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The Salmonella effector SptP dephosphorylates host AAA+ ATPase VCP to promote development of its intracellular replicative niche.

Humphreys D, Hume PJ, Koronakis V - Cell Host Microbe (2009)

Bottom Line: One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake.VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase.Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

View Article: PubMed Central - PubMed

Affiliation: Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK.

ABSTRACT
Virulence effectors delivered into intestinal epithelial cells by Salmonella trigger actin remodeling to direct pathogen internalization and intracellular replication in Salmonella-containing vacuoles (SCVs). One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake. SptP also harbors a C-terminal protein tyrosine phosphatase (PTPase) domain with no clear host substrates. Investigating SptP's longevity in infected cells, we uncover a late function of SptP, showing that it associates with SCVs, and its PTPase activity increases pathogen replication. Direct SptP binding and specific dephosphorylation of the AAA+ ATPase valosin-containing protein (VCP/p97), a facilitator of cellular membrane fusion and protein degradation, enhanced pathogen replication in SCVs. VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase. Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

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VCP Promotion of S. Typhimurium Intracellular Replication(A) Subcellular localization of VCP and SptPFLAG after cell infection. HeLa cells 4 hr postinfection with wild-type S. Typhimurium were mechanically fractionated into the pellet (P), internal membranes (IM), and host cytoplasm (C) before immunoblotting with antibodies against VCP, FLAG (SptP), AcrB, caveolin, histone, calnexin, and Hsp90.(B) Localization of VCP during wild-type S. Typhimurium cell infection. Noninfected and infected (WT) HeLa cells 4 hr postinfection were stained with DAPI (host nuclei and bacteria; blue) and antibodies against VCP (green). Inset shows VCP localization around bacteria. Scale bars, 5 μm.(C) Effect of VCP siRNA on LAMP1 acquisition by SCVs (closed symbols) and S. Typhimurium intracellular replication (open symbols). HeLa cells treated with VCP siRNA (triangles) or control siRNA (circles) were infected with wild-type S. Typhimurium. Infected cells were then stained with DAPI and antibodies against LAMP1, and LAMP1-positive SCVs were quantified (left axis). In parallel, intracellular replication (right axis) was measured. Following infection (0 hr), gentamicin was added at 1 hr to kill extracellular bacteria, and replication was quantified by colony counts at indicated times. Data points are shown as geometric means ± 95% confidence intervals. Asterisks indicate a significant difference from wild-type (p < 0.05, ANOVA; n ⩾ 3).
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fig4: VCP Promotion of S. Typhimurium Intracellular Replication(A) Subcellular localization of VCP and SptPFLAG after cell infection. HeLa cells 4 hr postinfection with wild-type S. Typhimurium were mechanically fractionated into the pellet (P), internal membranes (IM), and host cytoplasm (C) before immunoblotting with antibodies against VCP, FLAG (SptP), AcrB, caveolin, histone, calnexin, and Hsp90.(B) Localization of VCP during wild-type S. Typhimurium cell infection. Noninfected and infected (WT) HeLa cells 4 hr postinfection were stained with DAPI (host nuclei and bacteria; blue) and antibodies against VCP (green). Inset shows VCP localization around bacteria. Scale bars, 5 μm.(C) Effect of VCP siRNA on LAMP1 acquisition by SCVs (closed symbols) and S. Typhimurium intracellular replication (open symbols). HeLa cells treated with VCP siRNA (triangles) or control siRNA (circles) were infected with wild-type S. Typhimurium. Infected cells were then stained with DAPI and antibodies against LAMP1, and LAMP1-positive SCVs were quantified (left axis). In parallel, intracellular replication (right axis) was measured. Following infection (0 hr), gentamicin was added at 1 hr to kill extracellular bacteria, and replication was quantified by colony counts at indicated times. Data points are shown as geometric means ± 95% confidence intervals. Asterisks indicate a significant difference from wild-type (p < 0.05, ANOVA; n ⩾ 3).

Mentions: Our data indicate that the SptP PTPase activity promotes Sif formation and intracellular Salmonella replication, and that it can specifically dephosphorylate VCP in vitro. We therefore investigated whether the host VCP influences intracellular replication. We mechanically fractionated HeLa cells infected with wild-type S. Typhimurium, as in Figure 1C, and found (Figure 4A) that VCP was present in the pellet fraction containing plasma membranes, nuclei, and internalized bacteria, as well as in those containing internal membranes and those containing cytoplasm (the same pattern was seen in noninfected control HeLa cells [data not shown]). Parallel immunofluorescence, as exemplified in the inset boxes (Figure 4B), indicated that VCP localized to approximately 12% ± 4% of intracellular bacteria up to 8 hr postinfection. The low percentage of VCP-positive intracellular bacteria was similar to that seen for SptP (Figure 1B); however, no colocalization was observed.


The Salmonella effector SptP dephosphorylates host AAA+ ATPase VCP to promote development of its intracellular replicative niche.

Humphreys D, Hume PJ, Koronakis V - Cell Host Microbe (2009)

VCP Promotion of S. Typhimurium Intracellular Replication(A) Subcellular localization of VCP and SptPFLAG after cell infection. HeLa cells 4 hr postinfection with wild-type S. Typhimurium were mechanically fractionated into the pellet (P), internal membranes (IM), and host cytoplasm (C) before immunoblotting with antibodies against VCP, FLAG (SptP), AcrB, caveolin, histone, calnexin, and Hsp90.(B) Localization of VCP during wild-type S. Typhimurium cell infection. Noninfected and infected (WT) HeLa cells 4 hr postinfection were stained with DAPI (host nuclei and bacteria; blue) and antibodies against VCP (green). Inset shows VCP localization around bacteria. Scale bars, 5 μm.(C) Effect of VCP siRNA on LAMP1 acquisition by SCVs (closed symbols) and S. Typhimurium intracellular replication (open symbols). HeLa cells treated with VCP siRNA (triangles) or control siRNA (circles) were infected with wild-type S. Typhimurium. Infected cells were then stained with DAPI and antibodies against LAMP1, and LAMP1-positive SCVs were quantified (left axis). In parallel, intracellular replication (right axis) was measured. Following infection (0 hr), gentamicin was added at 1 hr to kill extracellular bacteria, and replication was quantified by colony counts at indicated times. Data points are shown as geometric means ± 95% confidence intervals. Asterisks indicate a significant difference from wild-type (p < 0.05, ANOVA; n ⩾ 3).
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fig4: VCP Promotion of S. Typhimurium Intracellular Replication(A) Subcellular localization of VCP and SptPFLAG after cell infection. HeLa cells 4 hr postinfection with wild-type S. Typhimurium were mechanically fractionated into the pellet (P), internal membranes (IM), and host cytoplasm (C) before immunoblotting with antibodies against VCP, FLAG (SptP), AcrB, caveolin, histone, calnexin, and Hsp90.(B) Localization of VCP during wild-type S. Typhimurium cell infection. Noninfected and infected (WT) HeLa cells 4 hr postinfection were stained with DAPI (host nuclei and bacteria; blue) and antibodies against VCP (green). Inset shows VCP localization around bacteria. Scale bars, 5 μm.(C) Effect of VCP siRNA on LAMP1 acquisition by SCVs (closed symbols) and S. Typhimurium intracellular replication (open symbols). HeLa cells treated with VCP siRNA (triangles) or control siRNA (circles) were infected with wild-type S. Typhimurium. Infected cells were then stained with DAPI and antibodies against LAMP1, and LAMP1-positive SCVs were quantified (left axis). In parallel, intracellular replication (right axis) was measured. Following infection (0 hr), gentamicin was added at 1 hr to kill extracellular bacteria, and replication was quantified by colony counts at indicated times. Data points are shown as geometric means ± 95% confidence intervals. Asterisks indicate a significant difference from wild-type (p < 0.05, ANOVA; n ⩾ 3).
Mentions: Our data indicate that the SptP PTPase activity promotes Sif formation and intracellular Salmonella replication, and that it can specifically dephosphorylate VCP in vitro. We therefore investigated whether the host VCP influences intracellular replication. We mechanically fractionated HeLa cells infected with wild-type S. Typhimurium, as in Figure 1C, and found (Figure 4A) that VCP was present in the pellet fraction containing plasma membranes, nuclei, and internalized bacteria, as well as in those containing internal membranes and those containing cytoplasm (the same pattern was seen in noninfected control HeLa cells [data not shown]). Parallel immunofluorescence, as exemplified in the inset boxes (Figure 4B), indicated that VCP localized to approximately 12% ± 4% of intracellular bacteria up to 8 hr postinfection. The low percentage of VCP-positive intracellular bacteria was similar to that seen for SptP (Figure 1B); however, no colocalization was observed.

Bottom Line: One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake.VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase.Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

View Article: PubMed Central - PubMed

Affiliation: Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK.

ABSTRACT
Virulence effectors delivered into intestinal epithelial cells by Salmonella trigger actin remodeling to direct pathogen internalization and intracellular replication in Salmonella-containing vacuoles (SCVs). One such effector, SptP, functions early during pathogen entry to deactivate Rho GTPases and reverse pathogen-induced cytoskeletal changes following uptake. SptP also harbors a C-terminal protein tyrosine phosphatase (PTPase) domain with no clear host substrates. Investigating SptP's longevity in infected cells, we uncover a late function of SptP, showing that it associates with SCVs, and its PTPase activity increases pathogen replication. Direct SptP binding and specific dephosphorylation of the AAA+ ATPase valosin-containing protein (VCP/p97), a facilitator of cellular membrane fusion and protein degradation, enhanced pathogen replication in SCVs. VCP and its adaptors p47 and Ufd1 were necessary for generating Salmonella-induced filaments on SCVs, a membrane fusion event characteristic of the pathogen replicative phase. Thus, Salmonella regulates the biogenesis of an intracellular niche through SptP-mediated dephosphorylation of VCP.

Show MeSH
Related in: MedlinePlus