzen et al., 1996; Taraska et al., 1996; van Ooij et al., 1997), does not contain markers of endosomes and lysosomes (Al-Younes et al., 1999). This indicates that chlamydiae-mediated avoidance of lysosomal fusion within an infected eukaryotic cell occurs by mechanisms common to all chlamydiae.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

C6-NBD-Cer labelling of C. pneumoniae-infected cells

To investigate the properties of C. pneumoniae acquisition of sphingomyelin from a eukaryotic host cell, C. pneumoniae-infected cells were labelled with C6-NBD-Cer and the delivery of the fluorescent analogue from the host to the parasite was followed (Fig. 1). Typically, C6-NBD-sphingomyelin and C6-NBD-glucocerebroside, synthesized endogenously from C6-NBD-Cer, are translocated to the plasma membranes of eukaryotic cells (Lipsky and Pagano, 1985; Pagano, 1990). Once incorporated into the plasma membrane, the fluorescent lipids can be extracted from the plasma membrane by incubation in the presence of an appropriate acceptor by a process referred to as back-exchange (Pagano, 1989). As previously described for C. trachomatis (Hackstadt et al., 1995), incubation with C6-NBD-Cer at 4°C resulted in diffuse fluorescent labelling of virtually all cellular membranes except the chlamydial inclusion membrane. Upon shifting the temperature to 37°C, the fluorescence rapidly redistributed first to the Golgi apparatus (Fig. 1A) and then, with continued incubation, to the intracellular chlamydiae. C. pneumoniae inclusions labelled at 40 h after infection were able to incorporate C6-NBD-sphingomyelin as soon as 15 min after back-exchange (Fig. 1B). By 1 h after temperature shift, the Golgi apparatus and the chlamydial inclusion stained with approximately equal intensity (Fig. 1D). Incubation for longer periods resulted in diminution of fluorescence from the Golgi apparatus but retention of the probe by C. pneumoniae within the inclusions.

image

Figure 1. C6-NBD-ceramide staining of C. pneumoniae-infected cells. HeLa cells were infected with C. pneumoniae and labelled at 40 h after infection with C6-NBD-ceramide. Labelled cells were visualized at different time points after back-exchange. At 0 min after back-exchange, only staining of Golgi apparatus was detected (A). By 15 min after back-exchange, faint labelling of chlamydial inclusion is visible (B). With longer incubation periods of 30 min (C) and 60 min (D), the probe is exported from the Golgi apparatus, whereas chlamydiae remain intensely fluorescent. Open arrows indicate the Golgi apparatus and filled arrows indicate the chlamydial inclusions. All fluorescent micrographs were taken with the same exposure and printed under identical conditions. Scale bar = 10 µm.

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Initiation of sphingomyelin uptake during the developmental cycle of C. pneumoniae

HeLa cells were infected with C. pneumoniae at a high multiplicity of infection (MOI ≈20) and were labelled with C6-NBD-Cer at various times after infection (Fig. 2). No fluorescent probe was observed within the intracellular chlamydiae at 0 or 2 h after infection (Fig. 2A and B). However, by 4 h after infection, chlamydiae which had incorporated C6-NBD-sphingomyelin were observed in the peri-Golgi region of the host cell (Fig. 2C). With time, increasing numbers of chlamydiae were visualized in the area of the Golgi apparatus, and by the later time points changes in size and shape of C. pneumoniae during the developmental cycle were apparent (Fig. 2D–F and G). Figure 2H depicts C6-NBD-Cer staining of C. pneumoniae-infected cells treated with chloramphenicol for 8 h immediately after infection. Chloramphenicol inhibits chlamydial protein synthesis, indicating a requirement for continued chlamydial protein synthesis to maintain fusogenicity with sphingomyelin-containing vesicles.

image

Figure 2. Initiation of sphingomyelin uptake during the developmental cycle of C. pneumoniae. At different time points after infection, infected HeLa cells were labelled with C6-NBD-ceramide and subjected to 60 min of back-exchange. C. pneumoniae did not uptake C6-NBD-sphingomyelin at 0 h (A) and 2 h (B) after infection. Incorporation of fluorescent probe by chlamydiae was observed by 4 h (C), 8 h (D), 12 h (E), 24 h (F) and 48 h (G) after infection. C. pneumoniae treated with chloramphenicol (200 µg ml−1) immediately after infection for 8 h did not incorporate C6-NBD-ceramide and only staining of Golgi apparatus of infected cells was visible. Arrows indicate the fluorescent C. pneumoniae. Scale bar = 10 µm.

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Effects of various inhibitors or treatments on sphingomyelin transport to the C. pneumoniae inclusions

Vesicle-mediated transport of endogenously synthesized sphingomyelin from the Golgi apparatus to C. trachomatis inclusions is ATP dependent and microtubule independent (Hackstadt et al., 1996). To characterize sphingomyelin transport to the C. pneumoniae inclusions, we have incubated C. pneumoniae-infected cells in the presence of various inhibitors that are known to interrupt vesicular trafficking or fusion (Fig. 3). Intracellular chlamydiae did not incorporate C6-NBD-sphingomyelin in ATP-depleted media, i.e. in the presence of 50 mM 2-deoxyglucose and 5 mM sodium azide (Fig. 3E). Monensin, a Na+/H+ ionophore known to block vesicular transport of newly synthesized sphingomyelin from the trans-Golgi to the plasma membrane (Lipsky and Pagano, 1985), caused vesiculation of the Golgi apparatus and reduced incorporation of fluorescent sphingolipid by chlamydiae (Fig. 3B). Nocodazole, an inhibitor of microtubule-mediated vesicular transport (Rogalski and Singer, 1984; Turner and Tartakoff, 1989), had no effect on C6-NBD-sphingomyelin uptake by C. pneumoniae (Fig. 3D). Brefeldin A, an inhibitor of anterograde transport from the Golgi apparatus (Misumi et al., 1986; Klausner et al., 1992), also inhibited chlamydial sphingolipid uptake (Fig. 3C).

image

Figure 3. Effects of various inhibitors or treatments on sphingomyelin transport to the C. pneumoniae inclusions. HeLa cells infected with C. pneumoniae were at 40 h after infection incubated with various inhibitors for 90 min, and were then labelled with C6-NBD-ceramide. All micrographs were taken at the same exposure and printed under identical conditions. Control cells were treated with 1 µl ml−1 ethanol (A), 10 µM monensin (B), 1 µg ml−1 brefeldin A (C), 10 µg ml−1 nocodazole (D), 50 mM 2-deoxyglucose and 5 mM NaN3 (E). A corresponding Nomarski image of infected cells in ATP-depleted cells is shown (F). Arrows indicate C. pneumoniae inclusions. Scale bar = 10 µm.

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Characterization of C. pneumoniae inclusions

Fluorescein isothiocyanate (FITC)-dextran, a fluid phase marker, is sequestered in early and late endosomes and lysosomes (Swanson, 1989). Fluid phase markers, including FITC-dextran, are excluded from C. trachomatis inclusions but were found to be delivered to Coxiella burnetii vacuoles in parallel experiments (Heinzen et al., 1996). HeLa cells infected with C. pneumoniae AR-39 were loaded for 12 h with FITC-dextran at 48 h after infection. FITC-dextran accumulation was detected in presumably endosomal and lysosomal compartments but not in chlamydial inclusions (Fig. 4).

image

Figure 4. Fluorescence localization of the endocytic tracer FITC-dextran in HeLa cells infected with Chlamydia pneumoniae. At 48 h after infection, infected cells were incubated for 12 h in the presence FITC-dextran. Fluorescent (A) and corresponding (B) Nomarski images of C. pneumoniae inclusions (indicated by arrows). Scale bar = 10 µm.

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To characterize the vacuolar membrane of C. pneumoniae inclusions, monoclonal antibody against human lysosomal glycoprotein (LAMP-1) and against mannose-6-phosphate receptor (CI-M6PR) were used in indirect immunofluorescence of infected HeLa cells (Fig. 5). CI-M6PR is an integral membrane protein that sorts lysosomal enzymes containing terminal mannose-6-phosphate moieties from the trans-Golgi network to late endosomal–prelysosomal compartments (Kornfeld and Mellman, 1989) and is considered to be a marker for late endosomes. LAMP-1 is a lysosomal glycoprotein observed in late endosomal and lysosomal compartments (Mane et al., 1989). Neither CI-M6PR (Fig. 5A) nor LAMP-1 (Fig. 5C) was detected on the inclusion membrane encompassing C. pneumoniae. These results confirm that chlamydial inclusions do not fuse with endosomes or lysosomes of infected host cells.

image

Figure 5. Immunofluorescence localization of CI-M6PR and LAMP-1. HeLa cells were infected with C. pneumoniae for 72 h and then fixed and stained by indirect immunofluorescence with anti-CI-M6PR (A) and anti-LAMP-1 (C) monoclonal antibody. Corresponding Nomarski images are shown (B and D). Arrows indicate localization of C. pneumoniae inclusions. Scale bar = 10 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Intracellular parasites have evolved diverse mechanisms to enhance their survival and replication within eukaryotic cells (Moulder, 1985; Finlay and Cossart, 1997; Sinai and Joiner, 1997). The inability to detect endosomal and lysosomal markers in the chlamydial inclusion membrane demonstrates that the inclusion is non-fusogenic with endosomes or lysosomes (Schramm and Wyrick, 1995; Heinzen et al., 1996; Taraska et al., 1996; Al-Younes et al., 1999). Instead, through processes that require the active participation of the chlamydiae (Scidmore et al., 1996), chlamydiae enter into a novel pathway characterized by fusion with sphingomyelin-containing vesicles. Interaction with this exocytic pathway has been proposed as a potential mechanism enabling chlamydiae to escape from lysosomal fusion (Hackstadt et al., 1997). These processes occur early in the developmental cycle and are necessary for successful replication of chlamydiae within a host cell. In this work, we demonstrate the ability of C. pneumoniae inclusions to incorporate sphingomyelin from their host. This suggests that a functional interaction between the Golgi apparatus and the inclusion is common to all members of the genus Chlamydia.

Ceramide is an amino alcohol produced in the endoplasmic reticulum and delivered to the Golgi apparatus, where it serves as the precursor for the synthesis of all cellular sphingolipids. Oligosaccharide chains are added to form glycosphingolipids, or phosphocholine head groups may be transferred from phosphatidylcholine to form sphingomyelin. Sphingomyelin-containing vesicles are exported from the trans-Golgi network and are delivered to the plasma membrane of a eukaryotic cell (Alberts et al., 1994).

To assess the properties of trafficking between the C. pneumoniae inclusion and the Golgi apparatus, the effect of various inhibitors of vesicular transport on uptake of sphingomyelin by C. pneumoniae was examined. As was described for C. trachomatis, vesicle-mediated transport of sphingomyelin from the Golgi apparatus to C. pneumoniae inclusions was ATP dependent and microtubule independent. Derivatives of C6-NBD-ceramide are trafficked to the C. pneumoniae AR-39 inclusions by processes similar to those described in C. trachomatis and C. psittaci GPIC (guinea pig inclusion conjunctivitis)-infected cells. C. pneumoniae undergoes a protracted developmental cycle in comparison with LGV (lymphogranuloma venereum) strains of C. trachomatis (Wolf et al., 2000). Relative to the L2 serovar of C. trachomatis, C. pneumoniae also seems to require more time to complete the initial cycle of differentiation from an EB to RB. A slower initiation of transcription or translation probably accounts for the initial observation of sphingomyelin incorporation by C. pneumoniae at 4 h after infection rather than at 2 h after infection, as was observed for C. trachomatis (Hackstadt et al., 1995). Even after interactions with this exocytic pathway are established, different kinetics in sphingomyelin delivery between C. trachomatis and C. pneumoniae are evidenced. The fluorescent probe was detected in C. pneumoniae inclusions as early as 15 min after back-extraction, whereas in C. trachomatis inclusions for at least 30 min were required (Hackstadt et al., 1995).

The chlamydial inclusion has been described as an aberrant Golgi-derived vesicle situated such that it receives host-derived sphingolipids from an exocytic pathway (Hackstadt et al., 1997). Sphingomyelin is at least transiently incorporated into the chlamydial inclusion membrane, from which it is incorporated by the chlamydiae. Once incorporated, the fluorescent lipids are apparently not exchanged from the host cell through recycling or export pathways (Hackstadt et al., 1995). However, it is not known how chlamydiae establish themselves in an intracellular site that intersects an exocytic pathway. Although chlamydial protein synthesis is required to establish and maintain interactions with this pathway, the chlamydial products required remain unknown. Vesicle fusion events are necessarily highly regulated and are specific processes in eukaryotic cells that are controlled though the activities of a number of integral membrane and soluble proteins. These include the v-SNARES, t-SNARES, SNAP and NSF (Rothman and Wieland, 1996) as well as members of the Rab family of small GTPases (Novick and Zerial, 1997; Schimmoller et al., 1998). A likely site for controlling the vesicular interactions of the chlamydial inclusion would be modification of the cytoplasmic face of the inclusion membrane (Hackstadt et al., 1997). The chlamydial genome (Stephens et al., 1998) has provided few clues to the proteins involved as homologues to eukaryotic proteins regulating vesicle fusion have not been detected. A class of chlamydial proteins that has attracted considerable interest are the inclusion membrane (or Inc) proteins (Rockey et al., 1995; Bannantine et al., 1998; Scidmore-Carlson et al., 1999). The chlamydial inclusion membrane proteins are a rapidly expanding group of what are believed to be integral membrane proteins. The functions of these are, for the most part, unknown. Surprisingly, there is relatively little similarity between orthologous Inc genes from the different chlamydial species (Stephens et al., 1998; Kalman et al., 1999; Read et al., 2000) considering that they would be expected to exert a common function. The only other known property of the products controlling interactions of the chlamydial inclusion is that they are functional by 2–4 h after infection for C. trachomatis (Hackstadt et al., 1996) and C. pneumoniae. Clearly, modification of the nascent inclusion membrane to create an environment suitable for chlamydial replication is one of the major initial activities of germinating EBs (Shaw et al., 2000), but identification of the protein or proteins conferring vesicular fusion remains one of the significant challenges of chlamydial research.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Cell culture and organisms

C. pneumoniae AR-39, purchased from American Type Culture Collection, were propagated in HeLa 229 cells (CCL 2.1; American Type Culture Collection). Confluent monolayers of HeLa 229 cells in 24-well plates, containing 12 mm glass coverslips, were infected with C. pneumoniae at a multiplicity of infection of ≈0.5 by centrifugation at 900 g for 1 h (Kuo and Grayston, 1988). Infected cells were incubated in RPMI-1640 medium (Gibco) supplemented with 10% FCS plus 10 µg of gentamicin and 0.9 µg ml−1 of cycloheximide at 37°C in an atmosphere of 5% CO2/95% humidified air.

{N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-derythro-sphingosine (C6-NBD-Cer) labelling

Fluorescent C6-NBD-Cer (Molecular Probes) was complexed with 0.034% defatted bovine serum albumin (dfBSA) in modified Eagle's medium (MEM), as described previously (Pagano and Martin, 1988), to yield complexes ≈5 µM in both dfBSA and C6-NBD-Cer. C. pneumoniae-infected HeLa cells were incubated with the dfBSA/NBD-Cer complex at 4°C for 30 min, washed with 10 mM HEPES-buffered calcium- and magnesium-free Puck's saline, pH 7.4 (HCMF), and incubated for various times in MEM/0.34% dfBSA to ‘back-exchange’ excess probe from plasma membrane. Cultures on coverslips were rinsed in HCMF solution before mounting for fluorescent microscopy (Hackstadt et al., 1995).

Immunofluorescence staining

Monoclonal antibody against human LAMP-1 (H4A3) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa, and monoclonal antibody 2G11 against bovine cation-independent mannose-6-phosphate receptor was generously provided by Suzanne Pfeffer, Department of Biochemistry, Stanford University, Stanford, CA, USA. C. pneumoniae-infected cells at 72 h after infection were fixed for 20 min with 4% paraformaldehyde in PBS and permeabilized for 10 min with 0.1% Triton X-100/0.05% SDS. After incubation with primary antibody, cultures were stained with secondary FITC/goat anti-mouse antibody (Zymed). Coverslips were then mounted onto glass slides using Vectashield (Vector Laboratories) mounting medium. Infected HeLa cells, 48 h after infection, were incubated with the fluid phase endocytic marker FITC-dextran (1 mg ml−1) (molecular weight 70 000 Da, Sigma Chemical) in Hanks' balanced salt solution (HBSS) for 12 h. Before viewing by fluorescence microscopy, the cultures were washed four times with HBSS.

ATP depletion and inhibitors

C. pneumoniae-infected HeLa cells at 40 h after infection were pretreated with inhibitors for 90 min, the cultures were then rinsed with MEM and were labelled with C6-NBD-Cer for 10 min at 37°C in the presence of inhibitor, rinsed with MEM and back-exchanged for 1 h in MEM plus 0.34% dfBSA in the presence of inhibitor. Inhibitors and the concentrations were: monensin, 10 µM; nocodazole, 10 µg ml−1; and brefeldin A, 1 µg ml−1. For the ATP depletion, control and C. pneumoniae-infected cells were rinsed with glucose-free MEM and incubated at 37°C in the presence of 50 mM 2-deoxy-d-glucose and 5 mM NaN3 for 90 min. All subsequent washings and incubations were carried out in glucose-free MEM with 50 mM 2-deoxy-d-glucose and 5 mM NaN3. Determination of ATP levels in a parallel set of cultures indicated that the ATP concentration of treated cells was reduced to 21% of the untreated controls.

Microscopy

Fluorescent and Nomarski differential interference contrast micrographs were taken on a Nikon FXA photomicroscope using a 60× Planapochromat objective. Photomicrographs were obtained using T-Max ASA 400 film (Kodak). All photographs within a particular experiment were taken at a fixed exposure and processed identically.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Drs H. Caldwell, M. Scidmore, R. Carabeo, E. Shaw and K. Fields for critical review of the manuscript.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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