Figure 1.
Rab6A and Rab11A are important factors for Chlamydia development.
A) Selected Rab proteins were transiently silenced by transfection of specific siRNAs. At 3 d p.t., cells were infected with C. trachomatis (MOI 3) and at 44 h p.i. cells were lysed and infectious bacteria titrated on HeLa cells. Numbers of infectious bacteria in KD cells are expressed as log IFU (inclusion forming units)/ml. siRNA against Luciferase was used as a control. Values were obtained from three independent experiments and are mean±SE. Student's t-test was used to determine p-value,*<0.05. B) Transient Rab KD cells were infected with C. trachomatis (MOI 1) for 24 h. Cells were then fixed and stained with an antibody specific for chlamydial LPS. Samples were analysed at a Laser scanning confocal microscope (LSCM). Overlay of LPS staining (green) and phase contrast are shown. Images are representative of n = 3. Scale bar, 10 µm.
Figure 2.
Less infectious bacteria are recovered from stable Rab6A and Rab11A KD cells compared to control KD cells.
A) Control, Rab6A (Rab6A KD) and Rab11A (Rab11A KD) stable KD cells were infected with C. trachomatis (MOI 1) for 24 h, 48 h, 72 h and 96 h, then infectious bacteria were titrated on HeLa cells. Numbers of infectious bacteria in KD cells were expressed as log IFU (inclusion forming units)/ml. shRNA against Luciferase was used as a control. Values were obtained from three independent experiments and are mean±SE. Student's t-test was used to determine p-value, **<0.01. (B–G) Electron microscopy pictures of inclusions at 24 h p.i. (B–D) and 48 h p.i (E–G) from B,E) Control, C,F) Rab6A KD and D,G) Rab11A KD cells. (n = 3). Scale bar, 10 µm. Images B′–G′ show an enlargement of inclusions, indicated by black frames, in B,E) Control, C,F) Rab6A KD and D,G) Rab11A KD cells. Scale bar, 1 µm.
Figure 3.
GA is not fragmented in C. trachomatis-infected Rab6A and Rab11A KD cells.
A) Control, Rab6A KD and Rab11A KD cells were either infected with C. trachomatis (MOI 1) or mock-infected (uninfected). At 24 h p.i., cells were fixed and stained with antibodies specific for GM130 (red channel) and LPS (blue channel). Stable KD cells express GFP as a marker of shRNA expression. shRNA against Luciferase was used as a control. Samples were analysed at a LSCM. Merge pictures are shown. Images are representative of n = 3. Scale bar, 10 µm. B) Quantification of Golgi elements in uninfected cells. Absolute numbers of Golgi elements/cell are shown. C) Quantification of Golgi elements in C. trachomatis-infected cells. Absolute numbers of Golgi elements/cell are shown. Values were obtained from three independent experiments and are mean±SE. Student's t-test was used to determine p-value, **<0.01.
Figure 4.
Silencing of Rab6A and Rab11A expression suppresses golgin-84-dependent Golgi fragmentation.
A) Control, Rab6A KD and Rab11A KD cells were transiently silenced for the expression of golgin-84 and p115 by siRNA transfection. At 4 d p.t., cells were fixed and stained for the Golgi marker GM130 (red channel). Stable KD cells express GFP as a marker of shRNA expression. shRNA against Luciferase was used as a control. Samples were analysed at a LSCM. Overlay of GM130 and GFP are shown. Images are representative of n = 3. Scale bar, 10 µm. B) Quantification of Golgi elements in the different double KD cells. Absolute numbers of Golgi elements/cell under the different experimental conditions are depicted. Values were obtained from three independent experiments and are mean±SE. Student's t-test was used to determine p-value, **<0.01.
Figure 5.
Loss of p115 induces Golgi fragmentation in infected cells independently of Rab6A and Rab11A.
A) Control, Rab6A KD and Rab11A KD cells were transiently silenced for the expression of p115 by siRNA transfection. Control cells were additionally transfected with an siRNA targeting Luciferase. At 3 d p.t. cells were infected with C. trachomatis (MOI 3). At 44 h p.i., cells were lysed and infectious bacteria were titrated on HeLa cells. IFU/ml expressed in log scale B) Control, Rab6A KD and Rab11A KD depleted cells were infected with C. trachomatis at an MOI 1. Infected cells were fixed and stained for GM130 (red channel) and chlamydial LPS (blue channel). shRNA against Luciferase was used as a control. Stable KD cells express GFP as a marker of shRNA expression. Samples were analysed at a LSCM. Merged pictures are shown. Images are representative of n = 3. Scale bar, 10 µm. C) Quantification of Golgi fragmentation. Absolute numbers of Golgi elements/cell are depicted. (A, C) Values were obtained from three independent experiments and are mean±SE.
Figure 6.
Efficient sphingolipid transport to Chlamydia depends on Rab6A and Rab11A expression.
A) Control, Rab6A KD and Rab11A KD cells were infected with C. trachomatis (MOI 1). At 1 d p.i., cells were labeled with BODIPY-FL-Ceramide (green channel) and lipid transport to the inclusion was analysed for about 60 min by live-cell microscopy. Images represent acquisition at the 48 min time point. Inclusions are seen as black holes as GFP does not cross inclusion membranes. B) Control, Rab6A KD and Rab11A KD cells were transiently transfected with siRNA against p115 and at 3 d p.t. infected with C. trachomatis (MOI 1). At 1 d p.i., cells were labeled with BODIPY-FL-Ceramide (green channel) and lipid transport to the inclusion was analysed for about 60 min by live-cell microscopy. Images represent acquisition at the 48 min time point. Inclusions are seen as black holes as GFP does not cross inclusion membranes. Images are representative of five independent experiments. Scale bar, 10 µm. C) Quantification of lipid transport in Control, Rab6A and Rab11A KD cells and in cells simultaneously silenced for the Control and p115, or Rab6 and p115, or Rab11 and p115. Diagram shows the fluorescence intensities of the inclusions relative to that of the Golgi apparatus at 48 min. Values were obtained from three inclusions and three Golgi areas and are mean±SD. Student's t-test was used to determine p-value, *<0.05, **<0.01.