Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane

Abstract

Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H₂O₂ and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncA_TM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncA _TM -APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.

Keywords: Chlamydia trachomatis; Inc; inclusion membrane; proximity labeling.

Figure 1

Induction of expression of APEX2 constructs from transformed Ctr L2. Ctr L2 was transformed with anhydrotetracycline (aTc)-inducible chlamydial expression vector pASK-L2 vector containing APEX2 (A), IncA_TM-APEX2 (TM = transmembrane domain only) (B), or IncF-APEX2 (C). HeLa cells were infected as described in Materials and Methods, and expression of constructs was induced 7 h post-infection with the indicated concentrations of aTc. Infected monolayers were fixed in methanol and processed for indirect immunofluorescence to detect expression of the construct with anti-FLAG antibody (red), the inclusion membrane with an anti-IncA antibody (green), or chlamydial organisms with an anti-Ctr L2 antibody (blue). Details for primary antibodies used in this study are found in Table 2. After incubation with the appropriate secondary antibodies, coverslips were mounted. Images were taken with Olympus Fluoview 1000 Laser Scanning Confocal Microscope (60x magnification with 2x zoom). Scale bars equal 10 μm; “a” indicates inclusions with aberrant bacteria; white arrows point to foci formed by secreted IncF-APEX2; the white asterisk indicates small inclusion formed by organisms expressing IncF-APEX2.

Figure 2

Effect of APEX2 construct expression on inclusion diameter. HeLa cells were infected with Ctr L2 transformants (detailed on the x-axis) as in Figure 1, and construct expression was induced with the indicated concentrations of anhydrotetracycline (aTc) at 7 h post-infection. Cells were fixed 36 h post-infection, processed for indirect immunofluorescence, and inclusion diameters (expressed in μm, y-axis) were determined as in Materials and Methods. Data shown include two independent experiments where 100 inclusions/coverslip were evaluated. Inclusion diameter mean and standard error of the mean are shown and were graphed using GraphPad Prism 7.0. Statistical analysis of the data included a one-way ANOVA with Tukey's multiple comparisons test; statistical significance is shown with ^**** equaling p < 0.0001, and # equaling p < 0.0001 for all conditions specifically compared to IncF-APEX2 induced with 5 nM aTc.

Figure 3

Effect of APEX2 construct expression during the primary infection on plasmid stability. HeLa cells were infected with Ctr L2 transformed (detailed on the x-axis) as in Figure 1, and construct expression was induced with the indicated concentrations of anhydrotetracycline (aTc) at 7 h post-infection. At 24 h post-infection duplicate coverslips were either fixed to determine construct expression or lysed to release infectious chlamydial organisms to infect a fresh monolayer (secondary infection) in the presence of penicillin to monitor retention of plasmid. Chlamydia that did not retain the expression plasmid would become susceptible to penicillin and become morphologically aberrant. This morphological form is readily distinguished from normal chlamydial development forms by indirect immunofluorescence microscopy. Treating Ctr L2 transformants of IncA_TM-APEX2 or APEX2 with 0.1 nM aTc did not result in construct expression, and, therefore, these conditions were eliminated from analysis. 100 inclusions per coverslip were determined to contain either normal or aberrant Chlamydia (see Supplemental Figure 3). This assay had three biological replicates within two independent experiments. Data are expressed as mean and standard error of the mean of the percentage of inclusions containing aberrant organisms, were graphed, and statistically analyzed using GraphPad Prism 7.0 software. Data were analyzed for statistical significance using two-way ANOVA with Tukey's multiple comparisons post-test, and are indicated with ^**** for p < 0.0001, and ^*** for p < 0.001.

Figure 4

Effect of Inc-APEX2 expression on acquisition of Golgi-derived sphingomyelin. HeLa cells were infected with Ctr L2 transformants of IncF-APEX2, IncA_TM-APEX2, and APEX2 only and construct expression was induced 7 h post-infection. Infected monolayers were labeled with NBD-ceramide and back-exchanged to remove fluorescent lipid trafficked to the plasma membrane as described in Materials and Methods. Live cell images were taken at 14.5 or 25 h post-infection using a 60x objective of an Olympus BX60 mounted with a Nikon DS-Qi1MC digital camera. Seven to ten fields of view were taken from duplicate coverslips in two independent experiments, and integrated density (brightness resulting from acquisition of fluorescent sphingomyelin) and inclusion area values were determined using ImageJ v1.48 software (National Institutes of Health, Bethesda, MD). Representative images of the matched graphed conditions are shown in (A). Scale bars are equal to 5 μm, and white arrows indicate inclusions. In (B), the fluorescent intensities (integrated density) divided by the areas of individual inclusions were graphed as mean and standard error of the mean using GraphPad Prism 7.0 software. Statistical significance was determined using an ordinary one-way ANOVA with Tukey's multiple comparisons post-test, with ^**** indicating p < 0.0001 and ^*** indicating p < 0.001. In statistical comparison of IncF-APEX2 induced with 5 nM aTc imaged at 25 hpi with indicated data points, & specifies p < 0.001 and && specifies p <0.0001.

Figure 5

Monitoring biotinylation of the inclusion membrane after expression of IncF-APEX2. HeLa cells were infected with Ctr L2 transformant IncF-APEX2 (A), or wild-type (not transformed) Ctr L2 (B) or mock infected (C). Expression of IncF-APEX2 was induced using 0.2 nM aTc at 7 h post-infection. As described in Materials and Methods, at 27 hpi cells were labeled with biotin-phenol, fixed, and processed for indirect immunofluorescence to detect (from left to right): construct expression with an anti-FLAG antibody (red), biotinylation using streptavidin-488 (green), and the inclusion membrane using an anti-IncA antibody (blue) or DAPI (blue) to detect nuclei in uninfected cells. Negative controls (conditions not supportive of biotinylation of the inclusion membrane) in this study included Ctr transformant IncF-APEX2 not induced for expression with biotin-phenol (A, top row), Ctr transformant IncF-APEX2 induced with 0.2 nM aTc without biotin-phenol (A, bottom row), and wild-type Ctr L2 treated with both aTc and biotin-phenol (B), and mock infected HeLa (C). All images were taken with an Olympus Fluoview 1000 Laser Scanning Confocal Microscope (60x magnification with 2x zoom). Scale bars equal 10 μm. These images were obtained from coverslips that were removed from the 6-well plates used to produce the data in Figure 6.

Figure 6

Lysis conditions compatible with solubilization of inclusion membrane proteins and pulldown of biotinylated proteins. HeLa cells seeded in 6-well plates were infected with Ctr L2 transformant IncF-APEX2, wild-type Ctr L2, or mock infected with the aTc induction/treatment conditions, followed by the biotin-phenol additions as indicated. The cells remaining in the 6-well plates were lysed as described in the Materials and Methods, and cleared lysates (lysates) and the insoluble pellets (pellets) were resolved by SDS-PAGE, transferred to a PVDF membrane, and Western blotted for a transmembrane Golgi protein GM130 (A), chlamydial heat shock protein 60 (cHsp60) (B), inclusion membrane protein IncA (C), eukaryotic cytoskeletal protein tubulin (D), and biotinylated proteins with streptavidin (E). Cleared lysates were normalized for protein content and equal amounts of protein were added to magnetic beads conjugated to streptavidin to pull down biotinylated proteins from lysates. Western blot analysis using a streptavidin conjugate of eluate and unbound fractions from the pulldowns are shown in (F) and Coomassie stain of the PVDF membrane showing total protein in (G).