|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02733
Research Article |
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA
e-mail: beatty{at}borcim.wustl.edu
Accepted 5 October 2005
 |
Summary |
|---|
 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
Key words: Chlamydia trachomatis, Multivesicular bodies, CD63, Trafficking
|
Introduction |
|---|
|
TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
). The spectrum of disease manifestations caused by Chlamydiae also encompasses C. pneumoniae, a causative agent of acute respiratory infections and implicated as a potential risk factor of cardiovascular disease (Grayston, 2000). Chlamydiae are obligate intracellular pathogens that reside exclusively in a membrane-bound organelle called the inclusion. Chlamydiae have evolved a unique biphasic developmental cycle alternating between two distinct morphological forms. Infection of a target host cell is initiated by the metabolically inactive elementary body (EB), which upon internalization differentiates into the noninfectious, metabolically active reticulate body (RB). After multiple divisions within the confines of the inclusion, the RBs redifferentiate back to EBs. Lysis of the inclusion membrane and the host cell results in the release of a multitude of infectious EBs, each capable of eliciting a new round of infection.
The mechanisms that allow chlamydiae to remain sequestered in a protected intracellular niche remain enigmatic. The use of contemporary cell-biological probes for early and late endocytic markers (transferrin and M6PR) (Heinzen et al., 1996; Scidmore et al., 2003), as well as lysosomal markers and endocytic tracers (LAMP-1, LAMP-2, Cathepsin D, ATPase, dextran, and lucifer yellow) (Heinzen et al., 1996; Scidmore et al., 2003; Taraska et al., 1996) have failed to demonstrate an effective interaction between host pathways and the chlamydial inclusion. However, within the confines of the inclusion, chlamydiae acquire amino acids and nucleotides, as well as eukaryotic lipids that are transported from the host cell and incorporated into the chlamydial inclusion (Hatch, 1975; McClarty, 1994; Wylie et al., 1997). Chlamydiae obtain host cell sphingomyelin and cholesterol from Golgi-derived vesicles destined for the plasma membrane; however, this pathway of acquisition is neither exclusive nor essential for inclusion development (Carabeo et al., 2003; Hackstadt et al., 1996). Acquisition of host cell protein and lipid constituents by other mechanisms or pathways remains elusive.
In the present study, the interaction between the chlamydial inclusion and multivesicular bodies (MVBs) was analyzed. MVBs are complex, heterogeneous late endocytic organelles, seminal in the segregation of proteins and lipids destined for lysosomal degradation, recycling to the Golgi, or plasma membrane exocytosis (Denzer et al., 2000; Piper and Luzio, 2001). Internal vesicles formed by invagination of the limiting membrane of MVBs provide the scaffold for transport and delivery of its contents (Pan et al., 1985; Vidal et al., 1997). These internal membranes are highly enriched in lysobisphosphatidic acid (LBPA) or phosphatidylinositol 3-phosphate with variable membrane compositions defining a diverse population (Gillooly et al., 2000; Kobayashi et al., 1998). LBPA-rich membranes are thought to play a role in sphingolipid hydrolysis and regulation of intracellular cholesterol transport (Kobayashi et al., 1999; Kolter et al., 1999). The tetraspanin protein CD63 is abundant in MVBs and cycles between these compartments and the exocytic pathway (Kobayashi et al., 2000). Restricted to the limiting membrane of CD63- and LBPA-positive late endosomes is the transmembrane protein metastatic lymph node 64 (MLN64) (Alpy et al., 2001). MLN64 shares homology with the StAR-related lipid transfer (START) domain of the steroidogenic acute regulatory protein (StAR) (Watari et al., 1997). The MLN64 START domain binds cholesterol and stimulates the mobilization of free cholesterol from sterol-rich donor vesicles to acceptor membranes (Kallen et al., 1998).
In this study, candidate MVB-related proteins and lipids were evaluated for their association with intracellular chlamydiae. Demonstration of an intersection between the chlamydial inclusion and MVB-related pathways, identifies these dynamic host cell compartments as a novel source for factors essential for intracellular bacterial growth and inclusion biogenesis.
|
Results |
|---|
|
TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
|
Orthogonal analysis confirms the presence of CD63 within the chlamydial inclusion
Immunolabeling studies indicated that protein and lipid constituents of MVBs colocalize with chlamydial inclusions. Potential trafficking of MVBs or MVB-related compartments to the chlamydial inclusion was further evaluated using CD63 as a surrogate marker for this interaction. HEp-2 cells were infected with C. trachomatis E for 48 hours and analyzed by indirect immunofluorescence. To precisely identify the boundary of the chlamydial inclusion, cells were labeled with antibody specific to the cytosolic C-terminal domain of the inclusion membrane protein IncA, using conditions that result in labeling of the inclusion membrane exclusively (see Materials and Methods). The inclusion membrane was then detergent-permeabilized and cells were labeled with mouse antibody specific to CD63. Confocal microscopy clearly identified the presence of CD63 within the inclusion (Fig. 2, upper panel) and this colocalized with TOPRO-3-labeled Chlamydia. Orthogonal views unequivocally demonstrated localization of CD63 with TOPRO-3-labeled Chlamydia within the confines of the IncA-labeled inclusion membrane (Fig. 2, lower panel).
|
|
|
Immunoelectron microscopy analysis of CD63-HA-transfected cells further confirmed the presence of CD63 within the chlamydial inclusion. Labeling of cryo sections of Chlamydia-infected cells with anti-HA antibody revealed the presence of CD63-HA in multivesicular organelles adjacent to the chlamydial inclusion (Fig. 4, right panel). In addition, immunolabeling clearly showed CD63-HA within the lumen of the inclusion and often associated with vesicles (Fig. 4, right lower panel insert), further implicating a direct fusion of MVBs with the chlamydial inclusion.
Internalized exogenous anti-CD63 antibody traffics to the chlamydial inclusion and perturbs intracellular chlamydial development
MVBs lie within the endocytic pathway providing a direct route for exogenously added tracers. This route can be exploited by following the fate of endocytosed antibodies that will selectively accumulate after binding to an antigen. Previous studies have demonstrated the accumulation of internalized anti-CD63 antibody in late endocytic compartments, with subsequent trafficking to secretory compartments (Kobayashi et al., 2000). Exogenously internalized anti-LBPA antibody traffics to late endocytic compartments, blocking subsequent transport (Kobayashi et al., 1999). In this study, the same approach was used to follow the fate of anti-CD63 antibodies in the Chlamydia-infected cells. Exogenously internalized anti-CD63 antibody accumulated in perinuclear vesicular compartments and in the chlamydial inclusion (Fig. 5). When anti-CD63 was added simultaneously with anti-LAMP-1 antibody or the fluid-phase marker dextran-FITC, anti-CD63 localized to the chlamydial inclusion, while anti-LAMP-1 and dextran were clearly excluded (Fig. 5). The distribution of these markers was shown quantitatively by generating a profile of the intensity distribution of each constituent along a line traversing the chlamydial inclusion (Fig. 5, lower panels). Anti-CD63 antibody clearly colocalized with TOPRO-3-labeled chlamydiae within the profile of the inclusion, while being excluded from the TOPRO-3-labeled host cell nuclei (Fig. 5, lower panels). Anti-LAMP-1 antibody and dextran-FITC displayed high-intensity values peripherally to the inclusion and the host cell nuclei but were clearly excluded from the inclusion itself, as revealed by background-intensity levels across this region of the profile (Fig. 5, lower panels). Orthogonal analysis of the entire Z-height of infected cells confirmed the presence of exogenous anti-CD63 within the inclusion (data not shown).
|
|
Inhibitors of MVB biogenesis disrupt chlamydial growth and development
Inhibitors of phosphoinositide 3-kinase (PI 3-kinase) disrupt the biogenesis of MVBs, resulting in swollen organelles that are impaired in membrane trafficking out of this compartment (Fernandez-Borja et al., 1999). The pharmacological agent U18666A inhibits cholesterol transport from late endocytic compartments (Liscum and Faust, 1992) and alters the trafficking of MVB-associated membrane proteins (Higgins et al., 2001; Kobayashi et al., 1999; Kobayashi et al., 2000). In the presence of U18666A and the PI 3-kinase inhibitors 3-methyladenine and LY-294002, maturation of the chlamydial inclusion was disrupted with a marked reduction in inclusion size and yield of infectious progeny (Fig. 7). Electron microscopy of the intracellular bacteria revealed primarily EBs in the untreated control cells but small inclusions that contain typical RBs (0.6 µm-1 µm) in inhibitor-treated cells, indicating an interruption in RB division and re-differentiation under these conditions. Infected cells treated with MVB inhibitors for 48 hours do not have detectable MVB constituents in their inclusions (data not shown). The concentrations of the inhibitors that disrupted inclusion development also induced swollen CD63-positive MVBs (data not shown), revealing a correlative inhibitory effect on MVB biogenesis and inclusion development. The disruption in inclusion maturation implies an essential role of MVBs or MVB-associated compartments in normal intracellular development of chlamydiae.
|
). Within the Golgi, ceramide is processed to sphingomyelin and glucosylceramide before being transported to the plasma membrane (Lipsky and Pagano, 1983). Hackstadt and colleagues used this fluorescent probe to demonstrate endogenous synthesis and transport of sphingomyelin from the Golgi complex to the inclusion and incorporation into the cell wall of chlamydiae (Hackstadt et al., 1995). Because MVBs are pivotal in intracellular sphingolipid transport, the role of these compartments in sphingomyelin delivery to the chlamydial inclusion was analyzed. HEp-2 cells, infected with C. trachomatis for 18 or 48 hours, incorporated the fluorescent probe into both the Golgi complex and the chlamydial inclusion (Fig. 8, left panels). Culturing in the presence of the MVB inhibitors 3-methyladenine and U18666A for 48 hours resulted in smaller inclusions, which failed to incorporate the fluorescent probe (Fig. 8, right panels). However, these cells displayed intense accumulation of the fluorescent probe in swollen perinuclear CD63-positive compartments. The disruption of sphingomyelin transport to the chlamydial inclusion by inhibition of trafficking from MVBs, identifies CD63-positive MVBs within a novel transport pathway of lipid delivery to intracellular chlamydiae.
|
|
Discussion |
|---|
|
TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
; Piper and Luzio, 2001). Two important features of MVBs make these compartments compelling candidates for an interaction with the chlamydial inclusion. First, MVBs are central to the intracellular trafficking of sphingolipids and cholesterol, two host-derived constituents acquired by the chlamydial inclusion (Carabeo et al., 2002; Hackstadt et al., 1995; Wylie et al., 1997). Second, MVBs are an active component of the exocytic pathway in various cell types (for reviews, see Denzer et al., 2000; Stoorvogel et al., 2002), the only intracellular trafficking pathway that has been shown to intersect the chlamydial inclusion (Hackstadt et al., 1996; Hackstadt et al., 1995). The present study demonstrates a dynamic interaction between MVB trafficking pathways and the chlamydial inclusion, essential for intracellular bacterial development. Rerouting of MVB-derived exocytic compartments to the chlamydial inclusion might provide an essential source of biosynthetic macromolecules vital to intracellular chlamydial development.
A hallmark of MVBs is the presence of lipid-enriched internal membranes, which have been proposed to be important in protein sorting and transport of cholesterol and sphingolipids (Denzer et al., 2000; Kobayashi et al., 1999; Piper and Luzio, 2001). Constituents of MVBs include LBPA, a lipid abundant in internal membranes of MVBs (Kobayashi et al., 1998), which is thought to play a role in sphingolipid hydrolysis and regulation of cholesterol transport (Kobayashi et al., 1999). The tetraspanin protein CD63 is present in internal membranes and at the limiting membrane of MVBs (Escola et al., 1998; Kobayashi et al., 2002). MLN64 is a cholesterol-binding protein restricted to the limiting membrane of CD63- and LBPA-positive late endosomes (Alpy et al., 2001).
Using a cell-biological approach, MVB markers, in addition to tracers and inhibitors, were used to analyze the interaction of these dynamic compartments with the chlamydial inclusion. This study demonstrates that CD63, MLN64 and LBPA localize to the chlamydial inclusion, as shown by indirect immunofluorescence (Fig. 1). Because the chlamydial inclusion has been proposed to intersect an exocytic pathway (Hackstadt et al., 1996; Hackstadt et al., 1995), CD63 – a host protein associated with both MVBs and the exocytic pathway (Kobayashi et al., 2000) – was used as a surrogate marker for additional characterization. Electron microscopy (Fig. 3) and orthogonal analyses obtained from confocal 3D-reconstruction (Fig. 2) substantiated the accumulation of CD63 within the chlamydial inclusion. Localization of HA-tagged CD63 in infected cells confirmed the distribution of CD63 to the chlamydial inclusion (Fig. 4).
The potential importance of MVB-associated CD63 was further explored in experiments based on internalization of exogenous CD63-specific antibodies. Previous studies have shown that endosomal CD63 protein is accessible to antibodies internalized by fluid-phase endocytosis. Exogenously added CD63-specific antibodies partition preferentially to and accumulate in LBPA-positive late endocytic compartments, with subsequent transport to secretory compartments (Kobayashi et al., 2000). Internalized anti-LBPA antibodies traffic to late endocytic compartments, resulting in the impairment of trafficking out of these compartments, as characterized by a massive accumulation of cholesterol (Kobayashi et al., 1999). Here, this approach was used to determine the fate and potential effect of anti-CD63 antibodies on Chlamydia-infected cells. Anti-CD63 antibodies that trafficked to and were incorporated into the chlamydial inclusion demonstrate a direct interaction between a CD63-positive compartment and intracellular chlamydiae (Fig. 5). The exclusion of anti-LAMP-1 antibody and dextran from the inclusion indicated that anti-CD63 acquisition was the result of selective vesicular-sorting and -transport from the endocytic pathway or subsequent MVB-related compartments. The disruption in chlamydial development by CD63-specific antibody further implies that this MVB constituent is important for inclusion-biogenesis and -maturation (Fig. 6).
Disruption of MVBs leads to detrimental accumulation of cholesterol and sphingolipids as shown experimentally and in disease states (Kobayashi et al., 1999; Vanier and Millat, 2003). Inhibitors of PI 3-kinases, key regulators of protein sorting, disrupt the biogenesis of MVBs, which results in swollen vesicles that are impaired in membrane trafficking out of these compartments (Fernandez-Borja et al., 1999; Kundra and Kornfeld, 1998). The pharmacological agent U18666A inhibits cholesterol transport from late endocytic compartments (Liscum and Faust, 1992) and alters the trafficking of CD63 (Higgins et al., 2001; Kobayashi et al., 1999; Kobayashi et al., 2000). Because inhibitors can pleiotropically effect cellular function, their potential effect on MVBs was initially confirmed in the HEp-2 cells used in this study. Culturing in the presence of defined concentrations of 3-methyladenine, LY-294002 or U18666A resulted in swollen CD63-positive MVBs. Altered morphology correlated with the disrupted maturation of inclusions when inhibitors where present (Fig. 7), further implicating a role of MVBs in the normal development of chlamydiae.
The PI 3-kinase inhibitor 3-methyladenine is also a modulator of autophagy (Seglen and Gordon, 1982); and in the pathogenesis of Chlamydia, a potential role of the host cell's autophagy pathway has been proposed (Al-Younes et al., 2004). Autophagy is a major mechanism for degradation and recycling of intracellular macromolecules (Klionsky and Emr, 2000). Although studies have indicated a lack of autophagosomal fusion with the chlamydial inclusion, the intracellular bacteria were sensitive to 3-methyladenine and other traditional inhibitors of autophagy (Al-Younes et al., 2004). The autophagy pathway is thought to intersect MVBs prior to lysosomal delivery (Lucocq and Walker, 1997). Thus, the effects of inhibitors of autophagy on chlamydial growth are potentially a result of downstream effects, subsequent to autophagosome-MVB interception.
Fundamental questions, regarding intracellular mediators and signals essential for inclusion formation, maturation and sequestration, remain unknown. In contrast to other intracellular pathogens that reside in membrane-bound vacuoles, an association between chlamydiae and endocytic pathways of its host cell has not been identified. However, common among all Chlamydiae species is the fusigenicity of the inclusion with an apparent exocytic pathway (Hackstadt et al., 1996; Rockey et al., 1996; Wolf and Hackstadt, 2001). Fluorescent analogs of ceramide demonstrate the direct trafficking of an exogenous substrate to the chlamydial inclusion. Ceramide is synthesized in the endoplasmic reticulum and enzymatically converted to sphingomyelin or glucosylceramide at the cis- or medial-Golgi before being transported to the plasma membrane (Futerman et al., 1990; Lipsky and Pagano, 1985a). Labeling infected cells with NBD-ceramide led to the diversion of fluorescent label to the chlamydial inclusion and the incorporation of NBD-sphingomyelin into the chlamydial cell wall (Hackstadt et al., 1995). The acquisition of endogenously synthesized sphingomyelin was disrupted by brefeldin A, an inhibitor of anterograde transport from the Golgi complex. These studies were expanded to analyze cholesterol, a lipid identified in purified EBs and the chlamydial inclusion membrane (Carabeo et al., 2003; Wylie et al., 1997). Acquisition of de novo-synthesized and low-density lipoprotein-derived cholesterol occurs through this brefeldin-A-sensitive, Golgi-dependent pathway (Carabeo et al., 2003). However, brefeldin A inhibition of sphingomyelin and cholesterol delivery failed to interrupt bacterial multiplication (Carabeo et al., 2003; Hackstadt et al., 1996), suggesting that, in addition to direct Golgi transport, chlamydiae possess an alternative pathway for lipid acquisition.
The phospholipid composition of purified Chlamydia trachomatis includes both typical prokaryotic lipids (phophatidylserine, phophatidylethanolamine and phophatidylglycerol) and lipids generally associated with eukaryotes (sphinogmyelin, cholesterol, phophatidylcholine, and phophatidylinositol) (Newhall, 1988; Wylie et al., 1997). In addition to de novo phopholipid synthesis, studies utilizing radiolabeled isoleucine demonstrated that host cell phospholipids are trafficked to and modified by chlamydiae, a process that occurs independently of Golgi transport (Wylie et al., 1997). Eukaryotic sphingolipids are required for intracellular bacterial growth and production of infectious progeny (Van Ooij et al., 2000), however, the transfer mechanism of host-derived lipids has not been identified. In the present study, the effect of MVB disruption on the direct delivery of sphingomyelin to the chlamydial inclusion was analyzed using the NBD-ceramide as previously described by Hackstadt (Hackstadt et al., 1995). Loading cells with the fluorescent probe resulted in the labeling of the Golgi complex and also of vesicles that occupied a larger perinuclear area beyond the Golgi that were subsequently identified as CD63-positive compartments. Culturing infected cells in the presence of 3-methyladenine and U18666A, the pharmacological inhibitors that prevent trafficking out of MVBs, resulted in loaded probe accumulating in enlarged CD63-positive MVBs. This accumulation was accompanied by the inhibition of delivery of synthesized sphingomyelin to the chlamydial inclusion (Fig. 8). These observations indicate that host lipids are trafficked to the chlamydial inclusion by a pathway that intercepts MVBs. Disruption of this pathway blocks both delivery and bacterial growth, hence, trafficking from MVBs is essential to productive intracellular development of the chlamydial inclusion.
Intersecting the endosome-lysosome- (Van Deurs et al., 1993), autophagy- (Lucocq and Walker, 1997) and exocytic-pathways (Denzer et al., 2000; Stoorvogel et al., 2002), MVBs are ideally positioned to interact with the chlamydial inclusion. In addition, the functional attributes of MVBs make these and related organelles an intriguing source of biosynthetic factors that might be essential for inclusion biogenesis and maturation. This study demonstrates a dynamic interaction between MVB-derived constituents and the chlamydial inclusion. It is not clear whether a direct interaction exists, or whether MVB-related or downstream compartments intersect the inclusion. Identification and characterization of chlamydial inclusion-influx pathways and corresponding requisite host factors, will have important implications on the general biology of Chlamydia and the potential development of therapeutic intervention targeting nutrient-acquisition pathways essential for Chlamydia propagation.
|
Materials and Methods |
|---|
|
TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
Cell culture and propagation of chlamydiae
C. trachomatis serovar E (obtained from Harlan Caldwell, Rocky Mountain Laboratories, NIH, NIAID) was propagated in HEp-2 cells (ATCC, Manassas, VA) and elementary bodies (EBs) were purified by Renografin gradient-centrifugation as previously described (Caldwell et al., 1981). HEp-2 cells were maintained at 37°C with 5.5% CO2 in Iscove's DME medium supplemented with 12.5 mM HEPES, 10% (vol/vol) FBS, and 10 µg/ml gentamicin. HEp-2 cells were infected by incubating monolayers with Chlamydia EBs at a multiplicity of infection of 0.5 for 1 hour at 37°C on a platform rocker. Cells were washed twice with PBS and incubated in Iscove's medium for the times indicated.
Plasmids and transfection
Full length cDNA for CD63 (Genbank accession no. BT007073) was obtained by PCR amplification from human cDNA with KlenTaq LA (Sigma). CD63 was cloned as a C-terminal HA9-epitope fusion in the pcDNA 3.1/V5 His vector (Invitrogen, Carlsbad, CA). HEp-2 cells at subconfluency were transfected with plasmid using FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN) as described in the manufacturer's protocol. Cells transfected for 24 hours were then infected with Chlamydia as described above.
Immunofluorescence and confocal microscopy
HEp-2 cells were grown on glass coverslips and infected with Chlamydia as described above. For immunofluorescence analysis, cells were fixed for 20 minutes at room temperature in 1% formaldehyde (37% stock contains 10-15% methanol, Fisher Scientific, Hanover Park, IL). Fixation with this concentration of formaldehyde permeabilized the host cell membrane, without permeabilizing intracellular vesicular membranes or the chlamydial inclusion membrane. To subsequently permeabilize these intracellular membranes, cells were treated for 1 minute with 1 mg/ml Zwittergent®. After blocking in 5% goat serum/PBS, cells were incubated with the indicated primary antibodies for 20 minutes. Cells were washed three times in blocking buffer and subsequently incubated with the indicated fluorophore-conjugated secondary antibody for 20 minutes. Controls omitting the primary antibody were consistently negative at the concentration of fluorophore-conjugated secondary antibodies used in these studies. Incubating with 0.2 µM TOPRO-3 (monomeric cyanine nucleic acid stain, Abs 642nm/Em 661nm) for 10 minutes at room temperature labeled both the intracellular bacteria and host cell nuclei. For colocalization studies of host cell markers to the chlamydial inclusion, TOPRO-3-labeling of intracellular bacteria eliminated any potential artifacts that may be encountered with multiple antibody labeling using both chlamydial- and host cell-specific antibodies. Coverslips were mounted on glass slides using ProLong Anti-Fade mounting medium (Molecular Probes). Images were acquired using a Zeiss LSM510 Meta laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a 63x, 1.4 numerical aperature Zeiss Plan Apochromat oil objective. Confocal Z-slices of 0.5 µm were obtained using the Zeiss LSM510 software. Orthogonal views were generated by acquiring 0.5 µm Z-slices, with an optimal interval of 0.32 µm, from the entire height of the combined Z-sections of infected cells.
Electron microscopy
For analysis of CD63 localization at the ultrastructural level, 48-hour Chlamydia-infected cells were prepared for cryo immunoelectron microscopy. Infected cells were fixed in 4% paraformaldehyde/0.1% glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM PIPES pH 7.2/0.5 mM MgCl2 for 1 hour at 4°C. Samples were then embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl2 at 4°C. Samples were trimmed, frozen in liquid nitrogen and sectioned with a Leica Ultracut UCT cryo-ultramicrotome (Leica Microsystems Inc., Bannockburn, IL). 70-nm-sections were blocked with 5% fetal bovine serum/5% goat serum for 30 minutes and subsequently incubated with the indicated primary antibody overnight at 4°C. Sections were then washed in blocking buffer and probed with the appropriate 18 nm colloidal gold-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 hour at room temperature. Sections were washed in PIPES buffer followed by an extensive water rinse and stained with 1% uranyl acetate/1.6% methyl cellulose (Ted Pella Inc., Redding, CA). Samples were viewed with a JEOL 1200EX transmission electron microscope (JEOL USA Inc., Peabody, MA). All labeling experiments were conducted in parallel with controls omitting the primary antibody. These controls were consistently negative at the concentration of colloidal gold-conjugated secondary antibodies used in these studies.
For quantification of anti-CD63 label by electron microscopy, the density of colloidal gold particles per cross-sectional area of the inclusion, MVBs, mitochondria or nucleus was determined. Twenty micrographs of Chlamydia-infected cells were analyzed. The cross-sectional area of the defined structures was determined by using a systematic array of test points as described by Griffiths (Griffiths, 1993).
For analysis of the effect of exogenous antibody and inhibitors on inclusion morphology at the ultrastructural level, infected cells were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc.) in 100 mM phosphate buffer pH 7.2 for 1 hour at room temperature. Following three washes in phosphate buffer, cells were postfixed in 1% OsO4 (Polysciences Inc.) for 1 hour. The samples were then rinsed extensively in dH2O before en-bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 hour. Following several rinses in dH2O, cells were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 70-80 nm were cut, stained with uranyl acetate and lead citrate and viewed by transmission electron microscopy.
Exogenous antibody loading
HEp-2 cells were grown on glass coverslips and infected with Chlamydia as described above. Twenty-four hours post infection, cells were washed with PBS and incubated with Iscove's medium containing 50 µg/ml of mouse anti-CD63 antibody (MX-49.129.5) and the same concentration of rabbit anti-LAMP-1 antibody (H-228) or 100 µg/ml dextran-FITC. At 48 hours post infection, cells were harvested for analysis of antibody uptake by indirect immunofluorescence with anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 antibodies. Effects of exogenous antibody on inclusion development and morphology were determined by infectivity assays and TOPRO-3 labeling, respectively.
Analysis of inhibitors
HEp-2 cells were infected with Chlamydia as described above. One hour post infection, cells were washed with PBS and incubated with Iscove's medium containing 3-methyladenine, LY-294002 hydrochloride or U18666A at the indicated concentrations for the 48-hour time-course of infection. Effects of inhibitors on inclusion development, morphology and sphingolipid trafficking were determined by infectivity assays, immunofluorescence labeling with anti-chlamydial hsp60 antibody and NBD-ceramide labeling, respectively.
Infectivity assays
At 48 hours post infection, infected monolayers cultured in the presence of inhibitors or exogenous antibodies were washed with PBS, scraped from the culture dishes into fresh Iscove's medium, and sonicated to disrupt the HEp-2 cells and release infectious EBs. Dilutions of the disrupted cell suspensions were inoculated onto fresh monolayers of HEp-2 cells as described above. At 48 hours post infection, the monolayers were fixed, immunolabeled with anti-chlamydial hsp60 antibody, and visualized with a Zeiss Axioskop Mot Plus fluorescence microscope to determine the number of infectious forming units.
Ceramide labeling
Chlamydia-infected monolayers were incubated with 5 µM fluorescent NBD-C6-ceramide complexed with BSA (1:1 mol:mol) for 30 minutes at 4°C as described previously (Hackstadt et al., 1995; Lipsky and Pagano, 1985a). After washing in HBSS, cells were incubated in Iscove's medium/0.034% dfBSA for 1 hour at 37°C to back-exchange excess label from the plasma membrane and incorporate the probe intracellularly. Cells were fixed, processed for indirect immunolabeling, and analyzed by confocal microscopy as described above.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
Al-Younes, H. M., Brinkmann, V. and Meyer, T. F. (2004). Interaction of Chlamydia trachomatis serovar L2 with the host autophagic pathway. Infect. Immun. 72, 4751-4762.
Alpy, F., Stoeckel, M. E., Dierich, A., Escola, J. M., Wendling, C., Chenard, M. P., Vanier, M. T., Gruenberg, J., Tomasetto, C. and Rio, M. C. (2001). The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein. J. Biol. Chem. 276, 4261-4269.
Caldwell, H. D., Kromhout, J. and Schachter, J. (1981). Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31, 1161-1176.
Carabeo, R. A., Grieshaber, S. S., Fischer, E. and Hackstadt, T. (2002). Chlamydia trachomatis induces remodeling of the actin cytoskeleton during attachment and entry into HeLa cells. Infect. Immun. 70, 3793-3803.
Carabeo, R. A., Mead, D. J. and Hackstadt, T. (2003). Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc. Natl. Acad. Sci. USA 100, 6771-6776.
Denzer, K., Kleijmeer, M. J., Heijnen, H. F., Stoorvogel, W. and Geuze, H. J. (2000). Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113, 3365-3374.[Abstract]
Escola, J. M., Kleijmeer, M. J., Stoorvogel, W., Griffith, J. M., Yoshie, O. and Geuze, H. J. (1998). Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273, 20121-20127.
Fernandez-Borja, M., Wubbolts, R., Calafat, J., Janssen, H., Divecha, N., Dusseljee, S. and Neefjes, J. (1999). Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity. Curr. Biol. 9, 55-58.[CrossRef][Medline]
Futerman, A. H., Stieger, B., Hubbard, A. L. and Pagano, R. E. (1990). Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus. J. Biol. Chem. 265, 8650-8657.
Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J., Gaullier, J. M., Parton, R. G. and Stenmark, H. (2000). Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577-4588.[CrossRef][Medline]
Grayston, J. T. (2000). Background and current knowledge of Chlamydia pneumoniae and atherosclerosis. J. Infect. Dis. 181, S402-S410.[CrossRef][Medline]
Griffiths, G. (1993). Fine Structure Immuno-Cytochemistry. Berlin, Heidelberg and New York: Springer-Verlag.
Hackstadt, T., Scidmore, M. A. and Rockey, D. D. (1995). Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc. Natl. Acad. Sci. USA 92, 4877-4881.
Hackstadt, T., Rockey, D. D., Heinzen, R. A. and Scidmore, M. A. (1996). Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. EMBO J. 15, 964-977.[Medline]
Hatch, T. P. (1975). Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis. J. Bacteriol. 122, 393-400.
Heinzen, R. A., Scidmore, M. A., Rockey, D. D. and Hackstadt, T. (1996). Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect. Immun. 64, 796-809.[Abstract]
Higgins, M. E., Davies, J. P., Chen, F. W. and Ioannou, Y. A. (2001). Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol. Genet. Metabol. 68, 1-13.
Kallen, C. B., Billheimer, J. T., Summers, S. A., Stayrook, S. E., Lewis, M. and Strauss, J. F., III (1998). Steriodogenic acute regulatory protein (StAR) is a sterol transfer protein. J. Biol. Chem. 273, 26285-26288.
Klionsky, D. J. and Emr, S. D. (2000). Autophagy as a regulated pathway of cellular degradation. Science 290, 1717-1721.
Kobayashi, T., Stang, E., Fang, K. S., de Moerloose, P., Parton, R. G. and Gruenberg, J. (1998). A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193-197.[CrossRef][Medline]
Kobayashi, T., Beuchat, M. H., Lindsay, M., Frias, S., Palmiter, R. D., Sakuraba, H., Parton, R. G. and Gruenberg, J. (1999). Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat. Cell Biol. 1, 113-118.[CrossRef][Medline]
Kobayashi, T., Vischer, U. M., Rosnoblet, C., Lebrand, C., Lindsay, M., Parton, R. G., Kruithof, E. K. and Gruenberg, J. (2000). The tetraspanin CD63/lamp3 cycles between endocytic and secretory compartments in human endothelial cells. Mol. Biol. Cell 11, 1829-1843.
Kobayashi, T., Beuchat, M. H., Chevallier, J., Makino, A., Mayran, N., Escola, J. M., Lebrand, C., Cosson, P. and Gruenberg, J. (2002). Separation and characterization of late endosomal membrane domains. J. Biol. Chem. 277, 32157-32164.
Kolter, T., Doering, T., Wilkening, G., Werth, N. and Sandhoff, K. (1999). Recent advances in the biochemistry of glycosphingolipid metabolism. Biochem. Soc. Trans. 27, 409-415.[Medline]
Kundra, R. and Kornfeld, S. (1998). Wortmannin retards the movement of the mannose 6-phosphate/insulin-like growth factor II receptor and its ligand out of endosomes. J. Biol. Chem. 273, 3848-3853.
Lipsky, N. G. and Pagano, R. E. (1983). Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc. Natl. Acad. Sci. USA 80, 2608-2612.
Lipsky, N. G. and Pagano, R. E. (1985a). Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J. Cell Biol. 100, 27-34.
Lipsky, N. G. and Pagano, R. E. (1985b). A vital stain for the Golgi apparatus. Science 228, 745-747.
Liscum, L. and Faust, J. R. (1992). The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one. J. Biol. Chem. 264, 11796-11806.
Lucocq, J. and Walker, D. (1997). Evidence for fusion between multilamellar endosomes and autophagosomes in HeLa cells. Eur. J. Cell Biol. 72, 307-313.[Medline]
McClarty, G. (1994). Chlamydiae and the biochemistry of intracellular parasitism. Trends Microbiol. 2, 157-164.[CrossRef][Medline]
Newhall, W. J. (1988). Macromolecular and Antigenic Composition of Chlamydiae. Boca Raton: CRC Press.
Pan, B.-T., Teng, K., Wu, C., Adam, M. and Johnstone, R. M. (1985). Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J. Cell Biol. 101, 942-948.
Piper, R. C. and Luzio, J. P. (2001). Late endosomes: sorting and partitioning in multivesicular bodies. Traffic 2, 612-621.[CrossRef][Medline]
Rockey, D. D., Fischer, E. R. and Hackstadt, T. (1996). Temporal analysis of the developing Chlamydia psittaci inclusion by use of fluorescence and electron microscopy. Infect. Immun. 64, 4269-4278.[Abstract]
Schachter, J. (1988). Overview of Human Diseases. Boca Raton: CRC Press.
Scidmore, M. A., Fischer, E. R. and Hackstadt, T. (2003). Restricted fusion of Chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection. Infect. Immun. 71, 973-984.
Seglen, P. O. and Gordon, P. B. (1982). 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl. Acad. Sci. USA 79, 1889-1892.
Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J. and Raposo, G. (2002). The biogenesis and functions of exosomes. Traffic 3, 321-330.[CrossRef][Medline]
Taraska, T., Ward, D. M., Ajioka, R. S., Wyrick, P. B., Davis-Kaplan, S. R., Davis, C. H. and Kaplan, J. (1996). The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins. Infect. Immun. 64, 3713-3727.[Abstract]
Van Deurs, B., Holm, P. K., Kayser, L., Sandvig, K. and Hansen, S. H. (1993). Multivesicular bodies in HEp-2 cells are maturing endosomes. Eur. J. Cell Biol. 61, 208-224.[Medline]
Van Ooij, C., Kalman, L., Van Ijzendoorn, S., Nishijima, M., Hanada, K., Mostov, K. and Engel, J. N. (2000). Host cell-derived sphingolipids are required for the intracellular growth of Chlamydia trachomatis. Cell. Microbiol. 2, 627-637.[CrossRef][Medline]
Vanier, M. T. and Millat, G. (2003). Niemann-Pick disease type C. Clin. Genet. 64, 269-281.[CrossRef][Medline]
Vidal, M., Mangeat, P. and Hoekstra, D. (1997). Aggregation reroutes molecules from a recycling to a vesicle-mediated secretion pathway during reticulocyte maturation. J. Cell Sci. 110, 1867-1877.[Abstract]
Watari, H., Arakane, F., Moog-Lutz, C., Kallen, C. B., Tomasetto, C., Gerton, G. L., Rio, M.-C., Baker, M. E. and Strauss, J. F., III (1997). MLN64 contains a domain with homology to the steriodogenic acute regulatory protein (StAR) that stimulates steriodogenesis. Proc. Natl. Acad. Sci. USA 94, 8462-8467.
Wolf, K. and Hackstadt, T. (2001). Sphingomyelin trafficking in Chlamydia pneumoniae-infected cells. Cell. Microbiol. 3, 145-152.[CrossRef][Medline]
Wylie, J. L., Hatch, G. M. and McClarty, G. (1997). Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis. J. Bacteriol. 179, 7233-7242.
This article has been cited by other articles:
|
|
 |
|
  J. L. Cocchiaro, Y. Kumar, E. R. Fischer, T. Hackstadt, and R. H. Valdivia From the Cover: Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole PNAS, July 8, 2008; 105(27): 9379 - 9384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. Beatty Late Endocytic Multivesicular Bodies Intersect the Chlamydial Inclusion in the Absence of CD63 Infect. Immun., July 1, 2008; 76(7): 2872 - 2881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Huang, S. Gilfillan, S. Kim, B. Thompson, X. Wang, A. J. Sant, D. H. Fremont, O. Lantz, and T. H. Hansen MR1 uses an endocytic pathway to activate mucosal-associated invariant T cells J. Exp. Med., May 12, 2008; 205(5): 1201 - 1211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Shivshankar, L. Lei, J. Wang, and G. Zhong Rottlerin Inhibits Chlamydial Intracellular Growth and Blocks Chlamydial Acquisition of Sphingolipids from Host Cells Appl. Envir. Microbiol., February 15, 2008; 74(4): 1243 - 1249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Brumell and M. A. Scidmore Manipulation of Rab GTPase Function by Intracellular Bacterial Pathogens Microbiol. Mol. Biol. Rev., December 1, 2007; 71(4): 636 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cortes, K. A. Rzomp, A. Tvinnereim, M. A. Scidmore, and B. Wizel Chlamydia pneumoniae Inclusion Membrane Protein Cpn0585 Interacts with Multiple Rab GTPases Infect. Immun., December 1, 2007; 75(12): 5586 - 5596. [Abstract] |
