Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole

The Chlamydia Inclusion Is Enveloped in Neutral Lipid-Rich Reticular Structures and Lipid Droplets (LDs).

The fluorescent neutral lipid dye BODIPY 493/503 labels reticular structures and scattered “classical” LDs in HeLa cells grown in standard tissue culture media. In contrast, when HeLa cells are infected with C. trachomatis genital serovars, ocular serovars, or the mouse pneumonitis strain MoPn, the periphery and lumen of inclusions are prominently labeled with BODIPY (Fig. 1A). Because HeLa cells do not accumulate the large LDs commonly observed in adipocytes and lipid-loaded cells, we enhanced LD formation by adding 100 μM oleic acid to growth media or overexpressing ADRP and assessed LD interaction with the inclusion. Neither of these treatments adversely affected chlamydial replication (data not shown). Both BODIPY- and EGFP-ADRP positive LDs preferentially associated with the periphery of inclusions as early as 18 h after infection (P < 0.01, Student's t test, for combined association vs. nonassociation phenotypes at 18 and 24 h) [Fig. 1 B–D and supporting information (SI) Fig. S1]. Based on these results, we postulate that the BODIPY-positive reticular structures enveloping the inclusion constitute sites of neutral lipid biosynthesis and, by extension, LD assembly.

LDs Are Translocated into the Lumen of the Chlamydia Inclusion.

We also observed BODIPY-positive droplets in close association with inclusion membranes and within the inclusion lumen, especially when LD formation was enhanced by oleic acid treatment (e.g., Fig. 1 A and B, arrows). To confirm that these neutral lipid-rich droplets were within the inclusion lumen, even in the absence of lipid-loading, we costained the inclusion membrane with antibodies against Inclusion membrane protein G (IncG) and assessed the subcellular localization of BODIPY-positive structures by laser scanning confocal microscopy (LSCM). Although the detergent permeabilization led to a reduction in BODIPY staining from reticular structures and RBs, droplet-like material was readily apparent in the inclusion lumen (Fig. 2A). Intrainclusion and inclusion membrane-associated BODIPY-positive droplets of various sizes were present in >70% of inclusions (data not shown).

Because we could not distinguish between bona fide LDs and aggregates of neutral lipid-rich membranes by light microscopy, we performed ultrastructural analysis of infected cells to determine the nature of these structures. LDs are disrupted by fixatives and organic solvents commonly used for electron microscopy applications. Therefore, we specifically preserved lipid-rich structures by fixing infected cells in the presence of malachite green (20) before processing the samples for transmission electron microscopy (TEM). This fixation methodology revealed previously unappreciated structural complexity inside the inclusion lumen, including intact LDs (Fig. 2 B and C). Intrainclusion LDs displayed all of the features of cytoplasmic LDs, including a thin phospholipid monolayer and weak staining of the lipid core. Intrainclusion LDs were often associated with the inclusion membranes and bacterial outer membranes (Fig. 2B). LDs entering the inclusion were occasionally surrounded by membrane blebs and vesicles (Fig. 2C), presumably originating from the inclusion membrane. In addition, the inclusion lumen displayed a significant amount of debris including electron dense material and membranous structures of unknown origin (Fig. 2B). Overall, our LSCM and TEM observations established the presence of LDs in the inclusion lumen and led us to hypothesize that these organelles are translocated from the cytoplasm of the infected cell.

Inclusion Membrane Protein IncA Cofractionates with LDs and Accumulates in the Inclusion Lumen.

TEM analysis revealed LDs at different stages of translocation into the inclusion lumen, with LDs occasionally remaining associated with inclusion membranes (Fig. 3A). Therefore, we predicted that a portion of inclusion membranes would cofractionate with LDs. To test this, we isolated LDs from infected and uninfected HeLa cells grown in 100 μM oleic acid for 12 h to obtain enough material for reliable biochemical analysis. Because Rab and 14–3-3 proteins have been reported to bind to inclusion membranes (21, 22) and copurify with LDs (18), we tested whether these proteins displayed differential association with LDs during infection. Rab1 and Rab11 cofractionated with LDs, but this association was independent of Chlamydia infection (Fig. 3B). In contrast, we were unable to detect 14–3-3β in LDs. Next, we assessed a variety of Inc proteins including IncG, IncA, CT223, CT229, and the nonclassical Inc protein, Cap1 for presence in LDs. Interestingly, only IncA significantly associated with purified LDs (Fig. 3B), suggesting that inclusion membranes do not cofractionate in bulk with translocating LDs.

The relative enrichment of IncA with LDs suggested that IncA may mark sites on the inclusion membrane permissive for LD translocation. As such, we predicted that IncA-positive membranes should accumulate in the inclusion lumen. To test this, HeLa cells were infected for 18 and 24 h, processed for immunofluorescence with anti-IncA and anti-IncG antibodies, and analyzed by LSCM. IncA localized to the inclusion membrane as described in ref. 23, to distinct intrainclusion aggregates (Fig. 3 C and D). These aggregates did not colocalize with chlamydial LPS (data not shown), suggesting that IncA in the lumen was not associated with bacterial membranes. Because these intrainclusion structures were recognized by two different sources of anti-IncA polyclonal antibodies (Fig. 3C and not shown) and an anti-IncA monoclonal antibody (Fig. 3D), it is unlikely that these structures are artifacts of immunostaining procedures. In contrast, IncG was primarily restricted to the inclusion membrane (Fig. 3 C and D). The association of IncA-positive membranes and LDs is likely transitory as intrainclusion BODIPY-positive droplets only show partial colocalization with lumenal IncA by LSCM (Fig. 3E). Based on these observations, we propose that LD-associated IncA represents segments of inclusion membrane that invaginate into the lumen and remain transiently associated with intrainclusion LDs.

Lda3 Binds to the Inclusion Membrane, and Lda3-Tagged LDs Are Translocated into the Inclusion Lumen.

We hypothesized that the chlamydial Lda proteins (12) might participate in the capture, translocation and eventual processing of LDs in the inclusion lumen. Lda3, in particular, is an attractive candidate as a mediator of LD recognition at the inclusion surface. Lda3 is conserved among Chlamydiae, anti-Lda3 antibodies label reticular structures closely associated with the cytoplasmic face of the inclusion membrane, and Lda3-EGFP expressed in mammalian cells localizes to LDs (12). To begin to evaluate the role of Lda3 in coopting LDs, we followed the fate of ectopically expressed Lda3 in Chlamydia-infected cells. HeLa cells were transfected with an Lda3-EGFP expression vector, infected for 18 and 32 h, and imaged by LSCM. Surprisingly, Lda3-EGFP prominently labeled the inclusion membrane in addition to LDs, especially at later stages in infection (Fig. 4A). These results indicated that Lda3 could potentially provide a physical link between LDs and the inclusion. We also detected Lda3-EGFP within the inclusion lumen (Fig. 4B), suggesting that cytoplasmic Lda3 had translocated across the inclusion membrane. When Lda3-DsRed was expressed in oleic acid-treated cells, LDs that were positive for both Lda3-DsRed and BODIPY were observed in the cytoplasm and inclusion lumen, suggesting that the intrainclusion Lda3-positive structures are likely LDs (Fig. 4C).

We took advantage of the dual tropism of Lda3-EGFP to monitor the interaction of LDs with the inclusion in real time. Selected frames from a time-lapse series show the translocation of Lda3-tagged LDs docked on the inclusion membrane into the lumen (Fig. 4D and Movie S1). Lda3-labeled LDs were present close to the inclusion and in contact with the inclusion membrane. Furthermore, time-lapse microscopy revealed that the association of Lda3-tagged LDs with inclusion membranes is dynamic, with docking and partial penetration stages that are reversible, especially in early- to mid-cycle inclusions (<24 h) (Movie S2). However, LDs bound to the inclusion membrane in mature inclusions (>30 h) either ceased to move or displayed restricted movement on the plane of the membrane (Movie S1).

LDs are heterogenous organelles with distinct protein compositions that may reflect different cellular functions (24). To determine whether Lda3 labels all LDs, we expressed EGFP and Lda3-EGFP in lipid-loaded HeLa cells and monitored the colocalization of Lda3 with ADRP, a pan-LD marker. Untransfected and EGFP-expressing cells displayed abundant ADRP-positive LDs. In contrast, Lda3-EGFP positive transfectants had a marked decrease in overall ADRP staining (Fig. 4E). The loss of ADRP was more prominent in Lda3 transfected cells compared with Lda1, the LD-binding domain of Lda2 (Lda2_LD), and a catalytic mutant of the LD-associated lipase ATGL (25) (Fig. 4F). Closer examination of LDs in transfected cells revealed that any remaining endogenous ADRP was restricted to distinct puncta on the surface of Lda3-EGFP positive LDs (Fig. 4G).

Overall, these results demonstrate that exogenously expressed Lda3 remains associated with LDs during the initial translocation into the inclusion lumen and that the association of ADRP with LDs decreases upon Lda3 overexpression.

Strains, Infections, and Cell Culture Reagents.

HeLa cells were obtained from ATCC and passaged in DMEM supplemented with 10% Fetal Bovine Serum (Invitrogen). C. trachomatis LGV-L2 was propagated and stored as EBs in SPG (0.25 M sucrose, 10 mM sodium phosphate, and 5 mM l-glutamic acid) as described in ref. 34. C. trachomatis serovar C and MoPn EBs were obtained from H. Caldwell (Rocky Mountain Laboratories, National Institutes of Health). For infections, EBs were diluted in DMEM, added to HeLa monolayers at an MOI of 0.5–1 and centrifuged at 1,600 × g for 30 min at 4°C. Cells were incubated at 37°C/5% CO₂ for 30 min. Then, cells were washed with PBS, media was replaced, and plates were returned to 5% CO₂ at 37°C for the indicated times. For lipid loading experiments, oleic acid (Sigma) was precomplexed with fatty acid-free BSA (Sigma) in PBS and emulsified by sonication. Oleic acid was added to growth media at final concentration of 100 μM.

Expression Constructs and Antibodies.

HeLa cells stably expressing EGFP-ADRP were obtained from P. Targett-Adams and J. McLauchlan (Medical Research Council Virology Unit, Institute of Virology, Glasgow, U.K.) and were derived as described in ref. 35. Lda3-EGFP and Lda3-DsRed were generated by inserting Lda3 (CT473) coding sequence (30) into pEGFP-N1 and pDsRed-N1 (Clontech). Transfections were performed with FuGene6 reagent (Roche) as detailed by the manufacturer. Antibodies were from the following sources: ADRP (ProGen Biotechnik), Nsdhl (M. Ohashi, Okasaki Institute, Okasaki, Japan), Rab1 and 14–3-3β (Santa Cruz Biotechnology), Rab11 (BD Biosciences), Omp2 (RDI) and Cap1 (M. Starnbach, Harvard Medical School, Boston, MA). Mouse monoclonal anti-IncA and anti-CT223 antibodies were obtained from D. Rockey (Oregon State University, Corvallis, OR). Anti-IncG antibodies have been described (36). Polyclonal antibodies to IncA and CT229 were generated by immunizing rabbits with purified GST-IncA (80–324 aa) and GST-CT229 (92–215 aa), respectively.

Fluorescence Microscopy.

For indirect immunofluorescence, infected cells were fixed in 3% formaldehyde and 0.025% glutaraldehyde in PBS for 20 min at room temperature. Cells were permeabilized and blocked in 0.05% saponin and 0.2% BSA/PBS (SBP) then incubated with primary antibodies to LD and bacterial proteins, followed by Alexa Fluor-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Invitrogen). For assessing intrainclusion accumulation of IncA and IncG, fixed cells were permeabilized in 0.1% Triton X-100 for 10 min and blocked in 5% BSA/PBS. For neutral lipid stains, fixed cells were incubated with a 1:1,000 dilution of a saturated acetone solution of BODIPY 493/503 (Invitrogen) in PBS. Nuclei and bacterial DNA were stained with Topro3 (Invitrogen). Fluorescence images were acquired with a Leica TCS Scanning Laser Confocal Microscope. P values shown in Figs. 3D and 4F were determined by one-way ANOVA Tukey–Kramer multiple comparisons test.

Live Cell Microscopy.

See SI Materials and Methods for experimental details.

Transmission Electron Microscopy.

Samples were fixed for 2 h at room temperature with 2.5% glutaraldehyde and 0.05% malachite green (EMS) in 0.1 M sodium cacodylate buffer, pH 6.8. Samples were post-fixed for 30 min with 0.5% osmium tetroxide and 0.8% potassium ferricyanide in 0.1 M sodium cacodylate, for 1 h in 1% tannic acid, and for 1 h in 1% uranyl acetate at room temperature. Specimens were dehydrated with a graded ethanol series, and embedded in Spurr's resin. Thin sections were cut with an RMC MT-7000 ultramicrotome (Ventana) stained with 1% uranyl acetate and Reynold's lead citrate before viewing at 80 kV on a Philips CM-10 transmission electron microscope (FEI). Digital images were acquired with an AMT digital camera system (AMT).

LD Analysis.

LDs were isolated from HeLa cells as described in ref. 37. Briefly, two T-175 flasks were either infected with LGV L2 (MOI≈5) or left uninfected. Gentamicin (100 μg/ml) and oleic acid (100 μM) were added to cells 12–14 h before harvesting LDs at the end of the infectious cycle (40 h). Cells were washed with PBS and harvested in 5 ml of TNE [20 mM Tris·Cl (pH 8.0), 120 mM NaCl, and 2 mM EDTA] containing protease inhibitors (Roche Diagnostics). The cells were lysed on ice with ≈40 strokes in a Dounce homogenizer, cell lysates were adjusted to 0.45 M sucrose; overlaid with 2 ml of each of 0.25 M, 0.15 M, and 0 M Sucrose/TNE; and centrifuged at 30,000 rpm for 90 min in an SW41 rotor (Beckman Coulter). The floating LD-enriched fat cake was collected, diluted in TNE, and refloated at 47,000 rpm for 45 min in a TLA55 rotor (Beckman Coulter). LDs were collected, and lipids were extracted with 4 vol of diethyl ether. De-lipidated proteins were precipitated with ice-cold acetone, solubilized in 0.1%SDS and 0.1N NaOH, and normalized for total protein content before SDS/PAGE and immunoblot analysis.