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Sequestration of host metabolism by an intracellular pathogen.

Gehre L, Gorgette O, Perrinet S, Prevost MC, Ducatez M, Giebel AM, Nelson DE, Ball SG, Subtil A - Elife (2016)

Bottom Line: We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion.Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase.Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biologie cellulaire de l'infection microbienne, Institut Pasteur, Paris, France.

ABSTRACT
For intracellular pathogens, residence in a vacuole provides a shelter against cytosolic host defense to the cost of limited access to nutrients. The human pathogen Chlamydia trachomatis grows in a glycogen-rich vacuole. How this large polymer accumulates there is unknown. We reveal that host glycogen stores shift to the vacuole through two pathways: bulk uptake from the cytoplasmic pool, and de novo synthesis. We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion. Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase. Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.

No MeSH data available.


Related in: MedlinePlus

Host glycogen imported in bulk from the host is not of autophagic origin.Wild-type (WT) or Atg5-/- mouse embryonic fibroblasts (MEFs) were infected for 30 hr with C. trachomatis. (A) PAS staining. Glycogen accumulation was identical in both cell lines. Scale bar: 10 µm. (B) DNA is stained in blue, Gys1 in green and the inclusion membrane in red (anti-CT813). While less abundant than in HeLa cells, Gys1 (white arrows) was detected inside inclusions of both cell lines. Scale bar: 10 µm. (C) Glycogen is visualized by TEM after PATAg stain. Glycogen-filled vesicles were observed in inclusions of both cell lines. Scale bar: 500 nm.DOI:http://dx.doi.org/10.7554/eLife.12552.009
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fig2s2: Host glycogen imported in bulk from the host is not of autophagic origin.Wild-type (WT) or Atg5-/- mouse embryonic fibroblasts (MEFs) were infected for 30 hr with C. trachomatis. (A) PAS staining. Glycogen accumulation was identical in both cell lines. Scale bar: 10 µm. (B) DNA is stained in blue, Gys1 in green and the inclusion membrane in red (anti-CT813). While less abundant than in HeLa cells, Gys1 (white arrows) was detected inside inclusions of both cell lines. Scale bar: 10 µm. (C) Glycogen is visualized by TEM after PATAg stain. Glycogen-filled vesicles were observed in inclusions of both cell lines. Scale bar: 500 nm.DOI:http://dx.doi.org/10.7554/eLife.12552.009

Mentions: Our conclusion raised an obvious question: how could a large polymer appear in the inclusion lumen? Two mechanisms are conceivable: bulk translocation of host glycogen, or transport of monomeric substrates (such as nucleotide-sugars or hexose phosphates) across the inclusion membrane for de novo luminal polymerization. Gys1, the host glycogen synthase, produces linear chains of Glc and is tightly bound to glycogen (Luck, 1961; Stapleton et al., 2010). Gys1 staining with specific antibodies gave a patchy pattern in the cytoplasm, as expected since it mirrors the distribution of glycogen. In addition, we observed that the enzyme accumulated within the inclusion lumen. Staining was specific since it disappeared when Gys1 expression was knocked down using siRNA prior to infection (Figure 2A). Observation of cytoplasmic markers in the inclusion can result from post-fixation artifact (Kokes and Valdivia, 2015). When we deprived the cells of Glc for two days before infection, and thus decreased cytoplasmic glycogen, Gys1 staining in the inclusion lumen was considerably reduced, while remaining abundant in the cytoplasm. This observation shows that Gys1 appearance in the inclusion lumen requires the presence of host glycogen, and is likely not the result of a post-fixation artifact. (Figure 2—figure supplement1). Altogether these data favored the scenario of bulk import of host glycogen and glycogen-bound Gys1 into the inclusion. TEM observations also came in support of this scenario, since we could observe one or more glycogen-filled vesicular structures in the inclusion lumen of about 20% of the glycogen-positive inclusions we examined (Figure 2B). Since in some cases several membranes were observed around luminal glycogen, we hypothesized that cytoplasmic glycogen might be entrapped by an autophagy-dependent process in the host cytoplasm, before delivery to the inclusion lumen. To test this hypothesis we used an Atg5-/- mutant mouse embryonic fibroblast (MEF) cell line, which is deficient in autophagy (Kuma et al., 2004). Inclusions of Atg5-/- MEFs still harboured glycogen, Gys1 and glycogen-filled vesicles, demonstrating that the pathway of bulk host glycogen uptake is independent of autophagy (Figure 2—figure supplement 2).10.7554/eLife.12552.007Figure 2.Bulk import of cytoplasmic glycogen contributes to the accumulation of glycogen in the inclusion.


Sequestration of host metabolism by an intracellular pathogen.

Gehre L, Gorgette O, Perrinet S, Prevost MC, Ducatez M, Giebel AM, Nelson DE, Ball SG, Subtil A - Elife (2016)

Host glycogen imported in bulk from the host is not of autophagic origin.Wild-type (WT) or Atg5-/- mouse embryonic fibroblasts (MEFs) were infected for 30 hr with C. trachomatis. (A) PAS staining. Glycogen accumulation was identical in both cell lines. Scale bar: 10 µm. (B) DNA is stained in blue, Gys1 in green and the inclusion membrane in red (anti-CT813). While less abundant than in HeLa cells, Gys1 (white arrows) was detected inside inclusions of both cell lines. Scale bar: 10 µm. (C) Glycogen is visualized by TEM after PATAg stain. Glycogen-filled vesicles were observed in inclusions of both cell lines. Scale bar: 500 nm.DOI:http://dx.doi.org/10.7554/eLife.12552.009
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Related In: Results  -  Collection

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fig2s2: Host glycogen imported in bulk from the host is not of autophagic origin.Wild-type (WT) or Atg5-/- mouse embryonic fibroblasts (MEFs) were infected for 30 hr with C. trachomatis. (A) PAS staining. Glycogen accumulation was identical in both cell lines. Scale bar: 10 µm. (B) DNA is stained in blue, Gys1 in green and the inclusion membrane in red (anti-CT813). While less abundant than in HeLa cells, Gys1 (white arrows) was detected inside inclusions of both cell lines. Scale bar: 10 µm. (C) Glycogen is visualized by TEM after PATAg stain. Glycogen-filled vesicles were observed in inclusions of both cell lines. Scale bar: 500 nm.DOI:http://dx.doi.org/10.7554/eLife.12552.009
Mentions: Our conclusion raised an obvious question: how could a large polymer appear in the inclusion lumen? Two mechanisms are conceivable: bulk translocation of host glycogen, or transport of monomeric substrates (such as nucleotide-sugars or hexose phosphates) across the inclusion membrane for de novo luminal polymerization. Gys1, the host glycogen synthase, produces linear chains of Glc and is tightly bound to glycogen (Luck, 1961; Stapleton et al., 2010). Gys1 staining with specific antibodies gave a patchy pattern in the cytoplasm, as expected since it mirrors the distribution of glycogen. In addition, we observed that the enzyme accumulated within the inclusion lumen. Staining was specific since it disappeared when Gys1 expression was knocked down using siRNA prior to infection (Figure 2A). Observation of cytoplasmic markers in the inclusion can result from post-fixation artifact (Kokes and Valdivia, 2015). When we deprived the cells of Glc for two days before infection, and thus decreased cytoplasmic glycogen, Gys1 staining in the inclusion lumen was considerably reduced, while remaining abundant in the cytoplasm. This observation shows that Gys1 appearance in the inclusion lumen requires the presence of host glycogen, and is likely not the result of a post-fixation artifact. (Figure 2—figure supplement1). Altogether these data favored the scenario of bulk import of host glycogen and glycogen-bound Gys1 into the inclusion. TEM observations also came in support of this scenario, since we could observe one or more glycogen-filled vesicular structures in the inclusion lumen of about 20% of the glycogen-positive inclusions we examined (Figure 2B). Since in some cases several membranes were observed around luminal glycogen, we hypothesized that cytoplasmic glycogen might be entrapped by an autophagy-dependent process in the host cytoplasm, before delivery to the inclusion lumen. To test this hypothesis we used an Atg5-/- mutant mouse embryonic fibroblast (MEF) cell line, which is deficient in autophagy (Kuma et al., 2004). Inclusions of Atg5-/- MEFs still harboured glycogen, Gys1 and glycogen-filled vesicles, demonstrating that the pathway of bulk host glycogen uptake is independent of autophagy (Figure 2—figure supplement 2).10.7554/eLife.12552.007Figure 2.Bulk import of cytoplasmic glycogen contributes to the accumulation of glycogen in the inclusion.

Bottom Line: We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion.Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase.Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biologie cellulaire de l'infection microbienne, Institut Pasteur, Paris, France.

ABSTRACT
For intracellular pathogens, residence in a vacuole provides a shelter against cytosolic host defense to the cost of limited access to nutrients. The human pathogen Chlamydia trachomatis grows in a glycogen-rich vacuole. How this large polymer accumulates there is unknown. We reveal that host glycogen stores shift to the vacuole through two pathways: bulk uptake from the cytoplasmic pool, and de novo synthesis. We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion. Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase. Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.

No MeSH data available.


Related in: MedlinePlus