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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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SseL reduces autophagic flux and can promote bacterial replication in macrophages.(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting. (B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments. (C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.
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ppat-1002743-g008: SseL reduces autophagic flux and can promote bacterial replication in macrophages.(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting. (B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments. (C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.

Mentions: Accumulation of protein aggregates has been shown to induce autophagy in models of Huntington's disease [41]. In addition, LPS-induced ALIS formation increases autophagic flux in macrophages [11], [42]. Since SseL deubiquitinates ALIS, we analysed autophagic flux by determining LC3-II levels in infected macrophages. When LC3 is conjugated with phosphatidylethanolamine (LC3-II) it localizes to autophagosomal membranes [43] and the amount of LC3-II correlates with the number of autophagosomes present in cells. Exposure of cells to NH4Cl to inhibit lysosomal degradation allows differentiation between an increase in autophagic flux and impaired autophagic degradation [43], [44]. Cells infected with sseL mutant bacteria had increased levels of LC3-II compared to wild-type infected cells and this was complemented when bacteria expressed wild-type SseL but not SseLC/A (Fig. 8A and B). Following exposure to NH4Cl, the levels of LC3-II were still higher in the absence of SseL (Fig. 8A and B), indicating that the increase in LC3-II observed in cells infected with bacteria lacking SseL did not result from a block in autophagosomal degradation. Therefore, the absence of SseL deubiquitinase activity stimulates autophagic flux, presumably by increasing the accumulation of ubiquitinated aggregates.


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 reduces autophagic flux and can promote bacterial replication in macrophages.(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting. (B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments. (C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375275&req=5

ppat-1002743-g008: SseL reduces autophagic flux and can promote bacterial replication in macrophages.(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting. (B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments. (C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.
Mentions: Accumulation of protein aggregates has been shown to induce autophagy in models of Huntington's disease [41]. In addition, LPS-induced ALIS formation increases autophagic flux in macrophages [11], [42]. Since SseL deubiquitinates ALIS, we analysed autophagic flux by determining LC3-II levels in infected macrophages. When LC3 is conjugated with phosphatidylethanolamine (LC3-II) it localizes to autophagosomal membranes [43] and the amount of LC3-II correlates with the number of autophagosomes present in cells. Exposure of cells to NH4Cl to inhibit lysosomal degradation allows differentiation between an increase in autophagic flux and impaired autophagic degradation [43], [44]. Cells infected with sseL mutant bacteria had increased levels of LC3-II compared to wild-type infected cells and this was complemented when bacteria expressed wild-type SseL but not SseLC/A (Fig. 8A and B). Following exposure to NH4Cl, the levels of LC3-II were still higher in the absence of SseL (Fig. 8A and B), indicating that the increase in LC3-II observed in cells infected with bacteria lacking SseL did not result from a block in autophagosomal degradation. Therefore, the absence of SseL deubiquitinase activity stimulates autophagic flux, presumably by increasing the accumulation of ubiquitinated aggregates.

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