Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments

Abstract

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER-PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.

Figure 1. Distribution of calnexin/GFP in transfected macrophages and recruitment to Leishmania PVs

The calnexin gene was cloned into the pShooter (pCMV/ER/GFP) plasmid to obtain the pCMV/calnexin/GFP plasmid. This plasmid was purified and transfected into Raw 264.7 macrophages. Transfected cells were then processed in immuno-fluorescence assays with 4’,6-diamidino-2-phenylindole (DAPI) to reveal nuclei (blue) and anti-LAMP 1 antibody to label late endosomes and lysosomes (red). A) A representative cell that shows the calnexin/GFP distribution pattern in cells transfected with the pCMV/calnexin/GFP plasmid. Transfected cell cultures were infected with Leishmania amastigotes. Infections were stopped at varying times and processed in immunofluorescence assays. Representative images of transfected Raw 264.7 cells infected with L. pifanoi amastigotes for 2 h (B) or 12 h (C) and L. donovani amastigotes for 2 h (D) or 24 h (E) are shown. Thin white arrows point to representative parasites while thick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z-series were combined to create the images shown. Scale bar = 5 micrometers (5µm) The proportion of PVs harboring L. pifanoi (F) or L. donovani (G) parasites that are positively displaying calnexin/GFP or LAMP-1 during a 24 h course of infection were enumerated and plotted. Also included is the proportion of Zymosan phagosomes displaying calnexin/GFP (F), the proportion of dead L. pifanoi parasites (F) or dead L. donovani parasites (G). Each data point is the mean value from at least three experiments in which at least 150 vacuoles were counted per experiment. Graphs were made using the Sigma plot software. The difference between the proportions of PVs that recruited calnexin as compared to Zymosan phagosomes was tested in a paired t-test at all time points. The p-value in each case was <0.001. .

Figure 1. Distribution of calnexin/GFP in transfected macrophages and recruitment to Leishmania PVs

The calnexin gene was cloned into the pShooter (pCMV/ER/GFP) plasmid to obtain the pCMV/calnexin/GFP plasmid. This plasmid was purified and transfected into Raw 264.7 macrophages. Transfected cells were then processed in immuno-fluorescence assays with 4’,6-diamidino-2-phenylindole (DAPI) to reveal nuclei (blue) and anti-LAMP 1 antibody to label late endosomes and lysosomes (red). A) A representative cell that shows the calnexin/GFP distribution pattern in cells transfected with the pCMV/calnexin/GFP plasmid. Transfected cell cultures were infected with Leishmania amastigotes. Infections were stopped at varying times and processed in immunofluorescence assays. Representative images of transfected Raw 264.7 cells infected with L. pifanoi amastigotes for 2 h (B) or 12 h (C) and L. donovani amastigotes for 2 h (D) or 24 h (E) are shown. Thin white arrows point to representative parasites while thick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z-series were combined to create the images shown. Scale bar = 5 micrometers (5µm) The proportion of PVs harboring L. pifanoi (F) or L. donovani (G) parasites that are positively displaying calnexin/GFP or LAMP-1 during a 24 h course of infection were enumerated and plotted. Also included is the proportion of Zymosan phagosomes displaying calnexin/GFP (F), the proportion of dead L. pifanoi parasites (F) or dead L. donovani parasites (G). Each data point is the mean value from at least three experiments in which at least 150 vacuoles were counted per experiment. Graphs were made using the Sigma plot software. The difference between the proportions of PVs that recruited calnexin as compared to Zymosan phagosomes was tested in a paired t-test at all time points. The p-value in each case was <0.001. .

Figure 1. Distribution of calnexin/GFP in transfected macrophages and recruitment to Leishmania PVs

The calnexin gene was cloned into the pShooter (pCMV/ER/GFP) plasmid to obtain the pCMV/calnexin/GFP plasmid. This plasmid was purified and transfected into Raw 264.7 macrophages. Transfected cells were then processed in immuno-fluorescence assays with 4’,6-diamidino-2-phenylindole (DAPI) to reveal nuclei (blue) and anti-LAMP 1 antibody to label late endosomes and lysosomes (red). A) A representative cell that shows the calnexin/GFP distribution pattern in cells transfected with the pCMV/calnexin/GFP plasmid. Transfected cell cultures were infected with Leishmania amastigotes. Infections were stopped at varying times and processed in immunofluorescence assays. Representative images of transfected Raw 264.7 cells infected with L. pifanoi amastigotes for 2 h (B) or 12 h (C) and L. donovani amastigotes for 2 h (D) or 24 h (E) are shown. Thin white arrows point to representative parasites while thick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z-series were combined to create the images shown. Scale bar = 5 micrometers (5µm) The proportion of PVs harboring L. pifanoi (F) or L. donovani (G) parasites that are positively displaying calnexin/GFP or LAMP-1 during a 24 h course of infection were enumerated and plotted. Also included is the proportion of Zymosan phagosomes displaying calnexin/GFP (F), the proportion of dead L. pifanoi parasites (F) or dead L. donovani parasites (G). Each data point is the mean value from at least three experiments in which at least 150 vacuoles were counted per experiment. Graphs were made using the Sigma plot software. The difference between the proportions of PVs that recruited calnexin as compared to Zymosan phagosomes was tested in a paired t-test at all time points. The p-value in each case was <0.001. .

Figure 2. Distribution of Sec22b/YFP in transfected macrophages and recruitment of Sec22b to Leishmania PVs

Sec22b was cloned into the pmVenus based plasmids and used to transfect Raw 264.7 macrophages (Hatsuzawa et al, 2006). Transfected cells were processed in immuno-fluorescence assays with DAPI (blue) to reveal nuclei; and anti-LAMP-1 (red) to reveal late endosomes and lysosomes. A) A representative transfected Raw 264.7 cell expressing Sec22/YFP is shown. Transfected cells were infected with L. pifanoi amastigotes for 2 h (B) and 24 h (C) or L. donovani amastigotes for 2 h (D) and 24 h (E) and then processed in immunofluorescence assays. The figures show representative images of PVs in infected cells. Images were captured on a Zeiss Axiovert 200M microscope with a plan neofluar 100x/1.3 oil immersion objective. Images series over a defined z-focus range were acquired and processed with 3D deconvolution software supplied with AxioVision. An extended focus function was used to merge optical sections to generate images presented in the figures. Scale bar = 5 micrometers (5µm). Thin arrows point to representative parasites; thick arrows point to the PV membrane. The proportion of L. pifanoi and L. donovani PVs that positively recruited Sec22b/YFP during a 48 h course of infection was estimated and plotted (F). More than 150 PVs containing live Leishmania parasites were screened. The experiments were repeated at least three times, graphs of the mean values were made using the Sigma plot software.

Figure 2. Distribution of Sec22b/YFP in transfected macrophages and recruitment of Sec22b to Leishmania PVs

Sec22b was cloned into the pmVenus based plasmids and used to transfect Raw 264.7 macrophages (Hatsuzawa et al, 2006). Transfected cells were processed in immuno-fluorescence assays with DAPI (blue) to reveal nuclei; and anti-LAMP-1 (red) to reveal late endosomes and lysosomes. A) A representative transfected Raw 264.7 cell expressing Sec22/YFP is shown. Transfected cells were infected with L. pifanoi amastigotes for 2 h (B) and 24 h (C) or L. donovani amastigotes for 2 h (D) and 24 h (E) and then processed in immunofluorescence assays. The figures show representative images of PVs in infected cells. Images were captured on a Zeiss Axiovert 200M microscope with a plan neofluar 100x/1.3 oil immersion objective. Images series over a defined z-focus range were acquired and processed with 3D deconvolution software supplied with AxioVision. An extended focus function was used to merge optical sections to generate images presented in the figures. Scale bar = 5 micrometers (5µm). Thin arrows point to representative parasites; thick arrows point to the PV membrane. The proportion of L. pifanoi and L. donovani PVs that positively recruited Sec22b/YFP during a 48 h course of infection was estimated and plotted (F). More than 150 PVs containing live Leishmania parasites were screened. The experiments were repeated at least three times, graphs of the mean values were made using the Sigma plot software.

Figure 3. Immuno-EM analysis of ER components recruitment to Leishmania PVs

Stably transfected J774 cells were infected with L. donovani parasites and then processed for immuno-EM analysis. Sections on Nickel grids were incubated with a rabbit anti-GFP antibody followed by a secondary antibody conjugated to 15nm gold particles. The grids were post-stained with 0.5% uranyl acetate and lead citrate and were examined with a Hitachi TEM H-7000 (Pleasanton, CA) operated at 80 kV. A-&B are representative infected J774 cells expressing Sec22b/YFP. A) PV with single parasite; B) A cell with multiple PVs; and Zoomed out area. Bold white arrows point to perinuclear ER and PV membrane lined with gold particles. Cells stably transfected with the pmVenus vector were infected as well. (C) shows a pmVenus/YFP transfected cell that is infected. Thin arrows point to gold particles randomly distributed in the cell. These images are representative of images obtained from at least 2 experiments. N= macrophage nucleus; P= Leishmania parasite

Figure 3. Immuno-EM analysis of ER components recruitment to Leishmania PVs

Stably transfected J774 cells were infected with L. donovani parasites and then processed for immuno-EM analysis. Sections on Nickel grids were incubated with a rabbit anti-GFP antibody followed by a secondary antibody conjugated to 15nm gold particles. The grids were post-stained with 0.5% uranyl acetate and lead citrate and were examined with a Hitachi TEM H-7000 (Pleasanton, CA) operated at 80 kV. A-&B are representative infected J774 cells expressing Sec22b/YFP. A) PV with single parasite; B) A cell with multiple PVs; and Zoomed out area. Bold white arrows point to perinuclear ER and PV membrane lined with gold particles. Cells stably transfected with the pmVenus vector were infected as well. (C) shows a pmVenus/YFP transfected cell that is infected. Thin arrows point to gold particles randomly distributed in the cell. These images are representative of images obtained from at least 2 experiments. N= macrophage nucleus; P= Leishmania parasite

Figure 4. Trafficking of ricin in non-infected Raw 264.7 macrophages

Raw 264.7 macrophages on coverslips were pulsed with fluoresceinated ricin at a concentration of 10ug/ml in complete RPMI medium. The cultures were rinsed and incubated with complete medium at 37°C and under 5% CO₂. Coverslips were removed after the pulse (0 min) (A) then after chase for 5 min (B); some coverslips were stained with anti-EEA1 to visualize early endosomes. Bold arrow in the merged image shows vesicles that are both EEA1 and ricin positive. Representative images after 30 min (C), and 1 h (D) and 3 h (E) show labeling of cell nucleus with DAPI dye (blue) and LAMP-1 using an anti-LAMP-1 antibody (red). Some samples were pre-treated with BFA at a concentration of 5µg/ml for 2 h before ricin treatment. Coverslips obtained after 2 h chase were labeled with anti EEA1 (F) or anti-GM130 (G) to localize compartment in which ricin was arrested after BFA treatment. Samples were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. These images are representative of images from 3 experiments. Scale bar = 5µm.

Figure 4. Trafficking of ricin in non-infected Raw 264.7 macrophages

Raw 264.7 macrophages on coverslips were pulsed with fluoresceinated ricin at a concentration of 10ug/ml in complete RPMI medium. The cultures were rinsed and incubated with complete medium at 37°C and under 5% CO₂. Coverslips were removed after the pulse (0 min) (A) then after chase for 5 min (B); some coverslips were stained with anti-EEA1 to visualize early endosomes. Bold arrow in the merged image shows vesicles that are both EEA1 and ricin positive. Representative images after 30 min (C), and 1 h (D) and 3 h (E) show labeling of cell nucleus with DAPI dye (blue) and LAMP-1 using an anti-LAMP-1 antibody (red). Some samples were pre-treated with BFA at a concentration of 5µg/ml for 2 h before ricin treatment. Coverslips obtained after 2 h chase were labeled with anti EEA1 (F) or anti-GM130 (G) to localize compartment in which ricin was arrested after BFA treatment. Samples were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. These images are representative of images from 3 experiments. Scale bar = 5µm.

Figure 5. Ricin accumulates in Leishmania PVs during infection in Raw 264.7 macrophages

Raw 264.7 macrophages were infected for at least 4h before the cultures were incubated with ricin at a concentration of 10µg/ml, and the reaction was chased at 37°C and under 5% CO₂ atmosphere. At 0 min (A), 1 h (B) and 3 h (C) the coverslips were recovered and processed to reveal nuclei (blue) and LAMP-1 (red). Thin white arrows point to parasites in the infected cells. Thick arrow points to ricin within a PV. Scale bar = 5µm. PVs harboring either L. pifanoi or L. donovani that were ricin positive were enumerated after 30 min, 1 h, 3 h and 6 h. The percentage of PVs that were ricin positive was plotted (E). Incubations were done in the presence of BFA and enumerated as well. The difference in the percentage of PVs that were positively displaying ricin was significantly different from the percentage of PV with ricin in cells incubated with BFA. * denotes a P value < 0.01. Each data point was compiled from at least 3 experiments.

Figure 5. Ricin accumulates in Leishmania PVs during infection in Raw 264.7 macrophages

Raw 264.7 macrophages were infected for at least 4h before the cultures were incubated with ricin at a concentration of 10µg/ml, and the reaction was chased at 37°C and under 5% CO₂ atmosphere. At 0 min (A), 1 h (B) and 3 h (C) the coverslips were recovered and processed to reveal nuclei (blue) and LAMP-1 (red). Thin white arrows point to parasites in the infected cells. Thick arrow points to ricin within a PV. Scale bar = 5µm. PVs harboring either L. pifanoi or L. donovani that were ricin positive were enumerated after 30 min, 1 h, 3 h and 6 h. The percentage of PVs that were ricin positive was plotted (E). Incubations were done in the presence of BFA and enumerated as well. The difference in the percentage of PVs that were positively displaying ricin was significantly different from the percentage of PV with ricin in cells incubated with BFA. * denotes a P value < 0.01. Each data point was compiled from at least 3 experiments.