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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.

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SCV-associated ubiquitinated aggregates are autophagy substrates.(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.
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ppat-1002743-g004: SCV-associated ubiquitinated aggregates are autophagy substrates.(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.

Mentions: To examine the role of autophagy in the regulation of these structures, HeLa cells infected with ΔsseL mutant bacteria were subjected to conditions that either inhibit or stimulate autophagy and examined for the presence of SCV-associated ubiquitinated aggregates (Fig. 4). 3-methyladenine (3-MA), ammonium chloride (NH4Cl) or bafilomycin A1 (BafA1) were used to inhibit autophagy at early (3-MA) and late (NH4Cl and BafA1) stages of the pathway; starvation and rapamycin were used to stimulate autophagy. To avoid any interference with the maturation of the SCV and early targeting of cytosolic bacteria, infected cells were maintained in normal medium until 8 h after invasion. Cells were then exposed to autophagy-altering conditions for 4 h before processing for confocal microscopy. The frequency of ubiquitinated aggregates was enhanced significantly upon inhibition of autophagosome formation (3-MA) or autophagic degradation (BafA1 and NH4Cl), whereas stimulation of autophagy by starvation or rapamycin strongly reduced their presence (Fig. 4A and 4B). The alterations in autophagic flux were confirmed by an increased presence of LC3-positive compartments around SCVs in starved cells (Fig. 4B); LC3-positive compartments were almost undetectable in cells exposed to 3-MA despite the increase in ubiquitin labelling (Fig. 4B). BafA1, which inhibits degradation of lysosomal contents, enhanced both ubiquitin and LC3 labelling of these compartments (Fig. 4B). These results show that the ubiquitinated aggregates induced by SPI-2 T3SS effectors are substrates of the autophagy pathway.


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)

SCV-associated ubiquitinated aggregates are autophagy substrates.(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002743-g004: SCV-associated ubiquitinated aggregates are autophagy substrates.(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.
Mentions: To examine the role of autophagy in the regulation of these structures, HeLa cells infected with ΔsseL mutant bacteria were subjected to conditions that either inhibit or stimulate autophagy and examined for the presence of SCV-associated ubiquitinated aggregates (Fig. 4). 3-methyladenine (3-MA), ammonium chloride (NH4Cl) or bafilomycin A1 (BafA1) were used to inhibit autophagy at early (3-MA) and late (NH4Cl and BafA1) stages of the pathway; starvation and rapamycin were used to stimulate autophagy. To avoid any interference with the maturation of the SCV and early targeting of cytosolic bacteria, infected cells were maintained in normal medium until 8 h after invasion. Cells were then exposed to autophagy-altering conditions for 4 h before processing for confocal microscopy. The frequency of ubiquitinated aggregates was enhanced significantly upon inhibition of autophagosome formation (3-MA) or autophagic degradation (BafA1 and NH4Cl), whereas stimulation of autophagy by starvation or rapamycin strongly reduced their presence (Fig. 4A and 4B). The alterations in autophagic flux were confirmed by an increased presence of LC3-positive compartments around SCVs in starved cells (Fig. 4B); LC3-positive compartments were almost undetectable in cells exposed to 3-MA despite the increase in ubiquitin labelling (Fig. 4B). BafA1, which inhibits degradation of lysosomal contents, enhanced both ubiquitin and LC3 labelling of these compartments (Fig. 4B). These results show that the ubiquitinated aggregates induced by SPI-2 T3SS effectors are substrates of the autophagy pathway.

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