Limits...
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates.

Mesquita FS, Thomas M, Sachse M, Santos AJ, Figueira R, Holden DW - PLoS Pathog. (2012)

Bottom Line: The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS).We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication.Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, Centre for Molecular Microbiology and Infection, Imperial College London, London, United Kingdom.

ABSTRACT
Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication. Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.

Show MeSH

Related in: MedlinePlus

SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3. (C–E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375275&req=5

ppat-1002743-g003: SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3. (C–E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.

Mentions: We analysed the distribution of p62 and LC3 in HeLa cells infected with wild-type or ΔsseL mutant bacteria. Dense punctate labelling of both p62 and LC3 was detected close to SCVs, and this partly co-localized with ubiquitin (Fig. 3A and 3B). To examine the influence of SseL on these proteins, confocal microscopy was used to measure the relative fluorescence intensity of ubiquitin, p62 and LC3 labelling in SCV-associated structures. The absence of SseL significantly increased the intensity of ubiquitin, p62 and LC3 labelling, compared to cells infected with wild-type bacteria (Fig. 3C–E). This was complemented by a plasmid expressing wild-type SseL, but not by expression of SseLC/A (Fig. 3C–E).


The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates.

Mesquita FS, Thomas M, Sachse M, Santos AJ, Figueira R, Holden DW - PLoS Pathog. (2012)

SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3. (C–E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375275&req=5

ppat-1002743-g003: SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3. (C–E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.
Mentions: We analysed the distribution of p62 and LC3 in HeLa cells infected with wild-type or ΔsseL mutant bacteria. Dense punctate labelling of both p62 and LC3 was detected close to SCVs, and this partly co-localized with ubiquitin (Fig. 3A and 3B). To examine the influence of SseL on these proteins, confocal microscopy was used to measure the relative fluorescence intensity of ubiquitin, p62 and LC3 labelling in SCV-associated structures. The absence of SseL significantly increased the intensity of ubiquitin, p62 and LC3 labelling, compared to cells infected with wild-type bacteria (Fig. 3C–E). This was complemented by a plasmid expressing wild-type SseL, but not by expression of SseLC/A (Fig. 3C–E).

Bottom Line: The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS).We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication.Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, Centre for Molecular Microbiology and Infection, Imperial College London, London, United Kingdom.

ABSTRACT
Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication. Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.

Show MeSH
Related in: MedlinePlus