The chlamydial inclusion preferentially intercepts basolaterally directed sphingomyelin-containing exocytic vacuoles

Abstract

Chlamydiae replicate intracellularly within a unique vacuole termed the inclusion. The inclusion circumvents classical endosomal/lysosomal pathways but actively intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To further examine this interaction, we developed a polarized epithelial cell model to study vectoral trafficking of lipids and proteins to the inclusion. We examined seven epithelial cell lines for their ability to form single monolayers of polarized cells and support chlamydial development. Of these cell lines, polarized colonic mucosal C2BBe1 cells were readily infected with Chlamydia trachomatis and remained polarized throughout infection. Trafficking of (6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)hexanoyl)sphingosine) (NBD-C(6)-ceramide) and its metabolic derivatives, NBD-glucosylceramide (GlcCer) and NBD-SM, was analyzed. SM was retained within L2-infected cells relative to mock-infected cells, correlating with a disruption of basolateral SM trafficking. There was no net retention of GlcCer within L2-infected cells and purification of C. trachomatis elementary bodies from polarized C2BBe1 cells confirmed that bacteria retained only SM. The chlamydial inclusion thus appears to preferentially intercept basolaterally-directed SM-containing exocytic vesicles, suggesting a divergence in SM and GlcCer trafficking. The observed changes in lipid trafficking were a chlamydia-specific effect because Coxiella burnetii-infected cells revealed no changes in GlcCer or SM polarized trafficking.

Figure 1

Measurement of TEER of epithelial cell monolayers. Epithelial cells were seeded for polarization, and TEER measurements were obtained. TEER values are expressed by raw ohm values minus the ohm value of a blank insert, multiplied by the area of the insert (0.33 cm2). Cells are considered weakly polarized at 150 Ωcm2 (dotted line) and fully polarized at 200 Ωcm2 or higher. Results are representative of at least two separate experiments with a minimum of n = 4. Mean and SEM are shown.

Figure 2

Transmission electron micrographs of polarized epithelial cell monolayers. Epithelial cells were polarized and processed for TEM. Polarized epithelial cell lines that routinely form multiple cells layers included: A) Caco-2 cells, B) HEC-1-A cells and C) T84 cells. Inset, higher magnification of a tight junction (TJ) found in polarized T84 cells. Polarized epithelial cell lines that formed a single monolayer of cells included: D) C2BBe1 cells and E) MDCK1 cells. All images are representative of multiple experiments. White stars indicate individual cells; black arrows denote TJs; the scale bar is equivalent to 2 microns.

Figure 3

Infectivity of C. trachomatis serovar L2. A) C2BBe1 and MDCK1 cells were either polarized on filters or seeded subconfluently onto coverslips. As a control, HeLa cells were seeded onto filters or coverslips. Eukaryotic cells were infected with a MOI of 10–20 for 24 h prior to fixation in ice-cold ethanol at −20°C for 40 min. Indirect immunofluorescence was used to enumerate inclusions per field of view. Ten fields of view from eight separate samples were counted at 40× magnification using a Nikon Micropot-FXA microscope (Nikon USA). Mean and SEM are shown. B) Development of L2 in polarized C2BBe1 cells. Polarized C2BBe1 cells or filter-grown HeLa cells were infected with an MOI of 13 EBs per eukaryotic cell at 4°C to synchronize the infection. Cells were shifted to 37°C and harvested at the indicated times. To harvest, the infected polarized monolayers were lysed in water and diluted in fresh HBSS, and the diluted lysate was then plated onto HeLa cells. Samples were processed and enumerated as described above. Ten fields of view from three separate samples were counted at 40× magnification. Mean and SD are shown. More noninfectious reticulate bodies (RBs) present within the cell at the time of lysis will result in a lower titer. As the conversion of RBs to EBs increases, the titer increases.

Figure 4

Examination of lipid trafficking in C. trachomatis serovar L2-infected polarized C2BBe1 cells. Polarized C2BBe1 cells were infected with serovar L2 for 24 h and then labeled with 5 µm NDB-ceramide. After a 7- to 8-h back-exchange, the majority of the fluorescent lipid remains within the chlamydial inclusion [(A) 60× magnification]. At this time, lipids were extracted and resolved by TLC. Densitometry was performed using a Typhoon Phosphorimager and Image Quant (v 5.0) software (Amersham). The density received from the apical, basolateral and cellular fractions for each replicate were totaled and used to derive percentages of GlcCer (B) and SM (C) found in each fraction for each replicate. Data are representative of five separate experiments with 21 total replicates; mean and SEM are shown. Representative TLC plates from a single experiment are shown in (D). L2, purified serovar L2 EBs. UI, uninfected cells.

Figure 5

Purification of C. trachomatis serovar L2 from polarized C2BBe1 cells. EBs were purified from NBD-C6-ceramide-labeled infected polarized C2BBe1 cells. Lipids were extracted from the purified EBs and aliquots of back-exchange medium. Samples were resolved by TLC and visualized with ultraviolet light. L2, purified serovar L2 EBs; M, back-exchange medium; Std, standards.

Figure 6

Examination of lipid trafficking in C. burnetii Nine Mile phase II-infected polarized C2BBe1 cells. Polarized cells were infected with C. burnetii Nine Mile phase II for 72–86 h, then labeled with 5 µm NBD-ceramide and processed as previously described. Data shown are a combination of seven independent experiments with 23 total replicates (n). Mean and SEM are shown. Subtle changes between mock and infected cells were determined to not be statistically significant.