pathway to acquire endogenously synthesized - Europe PMC

The EMBO Journal vol.15 no.5 pp.964-977, 1996

Chiamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane Ted Hackstadt', Daniel D.Rockey, Robert A.Heinzen and Marci A.Scidmore Host-Parasitc Inlterctiolns Section. 1..Lhor"itory ot IntnraCCHlalrk Parasites, Rockv MouniL1i i I-lhoraitoric,s NIAID. N il]. Ilamil Itoni. MT 59840. USA

'Corresponding au.th10or

Chlamydia trachomatis acquires C6-NBD-sphingomyelin endogenously synthesized from C6-NBD-ceramide and transported to the vesicle (inclusion) in which they multiply. Here we explore the mechanisms of this unusual trafficking and further characterize the association of the chlamydial inclusion with the Golgi apparatus. Endocytosed chlamydiae are trafficked to the Golgi region and begin to acquire sphingolipids from the host within a few hours following infection. The transport of NBD-sphingolipid to the inclusion is energy- and temperature-dependant with the characteristics of an active, vesicle-mediated process. Photooxidation of C5;-DMB-ceramide, in the presence of diaminobenzidine, identified DMB-lipids in vesicles in the process of fusing to the chlamydial inclusion membrane. C6-NBD-sphingomyelin incorporated into the plasma membrane is not trafficked to the inclusion to a significant degree, suggesting the pathway for sphingomyelin trafficking is direct from the Golgi apparatus to the chlamydial inclusion. Lectins and antibody probes for Golgi-specific glycoproteins demonstrate the close association of the chlamydial inclusion with the Golgi apparatus but do not detect these markers in the inclusion membrane. Collectively, the data are consistent with a model in which C.trachomatis inhabits a unique vesicle which interrupts an exocytic pathway to intercept host sphingolipids in transit from the Golgi apparatus to the plasma membrane.

Kevwords: Cilata Ylia trac(homtoti.s/exocytosis/Golgi apparatus/sphingolipids

Introduction Intracellular parasites have evolved means to avoid host defense mechanisms that involve adaptations for survival in distinct intracellular compartments (Moulder, 1985; Falkow et al.. 1992). Such parasites avoid lysosomal killing by one of three general mechanisms. Some, such as Rickettsiai, Shigellai or Listeria, escape from endocytic vesicles and replicate f-eely within the cytoplasm. Others, like Coxiellai buirtietii or Leishlmlallial, have adapted to survival within the harsh environment of the lysosome and even require the acidic conditions within that compartment to support trowth (Hackstadt and Williams, 1981; Mukkada et (al., 1985). There are a number of intracellular

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parasites, however, which inhabit vesicles that do not fuse with lysosomes and remain within these parasitophorous vacuoles for the duration of their intracellular replication cycle. Examples of organisms which replicate within non-lysosomal vesicles include Chlamvdia, Mvcobacteria, Legionella, Toxoplca.smla and Sailmoniella. For the most part, the biogenesis of these vacuoles is poorly understood. It is becoming increasingly clear, however, that the intracellular parasites which multiply within vacuoles may modify those vesicles to arrest their maturation at discrete stages of the endocytic pathway (Small et al., 1994). For example, MYcobacterium av,ium-containing vacuoles contain the lysosomal glycoproteins 1 and 2, but exclude the vacuolar-type, H+-ATPase (Sturgill-Koszycki et al., 1994). Vacuoles containing other intracellular parasites, such as Legioniella pneunmophil1a, do not display the LAMP proteins or other markers of the endocytic pathway (Clemens and Horwitz, 1992). Thus the vacuoles inhabited by intracellular parasites may form more of a continuum along the endocytic/lysosomal pathway than had been appreciated in the past. ChlamYdia traichomatits is the causative agent of several significant human diseases including trachoma, the leading cause of infectious blindness worldwide. Chlamydial infections are also the most common form of sexually transmitted disease in the US and developed countries

(Schachter, 1988). Chlamydiae are obligate intracellular bacteria with a biphasic life cycle that is characterized by cell types adapted for extracellular survival and intracellular multiplication (Moulder, 1991). Infection is initiated by a small, metabolically dormant cell type called the elementary body (EB). Immediately after endocytosis, EBs are found within tightly associated membrane vesicles. Within a few hours, EBs differentiate into larger, metabolically active cell types called reticulate bodies (RBs). As the chlamydiae multiply, the vesicles (termed inclusions) expand to accommodate an increasing number of bacteria. The vesicle occupied by this important human pathogen has remained particularly enigmatic. Of several probes of the endocytic pathway, none have been localized to the chlamydial inclusion membrane (Heinzen et cal., 1996) and until recently, no vesicles except those containing other chlamydiae (Ridderhof and Barnes, 1989) were known to fuse with the chlamydial inclusion. We have used a fluorescent analog of ceramide N-[7-(4-nitrobenzo-2-oxa- 1 ,3-diazole)]-6-aminocaproyll)-ervthro-sphingosine (C(6-NBD-Cer) to study trafficking of Golgi-derived lipids in cells infected with C.trachomatis (Hackstadt et al., 1995). This fluorescent probe provides a vital stain for the Golgi apparatus that has been used extensively in conjunction with either fluorescence or electron microscopy in the study of lipid trafficking in viable cells (Lipsky and Pagano, 1985a; Pagano et al., 1989; Rosenwald and Pagano, 1993). C6-NBD-Cer, like

Chiamydial interruption of sphingolipid export

endogenous ceramide, is processed to sphingomyelin or glucosylceramide within the Golgi apparatus prior to transport to the plasma membrane via a vesicle-mediated process (Lipsky and Pagano, 1985b). In cells infected with C.trachomatis, the trafficking of Golgi-derived lipid is interrupted and a significant proportion of the sphingomyelin endogenously synthesized from C6-NBD-Cer is instead directed to the chlamydial inclusion where it is subsequently incorporated into the cell wall of the intracellular bacteria. Once incorporated by chlamydiae, the probe is no longer exchanged or transported intracellularly (Hackstadt et al., 1995). Based upon these observations, we proposed a direct communication between the Golgi complex and the chlamydial inclusion in which the chlamydiae receive host-derived lipid for incorporation into their cell wall. This model hypothesizes that chlamydiae interrupt an exocytic pathway and suggests a potential mechanism for the growth and maintenance of the inclusion membrane through the incorporation of Golgi-derived lipid.

Results Transport of internalized EBs to the Golgi region and initiation of ceramide uptake Chlamydia trachomatis acquires C6-NBD-sphingomyelin endogenously synthesized from C6-NBD-ceramide and transported to the chlamydial inclusion (Hackstadt et al., 1995). C.trachomatis-infected HeLa cells were labeled with C6-NBD-cer at various times post-infection to determine: (i) how rapidly internalized chlamydiae were transported to the Golgi region, and (ii) how soon delivery of NBD-lipid to the intracellular chlamydiae began. Infection was initiated at a relatively high multiplicity of infection (MOI = 50). By 1 h post-infection (the earliest time point that could be taken), a few intracellular chlamydiae containing the fluorescent probe were detected in the region of the Golgi apparatus and elsewhere in the cytoplasm (Figure 1). With time, increasing numbers of chlamydiae were localized in the area of the Golgi apparatus. By 4 h post-infection, virtually all of the internalized chlamydiae were in close association with the Golgi apparatus and many were beginning to increase in diameter, signaling the beginning of differentiation of the input EBs to RBs. Chlamydiae therefore begin to acquire host-derived lipids rapidly after internalization as evidenced by distinct fluorescent labeling of their cell walls. This acquisition of host sphingolipid occurs before morphologically apparent differentiation of EBs to RBs.

Effect of various inhibitors and treatments on transport to the chiamydial inclusion C6-NBD-ceramide is concentrated in the Golgi apparatus even in fixed cells by what likely represents passive uptake and preferential intercalation into membranes of appropriate composition and physical properties (Pagano et al., 1989). We wished to determine whether C6-NBDceramide was incorporated by intravacuolar chlamydiae in glutaraldehyde fixed cells. As expected, the Golgi apparatus fluoresced brightly, however, the incorporation of C6-NBD-ceramide (or exogenously added NBD-sphingomyelin, not shown) by intracellular chlamydiae in glutaraldehyde-fixed cells was negligible (Figure 2). The

-

-

Fig. 1. Transport of endocytosed EBs to the Golgi region and incorporation of fluorescent sphingolipids during early stages of infection. At various times post-infection, cells were labeled with C6-NBD-cer and subjected to 1 h of back-exchange. Cells pulsed with C6-NBD-cer at 1 h (A), 2 h (B), 4 h (C), 8 h (D), 12 h (E) and 24 h (F) post-infection. The EBs appear as bright punctate spheres at the early time points (A, B and C). By 8 and 12 h, the EBs have begun to differentiate to RBs and are larger with a distinct rim-like staining pattern. By 24 h post-infection, the inclusion is filled with EBs which move rapidly by Brownian motion giving rise to a diffuse staining pattern in the micrograph. Some RBs remain within the inclusion. The Golgi apparatus is visible at the upper left of the inclusion in the center of (F). Bar = 10 gm.

preferential incorporation of fluorescent ceramide by the Golgi apparatus in glutaraldehyde-fixed cells is in contrast to the situation in viable cells in which the intracellular chlamydiae readily acquire the fluorescent probe. C6NBD-ceramide was incorporated by glutaraldehyde-fixed EBs in vitro (not shown), thus cross-linking of the bacterial cell wall did not interfere with insertion of the lipid probe. These results indicate that the inclusion membrane does not permit passive entry of C6-NBD-ceramide and imply an active process on the part of the infected cell. To characterize more fully the cellular processes involved in the translocation of endogenously synthesized sphingomyelin to the chlamydial inclusion, we examined a number of conditions and inhibitors known to interrupt vesicular trafficking or fusion (Figure 3). Depletion of cellular ATP pools by incubation in the presence of 50 mM 2-deoxyglucose + 5 mM sodium azide resulted in greatly reduced incorporation of fluorescent sphingolipid by the intracellular chlamydiae. This effect is apparent both in the absolute level of fluorescence of RBs as compared with the control, uninhibited cells and in the relative fluorescence of the Golgi apparatus to the chlamydial inclusion. Monensin, a Na+/H+ ionophore which blocks vesicular transport of newly synthesized sphingomyelin

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Fig. 2. Confocal micrographs of viable (A) and glutaraldehyde-fixed (B) HeLa 229 cells infected with C.trachomatis for 18 h before fixation and labeling with C6-NBD-ceramide. Both viable and fixed cells were back-exchanged for I h prior to microscopy. Arrows indicate chlamydial inclusions. Both images were taken with identical parameters on the confocal microscope and the images processed simultaneously in Adobe Photoshop. Bar = 5 gm.

from the trans-Golgi to the plasma membrane (Lipsky and Pagano, 1985b), caused a dramatic vesiculation of the Golgi apparatus, which remained intensely fluorescent, but only a moderate reduction in the absolute fluorescence of the chlamydiae. Although the morphology of the Golgi apparatus was dramatically altered, the resultant vesicles remained in a peri-nuclear location and clustered around the chlamydial inclusion. Nocodazole, an inhibitor of microtubule-mediated vesicular transport (Rogalski and Singer, 1984; Turner and Tartakoff, 1989), dispersed the Golgi apparatus but had a minimal effect on the incorporation of fluorescent sphingolipid by the chlamydiae. Brefeldin A, as shown previously (Hackstadt et al., 1995), dispersed the Golgi apparatus and inhibited chlamydial sphingolipid uptake. Incubation of C.trachomatisinfected cells at 20°C to block vesicle fusion resulted in essentially normal fluorescent labeling of the Golgi apparatus but a marked reduction in the labeling of the intracellular chlamydiae. Together, these results provide further support for a model of microtubule-independent vesicle mediated transport of endogenously synthesized sphingomyelin from the trans-Golgi network to the chlamydial inclusion. Plasma membrane sphingomyelin is not trafficked to the chiamydial inclusion C6-NBD-ceramide is believed to be metabolized and transported by cells identically to endogenous processes. Ceramide, which is synthesized in the endoplasmic reticulum, is enzymatically converted to sphingomyelin within the Golgi apparatus by the addition of phosphorylcholine (Futerman et al., 1990). Newly synthesized sphingomyelin is then transported from the trans-Golgi to the plasma membrane by a vesicle-mediated process (Pagano, 1990). Plasma membrane sphingomyelin may be converted back

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to ceramide, which is recycled to the Golgi apparatus, by the action of sphingomyelinases either at the plasma membrane or in lysosomes (Koval and Pagano, 1989, 1991). To address the possibility of indirect trafficking of sphingomyelin from the plasma membrane to the chlamydial inclusion versus direct trafficking of sphingomyelin from the Golgi apparatus, we analyzed the fate of C6-NBD-sphingomyelin incorporated into the plasma membrane. C6-NBD-sphingomyelin was inserted into the plasma membrane at 4°C as described. The fluorescent intensity of either C.trachomatis-infected or uninfected HeLa cells labeled with C6-NBD-sphingomyelin was comparable with that achieved using C6-NBD-ceramide as the probe (Figure 4). Upon shifting the temperature to 37°C by the addition of pre-warmed MEM + 0.34% dfBSA to back-exchange fluorescent lipids from the plasma membrane, there was a rapid loss of fluorescence from cells labeled with C6-NBD-sphingomyelin but a more gradual decline in fluorescence from cells loaded with C6-NBDceramide. Presumably, this reflects the characteristic metabolism of ceramide to sphingomyelin and transport to the plasma membrane from which it is extracted into the back-exchange medium. This result was confirmed microscopically (Figure 5). C6-NBD-ceramide was rapidly concentrated in the Golgi apparatus and with continued incubation, eventually in the cell walls of the intracellular chlamydiae within the inclusion. In contrast, C6-NBDsphingomyelin was readily observed in the plasma membrane after loading at 4°C but was virtually undetectable after 5 min of back-exchange at 37°C. Therefore, C6NBD-sphingomyelin was not internalized or trafficked to the chlamydial inclusion to an appreciable degree under the conditions in which these experiments were performed. To evaluate more rigorously the potential for trafficking


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Fig. 3. Fluorescent micrographs of the effects of various treatments and inhibitors on fluorescent sphingolipid acquisition by C.trachomatis at 18 h post-infection. HeLa 229 cells infected with C.trachomatis, L2, for 18 h were pre-incubated in the presence of inhibitor or under the relevant conditions for 90 min prior to labeling with C6-NBD-ceramide as described in Materials and methods. One hour of back-exchange with MEM plus 0.34% dfBSA in the presence or absence of inhibitor was allowed before photography. All micrographs were taken at the same exposure and printed under identical conditions. Control cells treated with 1 Rl/ml EtOH (A), 50 mM 2-deoxyglucose and 5 mM NaN3 (B), 10 1.M monensin (C), 10 ,ug/ml nocodazole (D), incubated at 18°C (E) and 1 tg/ml Brefeldin A. Additional control cells incubated without EtOH or with 1 gl/ml DMSO were indistinguishable from those incubated with EtOH. Arrows indicate the position of the inclusions. Bar = 10 gm.

of plasma membrane sphingomyelin to the chlamydial inclusion, the experiment was repeated with the omission of the dfBSA as a lipid acceptor (Figure 6). In the absence of back-exchange, C6-NBD-sphingomyelin remained detectable in the plasma membrane over the 2 h duration of the experiment. However, the intracellular chlamydiae were unlabeled. Thus even by biasing the experiment to favor potential trafficking of sphingomyelin from the plasma membrane to the inclusion we could not document significant acquisition of the probe from the plasma membrane. We conclude from these experiments that the primary route for trafficking of endogenously synthesized sphingomyelin to the chlamydial inclusion is direct from the Golgi apparatus and not an indirect trafficking via the plasma membrane. Such a proposal is consistent with the lack of trafficking of fluid phase markers or markers

of the endosomal pathway to the chlamydial inclusion (Heinzen et al., 1996).

Fusion of Golgi-derived vesicles with the

chiamydial inclusion

Photo-oxidation of C5-DMB-ceramide- or C6-NBDceramide-labeled cells in the presence of DAB results in the deposition of an electron-dense reaction product in the Golgi apparatus and adjacent small vesicles (Pagano et al., 1989, 1991; Takizawa et al., 1993). In an effort to visualize any interaction of Golgi-derived vesicles with the chlamydial inclusion we labeled C.trachomatis with C5-DMB-ceramide and allowed a sufficient period of time, 1 h, for the probe to be transported to the chlamydial inclusion. The cultures were then fixed and irradiated in the presence of DAB. Visible reaction product was observed 967

T.Hackstadt et al.

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TIME (min) Fig. 4. Retention of fluorescent sphingolipids by mock- or C.trachomatis-infected HeLa cells. Fluorescent lipids were back-exchanged from the cell surface by incubation in MEM plus BSA as described. Retained fluorescent lipid was quantified by photometry and is expressed in lux.

surrounding the chlamydial inclusions. No reaction product visible in surrounding areas of the coverslip which had not been irradiated. At an ultrastructural level, the osmiophilic DAB reaction product was detected in the Golgi apparatus, adjacent small vesicles, and the cell wall of the chlamydiae within the inclusion (Figure 7). These results were in agreement with observations using fluorescence microscopy. In addition, reaction product was visualized in regions of the inclusion membrane itself. Previous attempts to identify C6-NBD-ceramide or its metabolites in the inclusion membrane by either conventional or laser-scanning confocal microscopy had been unsuccessful. This was presumed to be due to its rapid incorporation by chlamydial developmental forms (Hackstadt et al., 1995). At the level of electron microscopy, reaction product was clearly present in the inclusion membrane but was localized to distinct areas of that membrane (Figure 7B) and small vesicles could occasionally be observed apparently in the process of fusing with the chlamydial inclusion membrane (Figure 7C). The points of contact of RBs with the inclusion membrane did not appear to be favored sites for fusion with the Golgi derived vesicles. was

Effect of Brefeldin A on chiamydial replication Brefeldin A, an inhibitor of anterograde vesicular traffic from the Golgi apparatus (Misumi et al., 1986; Klausner et al., 1992), inhibits transport of the fluorescent probe to the inclusion (Hackstadt et al., 1995). We therefore examined the effect of Brefeldin A on chlamydial replication to determine if interference with this pathway was detrimental to chlamydial growth. Chlamydial replication was not inhibited by the presence of Brefeldin A even at concentrations of 10 ,ug/ml (Table I). Brefeldin A was added to the cultures 2 h prior to infection, and the cells kept in the continuous presence of Brefeldin A by readdition of the drug at 0, 12 and 24 h post-infection. 968

Table I. Effect of Brefeldin A on C.trachomatis replication Brefeldin A

0 0.1 1 10

(gg/ml)

IFU/ml (x 105) 6.8 6.7 8.5 8.6 +

2.9 4.9 4.2 3.1

Brefeldin A was added at 2 h prior to infection and again at 0, 12 and 24 h post-infection. At 36 h post-infection cells were lysed and plated for IFUs.

When the cultures were lysed for determination of infectious progeny at 36 h post-infection, a parallel set of cultures was probed with NBD-ceramide to confirm disruption of the Golgi. All concentrations of Brefeldin A tested: 0.1, 1 and 10 ,ug/ml, caused typical disruption of the morphology of the Golgi apparatus (not shown). Although Brefeldin A inhibits the rate of transfer of fluorescent probe to the chlamydial inclusion (Hackstadt et al., 1995) it does not significantly interfere with chlamydial multi-

plication. The only effect of Brefeldin A on chlamydial development was an apparent effect on the morphology of the inclusion. Inclusions in the presence of Brefeldin A, at any of the concentrations tested, were smaller in size and appeared more densely packed with chlamydial developmental forms (Figure 8).

Incorporation of exogenous phospholipid probes into the inclusion membrane Uninfected and C.trachomatis-infected HeLa cells were incubated with other fluorescent lipid analogs to determine if exogenously added lipids could be transported and incorporated into the chlamydial inclusion membrane. NBD-phosphatidic acid (NBD-PA), NBD-phosphatidylcholine (NBD-PC), NBD-phosphotidylethanolamine (NBD-


0

60 min

Cer

Sph

Fig. 5. Rapid extraction of NBD-sphingomyelin from the plasma membrane by back-exchange medium (MEM plus 0.34% dfBSA) prevents delivery of fluorescent sphingomyelin to the chlamydial inclusion. HeLa 229 cells infected with C.trachomnatis, L2, for 18 h were loaded with C6-NBDceramide (A, B and C) or NBD-sphingomyelin (D, E and F) and incubated in MEM plus 0.34% dfBSA for 0 min (A and D), 5 min (B and E) or 60 min (C and F). Arrowheads indicate the approximate position of the chlamydial inclusions (identified where necessary by Nomarski differential contrast microscopy). All micrographs were taken at the same exposure and printed under identical conditions. Bar = 10 gim.

PE) and NBD-phosphatidylserine (NBD-PS) were incorporated into several intracellular membranes including the Golgi apparatus, plasma membrane, nuclear envelope, mitochondrial membranes and endoplasmic reticulum (Figure 9). In addition, each of these phospholipid probes appeared to be present in the inclusion membrane as assessed by confocal microscopy but, in contrast to NBD-ceramide, none were detected in the cell walls of C.trachomatis. Brefeldin A treatment did not inhibit insertion of these phospholipid probes into the inclusion membrane (not shown) thus we cannot ascribe the presence of these lipids in the inclusion membrane to any specific pathway. The extensive fluorescence of cellular membranes often interfered with the observation of the fluorescent lipid probes in the inclusion membrane.

To confirm the presence of the phospholipid probes in the chlamydial inclusion membrane, infected cells with incorporated NBD-lipids were disrupted and isolated inclusions examined for presence of the probe in the inclusion membrane. Each of the phospholipid probes was again detected in the inclusion membrane thus confirming that endogenous cellular phospholipids are incorporated into that membrane.

Additional Golgi markers in proximity to the chiamydial inclusion Other probes for resident Golgi proteins, anti-p58 and anti-a-mannosidase II, and for Golgi-derived vesicles, anti-s-COP antibodies, were tested for reactivity against the inclusion membrane (Figure 10). Each of these anti-

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Fig. 6. NBD-sphingomyelin is not delivered from the plasma membrane to the chlamydial inclusion. HeLa 229 cells infected with C.trachomatis, L2, for 18 h were loaded with C6-NBD-ceramide (A, B and C) or NBD-sphingomyelin (D, E and F) and incubated in MEM without dfBSA for O h (A and D), I h (B and E) or 2 h (C and F). After 2 h incubation, MEM plus 0.34% dfBSA was added for 10 min at 37°C to extract plasma membrane NBD-sphingomyelin (C and F) permitting visualization of the chlamydial inclusion without interference from fluorescence in the plasma membrane. Affowheads indicate the approximate position of the chlamydial inclusions (identified by Nomarski differential contrast microscopy). All fluorescent micrographs were taken at the same exposure and printed under identical conditions. Bar = 10 glm.

bodies specifically reacted with the Golgi apparatus or adjacent small vesicles and confirmed the close juxtaposition of the chlamydial inclusion with the Golgi apparatus. Occasionally, each of the markers could be observed surrounding the inclusion but none showed circumferential labeling of the inclusion membrane itself. 5'-nucleotidase activity was used as an ultrastructural marker for the trans-Golgi (not shown). Again, close association of the chlamydial inclusion with the Golgi apparatus was apparent but nucleoside diphosphatase activity was not detected in the inclusion itself nor within vesicles in the process of fusing with the inclusion. A series of fluorescently tagged lectins specific for the Golgi apparatus was also examined for reactivity with the chlamydial inclusion. These included Helix pomatia agglutinin, Ricinus communis agglutinin and Triticum vulgaris (wheat germ) agglutinin. WGA binds sialic acid residues on glycoproteins in the trans-Golgi region and at the cell surface (Virtanen et al., 1980). HPA and RCA bind to terminal N-acetylgalactosamine and galactose residues respectively. These sugars are exposed at intermediate stages of glycosylation in the Golgi apparatus (Kornfeld and Kornfeld, 1985; Campadelli et al., 1993) and are blocked at later stages by the addition of other

970

sugar residues. Distinct labeling of the Golgi apparatus to the chlamydial inclusion was evident with each of these lectins (Figure 11) but, as with the antibody markers, none of the lectins demonstrated distinct or convincing reactivity with the entire inclusion membrane.

adjacent

Discussion The biogenesis of the chlamydial inclusion has been an enigma in chlamydial biology. Based upon the transport of Golgi-derived sphingolipids to chlamydiae within an inclusion, we have suggested that the chlamydial inclusion may represent an aberrant Golgi-derived vesicle situated such that it receives host-derived sphingolipids from an exocytic pathway (Hackstadt et al., 1995). Here we demonstrate that the energy and temperature dependence of that process is similar to that of the normal Golgi to plasma membrane vesicular transport of sphingolipids and document the early entry of chlamydia into that cellular pathway. In addition, we confirm that ceramide or its

metabolites

are at

least transiently incorporated into the

chlamydial inclusion membrane. From the inclusion membrane, sphingomyelin is ostensibly acquired rapidly by chlamydiae within the inclusion. Once incorporated by


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Fig. 7. Electron micrograph of C.trachomatis infected HeLa 229 cells after C6-DMB-cer labeling and photo-oxidation in the presence of diaminobenzidine. (A) An inclusion showing reaction product associated with the Golgi apparatus, small Golgi-derived vesicles, the cell walls of chlamydial RBs within the inclusion and segments of the inclusion membrane. (B) A higher magnification showing reaction product associated with its an RB and a region of the inclusion membrane. (C) A vesicle containing reaction product apparently in the process of fusing with and exposing lumenal contents on the inner surface of the inclusion membrane. (D) A control, unirradiated cell demonstrating the absence of reaction product. Bars

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chlamydiae, the fluorescent lipids are apparently not exchanged out and are excluded from host cell recycling or export pathways (Hackstadt et al., 1995). Transport of plasma membrane sphingomyelin to the chlamydial

inclusion via endocytic vesicles does not appear to be a significant source of the sphingomyelin obtained by C.trachomatis. A working model for the relationship between the chlamydial inclusion and the Golgi apparatus

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Fig. 8. Effects of Brefeldin A on C.trachomatis inclusion morphology. HeLa 229 cells were infected with C.trachomatis and treated with Brefeldin A as described in the text. (A) No Brefeldin A treatment. (B) 0.1 ig/ml Brefeldin A. (C) 1.0 lg/ml Brefeldin A. (D) 10 .g/ml Brefeldin A. Cells were fixed and stained with anti-EB antiserum at 36 h post-infection. Bar = 20 ,um.

is depicted in Figure 12. This model is consistent with our inability to detect delivery of fluid phase markers or other markers of the endosomal/lysosomal pathway to the chlamydial inclusion (Heinzen et al., 1996) and hypothesizes that the chlamydial inclusion is disconnected from endocytic pathways. Instead, we believe the chlamydial inclusion interrupts an exocytic pathway from the transGolgi to the plasma membrane. As such, the mature chlamydial inclusion may be considered an aberrant

exocytic vesicle derived from the trans-Golgi network and potentially modified by the insertion of chlamydiaspecified proteins (Rockey et al., 1995). Such a proposal is supported by the observation that certain serovars of C.trachomatis do not lyse the host cell at the completion of replication but are released by a process in which the inclusion membrane fuses with the plasma membrane to release the contents of the inclusion to the environment (Todd and Caldwell, 1985). Functionally and topologically this process is consistent with our results and the view that the chlamydial inclusion represents an exocytic vesicle in which transport to, and fusion with the plasma membrane is inhibited or delayed. We have hypothesized that Golgi-derived lipids may serve as the biosynthetic source for the growth of the 972

inclusion membrane (Hackstadt et al., 1995). Although we can detect the presence of NBD-sphingolipid in the inclusion membrane by electron microscopy, these fluorescent lipids do not remain in the inclusion membrane but are instead accumulated by the chlamydiae. We suspect that other Golgi-derived lipids may contribute to the growth of the inclusion membrane. The mole percentage of ceramide and its metabolites in the Golgi-apparatus membranes has been estimated at 5-10% (Pagano et al., 1991). Assuming that the sphingomyelin-containing vesicles fusing to the inclusion membrane approximate that ratio, we would expect that other lipids in those vesicles would also be incorporated into the inclusion membrane and may not be acquired by the chlamydiae. Indeed, we have shown that exogenously-added NBD-phospholipids (PA, PC, PE and PS) are incorporated into multiple cellular membranes including the chlamydial inclusion membrane, although we cannot yet attribute their presence in the inclusion membrane to any specific pathway. In contrast to ceramide, these phospholipid probes were not incorporated by chlamydiae. Brefeldin A, which inhibits anterograde transport from the Golgi apparatus (Misumi et al., 1986; Klausner et al., 1992), inhibits the transfer of NBD-ceramide derivatives


I;.:

-.

Fig. 9. NBD-phospholipids are incorporated into the chlamydial inclusion membrane but not the chiamydial cell wall. (A) NBD-phosphatidic acid. (B) NBD-phosphatidylcholine. (C) NBD-phosphatidylethanolamine. (D) NBD-phosphatidylserine. Inserts are confocal images of cell-free C.trachomatis inclusions demonstrating incorporation of exogenous NBD-phospholipid into the inclusion membrane. Arrows indicate the location of the chlamydial inclusions. Bar = 10 gm.

to intracellular chlamydiae (Hackstadt et al., 1995). Paradoxically, inhibition of Golgi function by Brefeldin A does not inhibit chlamydial replication. Inhibition of sphingomyelin synthesis from ceramide and transport of sphingomyelin by Brefeldin A is not complete, however, but is reduced to a level of ~60% of normal (Slomiany et al., 1993). Indeed chlamydiae incubated with C6-NBDceramide in Brefeldin A treated cultures will eventually display fluorescent lipid in their cell walls (data not shown). It should also be considered that levels of endogenous sphingomyelin in host cells may be sufficient to support chlamydial growth for the relatively short (48-72 h) duration of their intracellular developmental cycle. Moreover, despite the fact that chlamydiae are known to contain sphingomyelin (Newhall, 1988), an absolute nutritional requirement for it has not been proven and may be difficult to establish. The one notable effect of Brefeldin A on chlamydial development was on inclusion morphology. Inclusions in Brefeldin A treated cells were distinctly smaller and the chlamydial developmental forms were apparently more densely packed. An interpretation of this result that is consistent with our hypothesis that Golgiderived lipids provide for the growth of the inclusion membrane would be that Brefeldin A treatment inhibits the delivery of lipid to the inclusion membrane, thus limiting its size.

Endocytosed chlamydiae are known to be transported to the perinuclear region of the host cell (Higashi, 1965). We confirmed the rapid accumulation of internalized chlamydiae in the perinuclear region and have identified this region as the peri-Golgi region using contemporary fluorescent probes. In addition, we have demonstrated an early functional interaction between the EBs and the Golgi apparatus in that C6-NBD ceramide or its metabolites are delivered to, and incorporated by, undifferentiated EBs. There is little or no metabolic activity in chlamydial EBs (Moulder, 1991). The nucleoid is compacted, presumably restricting transcriptional activity and there is no detectable energy metabolism. It is possible that the host cell may provide lipid for the growth of the bacterial membrane in an energy-independent manner (from the bacterial standpoint) as the EBs differentiate into the larger and fully metabolically active RBs. The possibility of energy independent incorporation of exogenous lipid by metabolically dormant EBs is evidenced by the uptake of C6NBD-ceramide or C6-NBD-sphingomyelin by extracellular EBs in vitro (Hackstadt et al., 1995; Heinzen et al., 1996). Acquisition of host-derived lipid may therefore contribute to the enlargement of EBs to RBs before endogenous chlamydial biosynthetic capabilities are fully active. The metabolically active RBs are typically observed juxtaposed to the inclusion membrane (Matsumoto, 1988).

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R A

WL3A Fig. 11. Association of Golgi-specific lectins with the chlamydial inclusion. C.trachomatis-infected cells were fixed at 18 h postinfection and stained by direct fluorescence for (A) Helix pomatia agglutinin, (B) Ricinus communis agglutinin and (C) Triticum vulgaris (wheat germ) agglutinin. Arrows indicate the position of the chlamydial inclusions. Bar = 10 gm.

association of RBs with the inclusion membrane appears tight with an apparent thickening of the inclusion membrane at the point of contact (Matsumoto, 1988; Todd and Caidwell, 1985). This tight junction possibly facilitates the transfer of lipid from the inclusion membrane to the RBs. It is of interest that it is almost exclusively RBs that associate with the inclusion membrane. This phenomenon suggests that loss of contact with the inclusion membrane may be a signal which triggers differentiation to EBs and would be consistent with the asynchrony that characterizes this stage of the developmental cycle. Although several other Golgi markers demonstrated the close association of the Golgi apparatus with the chlamydial inclusion, none showed appreciable staining of the inclusion membrane. A significant proportion (about half) of the sphingomyelin synthesized from C6-NBD-ceramide that would normally be exported to the plasma membrane is instead redirected to the chlamydial inclusion in C.trachomatis-infected cells (Hackstadt et al., 1995). Although none have yet been identified, we would expect that a sub-population of glycoproteins en route from the Golgi apparatus to the plasma membrane might also be redirected to the chlamydial inclusion. It is unclear, to be very

Fig. 10. Relationship of several Golgi-specific markers to the chlamydial inclusion. C.trachomatis-infected cells were fixed at 18 h post-infection and stained by indirect immunofluorescence for (A) Golgi p58 protein, (B) ,-COP and (C) ax-mannosidase II. Arrows indicate the position of the chlamydial inclusions. Bar = 10 gim.

Although the early stages of chlamydial growth are quite synchronous, after ~18 h post-infection the infection becomes asynchronous as the RBs continue to multiply while increasing numbers of EBs and intermediate developmental forms are observed unattached in the interior of the inclusion. Up until the point of secondary differentiation back to EBs, RBs are found circumscribing the inner margin of the inclusion with few or no organisms observed free within the center of the inclusion. The

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Chlamydial interruption of sphingolipid export Plasma Membrane

Golgi Apparatus

Cer Endoplasmic Reticulum

Fig. 12. Model of the interaction of the C.trachomatis inclusion with the Golgi apparatus. In this model the chlamydial inclusion is situated such that it fuses with sphingomyelin (SM)-containing vesicles en route from the trans-Golgi to the plasma membrane. Once these vesicles fuse with the chlamydial inclusion, the sphingomyelin is rapidly incorporated by the intracellular chlamydiae. The experimental evidence does not support a mechanism of sphingomyelin trafficking to the chlamydial inclusion via fusion of endocytic vesicles with the inclusion as indicated by the heavy line blocking that pathway. Instead, delivery of sphingomyelin, endogeously synthesized from ceramide (Cer), appears to be direct from the Golgi apparatus. As such, the chlamydial inclusion interupts an established exocytic pathway. Such a model is consistant with the failure of fluid phase markers to traffick to the inclusion and the absense of markers for early or late endosomes (Heinzen et al., 1996).

however, which host glycoproteins, if any, are present in the sphingomyelin-containing vesicles in transit to the plasma membrane or what the lumenal contents of the vesicles might be. As yet we have not identified any host glycoproteins in the inclusion membrane. A critical function of the trans-Golgi network is the segregation and sorting of glycoproteins bound for different cellular compartments. The trafficking of sphingomyelin to the chlamydial inclusion in the absence of host glycoproteins implies that at least a proportion of sphingomyelin en route to the plasma membrane is not associated with vesicles transporting glycoproteins to the cell surface. While such discrimination has not been described for sphingolipids exiting the Golgi apparatus, some precedence for sphingomyelin sorting in distinct vesicles has been documented for endocytic vesicles. Sphingomyelin has been found to colocalize with endocytic vesicles containing transferrin and its receptor. However, it was not observed in vesicles containing fluid phase markers (Koval and Pagano, 1989). The chlamydial inclusion may therefore provide a unique tool to investigate the sorting and trafficking of newly synthesized glycoproteins and glycolipids from the Golgi apparatus. Identification of those signals which govern trafficking to the chlamydial inclusion remains a significant challenge in understanding the interaction of chlamydiae with the host cell.

Materials and methods Reagents 6- {N-[(7-nitrobenzo-2-oxa- 1 ,3-diazol-4-yl)amino]caproyl } sphingosine (C6-NBD-ceramide), N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-

indacene-3-pentanoly)sphingosine (C5-DMB-ceramide), N-{6-[(7-nitrobenzo-2-oxa- 1 ,3-diazol-4-yl)amino]hexanoyl } sphingosylphosphocholine (C6-NBD-sphingomyelin) and Texas red-wheat germ agglutinin were obtained from Molecular Probes (Junction City, OR). 6:0-N-NBDphosphatidic acid (NBD-PA), 6:0-N-NBD-phosphatidylcholine (NBDPC), 6:0-N-NBD-phosphatidylethanolamine (NBD-PE), and 12:0-NNBD-phosphatidylserine (NBD-PS) were from Avanti Polar Lipids, Inc. (Alabaster, AL). Rhodamine-Helix pomatia agglutinin, rhodamineRicinus communis agglutinin, Anti-,-COP and anti-Golgi p58 were obtained from Sigma Chemical Co, St Louis, MO. The anti-cx-mannosidase II antibody was generously provided by Dr M.Farquhar.

Cells and cell culture Monolayer cultures of HeLa 229 epithelial cells (CCL 2.1: American Type Culture Collection) were grown in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum and 10 ztg/ml gentamicin or in RPMI-1640 supplemented with 10% FBS plus 10 tg/ ml gentamicin. Cells were seeded at a density of I X 105/well and grown at 37°C on 12 mm diameter glass coverslips (No. I thickness) in 24well plates in an atmosphere of 5% CO2 in humidified air.

Organisms Chlamvdia trachomatis, LGV-434, serotype L2, was grown in HeLa 229 cells as previously described (Caldwell et al., 1981). Infections were carried out by plating an appropriate dilution of bacteria in 0.22 M sucrose, 0.2 M sodium phosphate, 4 mM glutamic acid, pH 7.4 (SPG). Infectivity of C.trachomatis EBs was titrated by determination of inclusion-forming units (IFUs) on HeLa 229 cells and described by Fumess et al. (1960) except that inclusions were visualized by indirect immunofluorescence employing polyclonal antisera against formalinkilled C.trachomatis, L2, EBs and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobin serum (Zymed Laboratories, Inc., San Francisco, CA).

C6-NBD-ceramide labeling C6-NBD-ceramide was complexed with 0.034% defatted-bovine serum albumin (DFBSA) in Dulbecco's minimal essential medium (MEM) as described (Pagano et al., 1988) to yield complexes -5 gM in both

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T.Hackstadt et al. DFBSA and C6-NBD-ceramide. Mock- and C.trachomatis-infected HeLa cells were incubated with the DFBSA/NBD-ceramide 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 the plasma membrane. Cultures on coverslips were rinsed in HCMF prior to mounting for fluorescent microscopy.

0.05% SDS. The monolayers were blocked with 10% BSA in 50 mM NaPO4, 150 mM NaCl, pH 7.4 (PBS) plus 0.1% TX-100/0.05% SDS for 30 min at room temperature. Antibodies were diluted in 10% BSA in PBS plus 0.1 % TX-100/0.05% SDS and incubated on the monolayers for I h at room temperature. The coverslips were rinsed twice with PBS and incubated with FITC-conjugated anti-mouse immunoglobin for 1 h at room temperature. The coverslips were then rinsed three times with PBS, once with distilled water, and mounted in buffered glycerol for

C6-NBD-sphingomyelin labeling Complexes of C6-NBD-sphingomyelin were prepared in MEM + 0.034% DFBSA as described above for C6-NBD-ceramide (Pagano and Martin, 1988; Martin and Pagano, 1994). Uninfected or C.trachomatis-infected HeLa 229 cells at 18 h post-infection were chilled to 4°C for 30 min, rinsed once with Hank's buffered salts solution (HBSS) and incubated with the DFBSA/C6-NBD-sphingomyelin complex for 30 min at 4°C. The fluorescent lipid complexes were removed, the cultures washed once with HBSS and pre-warmed (37°C) MEM with or without 0.34% DFBSA was added. The cultures were incubated at 37°C for selected intervals and washed three times with HCMF prior to microscopy or photometric quantitation of retained fluorescence as described (Hackstadt et al., 1995). Briefly, monolayer cultures of HeLa 229 cells on 12 mm glass coverslips were infected with C.trachomatis, L2, EBs at an MOI of -2, or mock-infected, and incubated at 37°C for 18 h. The coverslips

microscopy.

were rinsed once with HBSS and incubated with 5 .M C6-NBDceramide or C6-NBD-sphingomyelin in MEM plus 0.034% DFBSA for 30 min at 4°C. The cultures were rinsed three times with HBSS and the medium replaced with pre-warmed MEM ±0.34% DFBSA. The cultures were incubated at 37°C and at various times coverslips were rinsed three times with HCMF. Coverslips were inverted onto glass microscope slides and areas for quantitation of retained fluorescence were identified by visualization using Nomarski differential interference contrast optics on a Nikon FXA photomicroscope with a Nikon 20X planapochromat objective. The selected area was demarcated using the photomicrographic spotmeter and the transmitted light was extinguished. The photometric function of the microscope was used to quantify (in Lux) the fluorescence intensity of the specimens under epifluorescent illumination using the fluorescein filter set. Five readings from each time point were averaged and the standard errors of the mean calculated.

Other NBD-phospholipids

Small unilaminar vesicles containing NBD-PA, NBD-PC, NBD-PE or NBD-PS (1:1.5 mol/mol in dioleoyl phophatidylcholine) were prepared by ethanol injection as described (Kremer et al., 1977). The lipids were mixed and dried under argon. The dried lipids were then solubilized in 50 1g of absolute ethanol and injected into MEM at 42°C with vortexing to create a 50 gM suspension. Uninfected or Ctrachomatis-infected HeLa 229 cells at 18 h post-infection were chilled to 4°C for 30 min, rinsed once with HBSS and incubated with the NBD-lipid vesicles for 30 min at 4°C. The cultures were then washed three times with cold MEM before addition of pre-warmed MEM. The cells were incubated for I h at 37°C, chilled to 4°C, and incubated three times for 10 min each in MEM plus 2 mM dioleoyl phophatidylcholine. The coverslips were then rinsed once with HCMF prior to mounting for microscopy.

ATP depletion and inhibitors

For all experiments involving C6-NBD-ceramide trafficking in the presence of various treatments and inhibitors, control and C.trachomatisinfected monolayers at 18 h post-infection were pretreated for 90 min, the cultures rinsed with glucose-free MEM, and labeled with C6-NBDceramide for 10 min at the appropriate temperature in the presence of inhibitor, rinsed with glucose-free MEM, and back-exchanged for I h in MEM plus 0.34% dfBSA in the presence of inhibitor. Inhibitors and the concentrations were: carbonyl cyanide chlorophenylhydrazone, 10 ,uM; monensin, 10 gM; nocodazole, 10 jig/ml; and Brefeldin A, 1 ,ug/ml. For the depletion of ATP, control and C.trachomatis-infected monolayers 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 in glucose-free MEM with 50 mM 2-deoxy-D-glucose and 5 mM NaN3. Determination of ATP levels in a parallel set of culture indicated that the ATP concentration of treated cells was reduced to 16% of the untreated controls.

Immunofluorescence For indirect immunofluorescence, monolayers were fixed at 4°C with 4% paraformaldehyde in 25 mM NaPO4, 150 mM NaCI, pH 7.4. The cells were permeabilized by 3 min incubation in 0.1% Triton X-100/

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Microscopy Fluorescent and Nomarski differential interference contrast micrographs were taken on a Nikon FXA photomicroscope using either 20X or 60X Planapochromat objectives. Photomicrographs were obtained using T-Max ASA 400 film (Eastman Kodak Co., Rochester, NY). Some images were recorded digitally using a Dage-MTI CCD 72 camera attached to a DSP2000 image processor (Dage-MTI, Inc., Michigan City, IN). A Bio-Rad MRC-1000 confocal imaging system equipped with a krypton-argon laser (Bio-Rad Laboratories, Hercules, CA) on a Zeiss inverted microscope with a 63 x objective was utilized for laser-scanning confocal microscopy. Confocal and digital images were processed using Adobe Photoshop 2.5.1 (Adobe Systems, Inc., Mountain View, CA). Each experiment was repeated at least three times and multiple fields of view examined. Micrographs were chosen as representative of typical fields.

Electron microscopy Chlamydia trachomatis-infected HeLa 229 cells on Thermanox cover slips (Nunc, Inc.) were labeled by incubation in 5 lM C5-DMB-ceramide in MEM + 0.034% DFBSA as described above for C6-NBD-ceramide. The cultures were then back-exchanged with MEM plus 0.34% dfBSA for I h at 37°C and washed twice with HBSS. The monolayers were processed for electron microscopy essentially as described (Takizawa et al., 1993) once with 0.1 M NaCacodylate buffer, pH 7.4, and fixed with 4% paraformaldehyde-2% glutaraldehyde in 0.1 M NaCacodylate buffer, pH 7.4, for 60 min at 4°C. The monolayers were then washed five times with 0.1 M NaCacodylate buffer, pH 7.4, then incubated for 10 min at room temperature in 2.5 mg/ml diaminobenzidine in 0.1 M NaCacodylate buffer, pH 7.4. An 1 mm diameter area of the coverslips was illuminated for 30 min at room temperature with a 100 W mercury bulb with a lOx objective and the fluorescein filter set. The monolayers were washed again five times with 0.1 M NaCacodylate buffer, pH 7.4, and post-fixed with 1% Os04 for 30 min at 4°C, rinsed once with distilled water, and dehydrated and embedded for electron microscopy.

Acknowledgements We are grateful to Drs H.Caldwell and W.Cieplak for critical review of the manuscript and Dr R.E.Pagano for helpful discussions. We thank S.F.Hayes for electron microscopy, J.Sager and J.Simmons for technical assistance, and G.Hettrick and B.Evans for graphic arts.

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Received oni September 25. 1995; revised on Nov ember 13, 1995

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