Chlamydiae assemble a pathogen synapse to hijack the host endoplasmic reticulum

Abstract

Chlamydiae are obligate intracellular bacterial pathogens that replicate within a specialized membrane-bound compartment, termed an 'inclusion'. The inclusion membrane is a critical host-pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. Here we show that the host endoplasmic reticulum (ER) is specifically recruited to the inclusion, and that key rough ER (rER) proteins are enriched on and translocated into the inclusion. rER recruitment is a Chlamydia-orchestrated process that occurs independently of host trafficking. Generation of infectious progeny requires an intact ER, since ER vacuolation early during infection stalls inclusion development, whereas disruption post ER recruitment bursts the inclusion. Electron tomography and immunolabelling of Chlamydia-infected cells reveal 'pathogen synapses' at which ordered arrays of chlamydial type III secretion complexes connect to the inclusion membrane only at rER contact sites. Our data show a supramolecular assembly involved in pathogen hijack of a key host organelle.

Figure 1. The Chlamydia inclusion specifically recruits ER markers

A) Confocal xy-sections of HeLa cells after infection with C. trachomatis LGV2 and fixed 24 hpi. Cells were immunolabelled for ER, intermediate compartment or Golgi markers, or incubated with Mitotracker Orange prior to fixation as indicated (red), and co-stained for Chlamydia (green). Scale bars, 5 µm. B) Confocal xz sections as (A), acquired with a y-step of 5 µm. Scale bars, 5 µm. C) Confocal xy-sections of HeLa HA-calreticulin, DP1-HA and Rtn4b-HA transfectants fixed 24 hpi with C. trachomatis LGV2. Cells were immunolabelled for HA (green) and stained with Hoechst. Right panels show corresponding xz-stacks acquired with a y-step of 5 µm. Scale bars, 5 µm.

Figure 2. DsRed-ER is recruited to the chlamydial inclusion in live cells

HeLa DsRed-ER transfectants were infected with C. trachomatis LGV2 and individual cells imaged by confocal microscopy beginning 24 hpi. Panels show maximum intensity projections of confocal stacks of a typical infected cell imaged every 30 min. Cell periphery (outline), inclusion (I) and cell nucleus (N) are indicated in the first two panels for clarity. DsRed-ER (greyscale) accumulates at the inclusion periphery (an example indicated with an arrow in panel 10), and translocates into the inclusion lumen (an example indicated with an arrowhead in panel 12). Scale bar, 5 µm.

Figure 3. Calreticulin recruitment is independent of cell type and Chlamydia species/serovar

For each cell type and Chlamydia strain, fixation was performed at 24 hpi followed by immunolabelling for calreticulin (red) and Chlamydia (green). xy- and xz-stacks were acquired by confocal microscopy. Scale bars, 5 µm. A) Endometrial cell line RL95-2 infected with C. trachomatis LGV2. B) Cervical cell line HeLa infected with C. trachomatis serovar D. C) HeLa cells infected with Chlamydia muridarum.

Figure 4. Biphasic recruitment of ER during the Chlamydia infection cycle

A) Quantitative analysis of calreticulin recruitment during the Chlamydia infection cycle. HeLa cells were infected with C. trachomatis LGV2 and fixed at different times post-infection. Left plot shows early time points when bacteria are not yet organized as an inclusion. The behaviour of live (black) and heat-killed (white) bacteria is not statistically different (p < 0.05). Right plot shows later stages, when bacteria are progressively organized into a mature inclusion. B) Quantitative analysis of Mitotracker Orange recruitment during the Chlamydia infection cycle. Left plot shows early time points when bacteria are not yet organized as an inclusion. Right plot shows later stages, when bacteria are progressively organized into a mature inclusion.

Figure 5. Calreticulin recruitment requires bacterial protein synthesis, but occurs independently of ER-Golgi trafficking

A) HeLa cells were infected with C. trachomatis LGV2, treated with chloramphenicol 12 hpi (5 or 15 µg/mL; CHP-5, CHP-15), and fixed 24 hpi. Calreticulin (red) and Chlamydia (green) were immunolabelled and imaged by confocal microscopy. Scale bar, 5 µm. Quantification of calreticulin at the inclusion following chloramphenicol treatment was performed using the second Manders coefficient (red pixels colabelled green) from confocal z-sections. Error bars represent standard deviation (***p < 0.001). B) HeLa cells were infected with C. trachomatis LGV2. Brefeldin A (14 hpi; BFA, 10 or 20 µg/mL; BFA-10, BFA-20) or nocodazole (14 hpi; NZ, 10 or 20 ng/mL; NZ-10, NZ-20) was added to the media and cells fixed 24 hpi. Cells were immunolabelled for calreticulin (red) and Chlamydia (green) and z-stacks acquired by confocal microscopy. Upper panels show confocal xy-section of a typical infected cell treated with 20 µg/mL BFA or 20 ng/mL NZ. Scale bars, 5 µm. Quantification of calreticulin at the inclusion was performed using the second Manders coefficient (red pixels colabelled green) from z-sections. No significant differences were observed between the control and the treated cells (p < 0.05). Error bars represent the standard deviation. C) HeLa cells were fixed 24 hpi with C. trachomatis LGV2 and immunolabelled for CERT (green), calreticulin (red) and Chlamydia (AlexaFluor 633 pseudocoloured blue). Left panel shows a representative confocal xy-section through a chlamydial inclusion containing typical calreticulin and CERT patches. The contour of the inclusion used to analyse the grey levels in each channel is shown in yellow. Scale bar, 5 µm. Histograms show pairwise comparisons of each channel following analysis of multiple inclusions, illustrating the percentage of inclusion membrane pixels stained in each channel, alone or in combination as indicated.

Figure 6. Aerolysin treatment disturbs inclusion biogenesis and abolishes infectivity

A) HeLa cells were infected with C. trachomatis LGV2. At 12 or 24 hpi, cells were treated (30 min) with pro-aerolysin (0.5 nm) and fixed 6 h later for immunolabelling for calreticulin (red) and Chlamydia (green). When aerolysin is added 12 hpi (upper), vacuolization inhibits growth of the inclusion and prevents calreticulin recruitment. When added 24 hpi (lower), the inclusion bursts and RBs disseminate into the cytosol. Scale bars, 5 µm. B) Cell layers collected after infection and treatment with aerolysin (A) were diluted in fresh medium to infect a new layer of HeLa cells. After 24 h, freshly infected cells were fixed and stained for Chlamydia and DNA. Random fields were scored to quantify inclusion-forming units per mL (IFU/mL). Error bars represent standard deviation (*p < 0.05). It was not possible to assay the effect on bacterial infectivity 12 hpi as all the intracellular bacteria in the control and treated cells were in the non-infectious RB state.

Figure 7. Aerolysin treatment arrests inclusion growth but maintains cell integrity

HeLa cells were infected with C. trachomatis LGV2. At 12 or 24 hpi, cells were treated 30 min with 0.5 nm pro-aerolysin and fixed for immunolabelling 6 h later. AlexaFluor 594-coupled wheat germ agglutinin (WGA) (red) was incubated with cells 15 min prior to Chlamydia immunolabelling (green). WGA recognizes carbohydrates present predominantly at the plasma membrane and in early endocytic vesicles. Aerolysin treatment inhibits Chlamydia inclusion development when cells are treated 12 hpi. When aerolysin is added 24 hpi, once the ER is already recruited, the inclusion membrane is disrupted and bacteria released into the cell cytoplasm. Scale bars, 5 µm. Images are average projections of 4 xy-views in z to facilitate complete visualization of the plasma membrane, representing a thickness of 1.32 µm.

Figure 8. Ionophores do not affect Chlamydia growth or inclusion membrane integrity

HeLa cells were infected with C. trachomatis LGV2. At 12 or 24 hpi, cells were treated 30 min with 2 μm of ionomycin or valinomycin, calcium and potassium ionophores, respectively. After 6 h, cells were fixed and immunolabelled for calreticulin (red) and Chlamydia (green). Scale bars, 5 µm.

Figure 9. Electron tomography reveals a pathogen synapse involving the ER

HeLa cells were infected with C. trachomatis LGV2 and high pressure frozen at 24 hpi. Tomograms were recorded from 200 nm thick sections of the freeze-substituted, embedded samples. A) Left panel shows an average of 10 z-sections after reconstruction, alignment and de-noising. The tomogram shows an example of an rER-inclusion contact. Manual segmentation (right panel) reveals the 3D organization. There is widespread contact between the inclusion membrane (bright blue) and rER (dark blue; eukaryotic ribosomes in red). RBs proximal to the inclusion membrane contain structures (brown) originating in the inner membrane (dark grey) and traversing the outer membrane (purple) to contact the inclusion membrane. Smaller, prokaryotic ribosomes are violet. Golgi and intermediate compartments (IC; green) do not contact the inclusion. Scale bars, 200 nm. B) Enlargement of the blue-boxed area in (A) showing an extensive rER-inclusion contact. Scale bar, 200 nm. (C) Enlargement of the purple-boxed area in (A) showing structures traversing the chlamydial membranes. Scale bar, 50 nm. D) Cross-section of the structures in (C) traversing the periplasm. E) Sections of two pathogen synapses showing needles in contact with the inclusion membrane–host ER contact sites and tip complexes (arrow). Scale bar, 50 nm. F) Immunogold CdsF labelling showing an RB involved in a pathogen synapse (left) and an EB (right). Scale bars, 250 nm (left) and 100 nm (right).

Figure 10. Immunogold labelling reveals a pathogen synapse with the inclusion membrane apposed to the rER

A) Lower magnification overview of part of a Chlamydia inclusion showing immunogold labelling of calreticulin (10 nm). Labelling is specifically observed at the ER membrane (blue tracing; panels 2, 3 and 5), at the bacterial membrane (panels 4, 6 and 7) and on free membrane fragments in the inclusion lumen (pink tracing; 1, 6 and 8). Scale bar, 1 µm. B) 50-nm thickness sections were immunolabelled with anti-calreticulin or anti-CdsF primary and a gold-conjugated secondary antibody. Upper panels show typical anti-calreticulin immunogold labelling on rER membranes (dark blue) contacting the inclusion membrane, on the bacterial membrane, on free membrane fragments in the inclusion (outlined in pink) and on the inclusion membrane (light blue). Scale bars, 50 nm. Lower panels show additional examples of immunogold labelling of CdsF (10 nm) showing an RB involved in a ‘pathogen synapse’ (left) and an EB (right). CdsF is a major component of the T3SS. Scale bars, 500 nm (left) and 250 nm (right).

Figure 11. T3SS assemblies at rER-inclusion contact sites with luminal RBs

HeLa cells were infected with C. trachomatis LGV2 and high pressure frozen at 24 hpi. Tomograms of 200 nm sections, showing RBs in contact with the inclusion membrane. T3SS complexes are observed when RBs contact an inclusion membrane connected to the rER (A and B), but not in the absence of ER-inclusion interaction (C) or when a smooth membrane contacts the inclusion (D). Scale bars, 100 nm.