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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 interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.
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ppat-1002743-g007: SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.

Mentions: The catalytically inactive SseL mutant (SseLC/A) binds stably to ubiquitinated substrates and allows their immunoprecipitation [7]. To determine if SseL can co-immunoprecipitate autophagic substrates, macrophages were infected with the sseL deletion mutant expressing either wild-type SseL-HA or SseLC/A-HA from a plasmid, and after 10 h of infection, lysates were immunoprecipitated with an anti-HA antibody (Fig. 7A). Immunoblotting revealed that p62 was specifically co-immunoprecipitated by SseLC/A-HA (Fig. 7A). A similar result was obtained using infected HeLa cells (data not shown). This shows that SseL and p62 might bind to the same ubiquitinated substrates. Furthermore, since p62 was immunoprecipitated by SseLC/A but not wild-type SseL (Fig. 7A), it is likely that this interaction occurs indirectly through the ubiquitinated substrates. Next we examined the effects of autophagy on the accumulation of SseL-interacting ubiquitinated substrates. Infected macrophages were subjected to 3 h of 3-MA or starvation treatment to inhibit or stimulate autophagic flux respectively before cell lysis. 3-MA markedly increased the levels of ubiquitinated proteins interacting with SseLC/A (Fig. 7B). In contrast, starvation conditions virtually eliminated these substrates (Fig. 7B). Therefore, SseL deubiquitinates substrates targeted by p62 and destined for autophagic degradation.


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 interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375275&req=5

ppat-1002743-g007: SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.
Mentions: The catalytically inactive SseL mutant (SseLC/A) binds stably to ubiquitinated substrates and allows their immunoprecipitation [7]. To determine if SseL can co-immunoprecipitate autophagic substrates, macrophages were infected with the sseL deletion mutant expressing either wild-type SseL-HA or SseLC/A-HA from a plasmid, and after 10 h of infection, lysates were immunoprecipitated with an anti-HA antibody (Fig. 7A). Immunoblotting revealed that p62 was specifically co-immunoprecipitated by SseLC/A-HA (Fig. 7A). A similar result was obtained using infected HeLa cells (data not shown). This shows that SseL and p62 might bind to the same ubiquitinated substrates. Furthermore, since p62 was immunoprecipitated by SseLC/A but not wild-type SseL (Fig. 7A), it is likely that this interaction occurs indirectly through the ubiquitinated substrates. Next we examined the effects of autophagy on the accumulation of SseL-interacting ubiquitinated substrates. Infected macrophages were subjected to 3 h of 3-MA or starvation treatment to inhibit or stimulate autophagic flux respectively before cell lysis. 3-MA markedly increased the levels of ubiquitinated proteins interacting with SseLC/A (Fig. 7B). In contrast, starvation conditions virtually eliminated these substrates (Fig. 7B). Therefore, SseL deubiquitinates substrates targeted by p62 and destined for autophagic degradation.

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