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Inhibition of phagolysosomal biogenesis by the Leishmania lipophosphoglycan.

Desjardins M, Descoteaux A - J. Exp. Med. (1997)

Bottom Line: Here, evidence is presented that interaction of these parasitophorous vacuoles with endocytic organelles is very limited.In contrast, vacuoles formed around L. donovani mutants lacking the cell surface lipophosphoglycan (LPG) fuse extensively with endosomes and lysosomes.Inasmuch as LPG is essential for infecting macrophages, these results suggest that inhibition of phagolysosomal biogenesis by LPG repeating units represents an intramacrophage survival strategy used by promastigotes to establish infection.

View Article: PubMed Central - PubMed

Affiliation: Département d'anatomie,Université de Montréal, Montréal, Québec, Canada, H3C 3J7.

ABSTRACT
Whereas amastigotes of the protozoan parasite Leishmania proliferate inside acidic phagolysosomal vacuoles of the macrophage, vacuoles induced by Leishmania donovani promastigotes during initiation of infection are poorly characterized. Here, evidence is presented that interaction of these parasitophorous vacuoles with endocytic organelles is very limited. In contrast, vacuoles formed around L. donovani mutants lacking the cell surface lipophosphoglycan (LPG) fuse extensively with endosomes and lysosomes. The role of LPG repeating units in the inhibition of phagosome-endosome fusion was demonstrated using two different approaches. First, genetic complementation of the LPG-defective C3PO mutant restored its ability to inhibit phagosome-endosome fusion to a degree similar to that of wild-type promastigotes. Second, opsonization of C3PO mutant cells with purified L. donovani LPG also conferred to this mutant the ability to inhibit phagosome-endosome fusion. Inasmuch as LPG is essential for infecting macrophages, these results suggest that inhibition of phagolysosomal biogenesis by LPG repeating units represents an intramacrophage survival strategy used by promastigotes to establish infection.

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Quantitative analysis of the fusion between phagosomes containing L. donovani parasites and endocytic organelles. (A) Parasites and  BSA–gold were internalized as described in Fig. 1. Macrophages were  prepared for electron microscopy and quantitative analysis of phagosome– endosome fusion performed on thin sections. The presence of a single  gold particle in a parasitophorous vacuole was scored as a fusion event.  These results represent the average of three to six experiments. Ops  C3PO, LPG-opsonized C3PO. (B) Macrophages were fed a 5- and 16-nm  BSA–gold particles mixture for 30 min, followed by either a 15-min or a  4-h incubation period to fill endosomes and lysosomes, respectively. Labeled macrophages were then infected with promastigotes for 60 min and  further incubated for 60 min. Fusion was assessed as above. These results  represent the average of two experiments. (C) Fusion properties of latex  bead–containing phagosomes present in cells coinfected with various parasites (see Fig. 4). Latex bead phagosomes were analyzed only in cells  showing the presence of a parasite on the section profile. These results  represent the average of three to four experiments.
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Figure 2: Quantitative analysis of the fusion between phagosomes containing L. donovani parasites and endocytic organelles. (A) Parasites and BSA–gold were internalized as described in Fig. 1. Macrophages were prepared for electron microscopy and quantitative analysis of phagosome– endosome fusion performed on thin sections. The presence of a single gold particle in a parasitophorous vacuole was scored as a fusion event. These results represent the average of three to six experiments. Ops C3PO, LPG-opsonized C3PO. (B) Macrophages were fed a 5- and 16-nm BSA–gold particles mixture for 30 min, followed by either a 15-min or a 4-h incubation period to fill endosomes and lysosomes, respectively. Labeled macrophages were then infected with promastigotes for 60 min and further incubated for 60 min. Fusion was assessed as above. These results represent the average of two experiments. (C) Fusion properties of latex bead–containing phagosomes present in cells coinfected with various parasites (see Fig. 4). Latex bead phagosomes were analyzed only in cells showing the presence of a parasite on the section profile. These results represent the average of three to four experiments.

Mentions: Intermixing of BSA–gold particles and L. donovani was then recorded for each combination of incubations performed. The presence of a single gold particle inside a phagosome was scored as a fusion event. For the analysis of fusion occurrence (Fig. 2), each experiment was done at least twice and a minimum of 25 Leishmania-containing phagosomes per timepoint were recorded, while extreme care was taken to avoid serial sections. For the quantitative analysis of the transfer of BSA–gold particles from endosomes to Leishmania-containing phagosomes (Table 1), the number of 5- and 16-nm gold particles was counted on at least 12–20 of the BSA–gold positive phagosome profiles at the electron microscope.


Inhibition of phagolysosomal biogenesis by the Leishmania lipophosphoglycan.

Desjardins M, Descoteaux A - J. Exp. Med. (1997)

Quantitative analysis of the fusion between phagosomes containing L. donovani parasites and endocytic organelles. (A) Parasites and  BSA–gold were internalized as described in Fig. 1. Macrophages were  prepared for electron microscopy and quantitative analysis of phagosome– endosome fusion performed on thin sections. The presence of a single  gold particle in a parasitophorous vacuole was scored as a fusion event.  These results represent the average of three to six experiments. Ops  C3PO, LPG-opsonized C3PO. (B) Macrophages were fed a 5- and 16-nm  BSA–gold particles mixture for 30 min, followed by either a 15-min or a  4-h incubation period to fill endosomes and lysosomes, respectively. Labeled macrophages were then infected with promastigotes for 60 min and  further incubated for 60 min. Fusion was assessed as above. These results  represent the average of two experiments. (C) Fusion properties of latex  bead–containing phagosomes present in cells coinfected with various parasites (see Fig. 4). Latex bead phagosomes were analyzed only in cells  showing the presence of a parasite on the section profile. These results  represent the average of three to four experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196352&req=5

Figure 2: Quantitative analysis of the fusion between phagosomes containing L. donovani parasites and endocytic organelles. (A) Parasites and BSA–gold were internalized as described in Fig. 1. Macrophages were prepared for electron microscopy and quantitative analysis of phagosome– endosome fusion performed on thin sections. The presence of a single gold particle in a parasitophorous vacuole was scored as a fusion event. These results represent the average of three to six experiments. Ops C3PO, LPG-opsonized C3PO. (B) Macrophages were fed a 5- and 16-nm BSA–gold particles mixture for 30 min, followed by either a 15-min or a 4-h incubation period to fill endosomes and lysosomes, respectively. Labeled macrophages were then infected with promastigotes for 60 min and further incubated for 60 min. Fusion was assessed as above. These results represent the average of two experiments. (C) Fusion properties of latex bead–containing phagosomes present in cells coinfected with various parasites (see Fig. 4). Latex bead phagosomes were analyzed only in cells showing the presence of a parasite on the section profile. These results represent the average of three to four experiments.
Mentions: Intermixing of BSA–gold particles and L. donovani was then recorded for each combination of incubations performed. The presence of a single gold particle inside a phagosome was scored as a fusion event. For the analysis of fusion occurrence (Fig. 2), each experiment was done at least twice and a minimum of 25 Leishmania-containing phagosomes per timepoint were recorded, while extreme care was taken to avoid serial sections. For the quantitative analysis of the transfer of BSA–gold particles from endosomes to Leishmania-containing phagosomes (Table 1), the number of 5- and 16-nm gold particles was counted on at least 12–20 of the BSA–gold positive phagosome profiles at the electron microscope.

Bottom Line: Here, evidence is presented that interaction of these parasitophorous vacuoles with endocytic organelles is very limited.In contrast, vacuoles formed around L. donovani mutants lacking the cell surface lipophosphoglycan (LPG) fuse extensively with endosomes and lysosomes.Inasmuch as LPG is essential for infecting macrophages, these results suggest that inhibition of phagolysosomal biogenesis by LPG repeating units represents an intramacrophage survival strategy used by promastigotes to establish infection.

View Article: PubMed Central - PubMed

Affiliation: Département d'anatomie,Université de Montréal, Montréal, Québec, Canada, H3C 3J7.

ABSTRACT
Whereas amastigotes of the protozoan parasite Leishmania proliferate inside acidic phagolysosomal vacuoles of the macrophage, vacuoles induced by Leishmania donovani promastigotes during initiation of infection are poorly characterized. Here, evidence is presented that interaction of these parasitophorous vacuoles with endocytic organelles is very limited. In contrast, vacuoles formed around L. donovani mutants lacking the cell surface lipophosphoglycan (LPG) fuse extensively with endosomes and lysosomes. The role of LPG repeating units in the inhibition of phagosome-endosome fusion was demonstrated using two different approaches. First, genetic complementation of the LPG-defective C3PO mutant restored its ability to inhibit phagosome-endosome fusion to a degree similar to that of wild-type promastigotes. Second, opsonization of C3PO mutant cells with purified L. donovani LPG also conferred to this mutant the ability to inhibit phagosome-endosome fusion. Inasmuch as LPG is essential for infecting macrophages, these results suggest that inhibition of phagolysosomal biogenesis by LPG repeating units represents an intramacrophage survival strategy used by promastigotes to establish infection.

Show MeSH
Related in: MedlinePlus