Sequestration of host metabolism by an intracellular pathogen
- Institut Pasteur, France
- CNRS UMR3691, France
- Université de Lille, France
- Indiana University Bloomington, United States
- Indiana University School of Medicine, United States
Figures
Figure 1 with 3 supplements
Glycogen accumulation in C. trachomatis inclusions.
(A) Glycogen metabolism in bacteria. In green: glycogen synthesis. In blue: glycogen degradation. Glc1P is the substrate of GlgC for ADP-Glc synthesis. GlgA (glycogen synthase) produces linear …
Figure 1—figure supplement 1
Relocation of glycogen stores to the inclusion during infection.
Cells were (A) non-infected, or infected with C. trachomatis for (B) 24 hr or (C) 48 hr, fixed in PFA and processed for PAS stain. (D) Enlargement of the boxed region in (B) Note that glycogen …
Figure 1—figure supplement 2
Luminal and cytoplasmic glycogen differs in size.
Cells were infected for 30 hr with C. trachomatis. The picture on the right shows an enlargement of the boxed region. Glycogen is visualized by TEM after PATAg stain. Glycogen deposits in the …
Figure 1—figure supplement 3
Kinetics of glycogen accumulation.
HeLa cells were infected with C. trachomatis for 8, 16, 20, 24 or 48 hr. Glycogen is visualized by TEM after PATAg stain. White arrows point to inclusions. Glycogen is first detected in the …
Figure 2 with 2 supplements
Bulk import of cytoplasmic glycogen contributes to the accumulation of glycogen in the inclusion.
(A) Gys1 is imported into the inclusion lumen. Cells were treated with siRNA control or against Gys1 prior to infection for 30 hr. DNA was stained in blue, Gys1 in green and the inclusion membrane …
Figure 2—figure supplement 1
Luminal location of Gys1 strongly decreases in glucose-deprived cells.
HeLa cells were Glc-deprived (A) or not (B, same medium supplemented with 4.5 mg/ml Glc) for 48 hr, prior to infection with C. trachomatis. Gys1 was stained in green and the inclusion membrane in …
Figure 2—figure supplement 2
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. …
Figure 3 with 3 supplements
Bacterial glycogen metabolism enzymes are secreted in the inclusion lumen.
(A) HeLa cells were infected for 24 hr or 48 hr with GFP expressing L2 (GFP-Chlam, green) and stained with an anti-GlgX antibody (red) and Hoechst (blue). Insets to the right show enlargements of …
Figure 3—figure supplement 1
Expression profiles of genes related to glycogen metabolism.
qRT-PCR of selected genes related to glycogen metabolism was performed. Values were plotted against standard curves and cDNA was normalized with the overall chlamydial genomic DNA (gDNA) quantified …
Figure 3—figure supplement 2
Specificity of the anti-GlgX antibody.
(A) Western Blot of HeLa cells non-infected (nI) or infected (I) with LGV for 24 hr. The anti-GlgX antibody detected a band of the expected molecular weight (73 kDa), only present in the infected …
Figure 3—figure supplement 3
GlgX accumulates at the inclusion membrane.
HeLa cells were infected for 24 hr with LGV, fixed with 3% PFA and stained with the anti-GlgX antibody (green), an antibody against the inclusion protein CT813 (red) and Hoechst (blue). Insets to …
Figure 4
C. trachomatis takes up Glc6P and secretes phosphoglucomutase (MrsA).
(A) Purified EBs were incubated for 2 hr with [14C]-Glc, [14C]-Glc6P or [14C]-Glc1P in absence or presence of a 50-fold excess of non-radioactive Glc, Glc6P or Glc1P. Bacteria were subsequently …
Figure 5 with 2 supplements
Host UDP-Glc is the substrate for intraluminal glycogen synthesis.
(A) PAS staining was performed 24 hr after transfection with chlamydial Flag-GlgA. (B) Zymogram analysis. Serial dilutions of lysates of E. coli lacking endogenous glgA and transformed with …
Figure 5—figure supplement 1
Flag-GlgA is imported into the inclusion lumen and enhances luminal glycogen accumulation.
(A) HeLa cells were transfected (top right) or not (top left) with Flag-GlgA before infection, and fixed 24 hpi. PAS staining revealed an increase of intraluminal glycogen (arrowheads) in Flag-GlgA …
Figure 5—figure supplement 2
The plasmid-less strain accumulates glycogen in EBs and - to a lesser extent than the wild-type strain - in the inclusion lumen.
Cells were infected for 40 hr with the plasmid-less strain LGV 25667R. Glycogen is visualized by TEM after PATAg stain and is found in EBs (black arrow heads) and free in the inclusion lumen (white …
Figure 6 with 1 supplement
SLC35D2 imports UDP-Glc into the inclusion lumen.
(A, B) Prior to infection cells were transfected with SLC35D2-HA, and fixed 24 hpi. (A) Labelling of the HA tag (green), inclusion membrane marker Cap1 (red) and DNA (blue) show recruitment of …
Figure 6—figure supplement 1
Depletion of Gys1 does not further decrease luminal glycogen content in cells depleted for SLC35D2.
Cells were treated with control siRNA, or siRNA against SLC35D2, or SLC35D2 and Gys1 48 hr and 4 hr prior to infection. (A) Cells were fixed 48 hr after infection and PAS staining was applied. Mean …
Figure 7 with 1 supplement
Mutations in GlgA result in defect in glycogen storage capacity and loss of infectivity.
Cells were treated with either siRNA control or siRNA against Gys1 48 hr and 4 hr prior to infection with glgA mutants P2B10, P3B4 or with the parental wild-type strain. (A) PAS staining was …
Figure 7—figure supplement 1
Alignment of GlgA sequences of different bacteria.
Alignments were obtained using Clustal W. An asterisk indicates positions which have a single, fully conserved residue. A colon points to conservation between groups of strongly similar properties …
Figure 8
Glc flux in C. trachomatis infected cells.
Early during the infectious cycle (left) the inclusion contains mostly RBs. This developmental form does not accumulate glycogen and uses ATP rather than Glc6P (Omsland et al., 2012). SLC35D2, and …
Additional files
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Supplementary file 1
Primers used for cloning purposes.
- https://doi.org/10.7554/eLife.12552.023
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Supplementary file 2
List of siRNAs.
- https://doi.org/10.7554/eLife.12552.024
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Supplementary file 3
List of primers used in qRT-PCR and RT-PCR.
- https://doi.org/10.7554/eLife.12552.025
