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

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.

Figure 1

SptP Persistence and Promotion of Intracellular Replication (A) Persistence of SptP after cell infection. Lysates of HeLa cells infected with wild-type S. Typhimurium sptP^FLAG were immunoblotted with antibodies against FLAG (SptP) and control Hsp90. No SptP^FLAG was detected following infection with T3SS-deficient S. Typhimurium ΔinvG expressing SptP^FLAG (ΔT3). (B) Intracellular localization of SptP. HeLa cells infected as in (A) were stained with DAPI (host nuclei and bacteria; blue) and antibodies against FLAG (SptP; green). Scale bars, 5 μm. (C) Subcellular localization of SptP. HeLa cells infected as in (A) were fractionated into the pellet (P), internal membranes (IM), and host cytoplasm (C). Samples were immunoblotted with antibodies against FLAG (SptP), AcrB, caveolin, histone, calnexin, and Hsp90. No SptP^FLAG was detected following infection with T3SS-deficient S. Typhimurium ΔinvG expressing SptP^FLAG (ΔT3). (D) SptP influence on LAMP1 acquisition by SCVs (closed symbols) and intracellular S. Typhimurium replication (open symbols). HeLa cells were infected in parallel with WT (circles), ΔsptP (squares), and ΔsptP pSPTP (triangles) strains. Infected cells were stained with DAPI and antibodies against LAMP1 at indicated times postinfection, 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).

Figure 2

SptP Interaction with the AAA+ ATPase VCP (A) Isolation of a stabilized SptP substrate intermediate from porcine brain extract. Purified GST control (−), GST-SptP (SptP^WT), and GST-SptP^D441A (SptP^D441A) were immobilized on glutathione-Sepharose beads and incubated with brain extract. Beads were washed (W) before eluting bound eukaryotic proteins with 1 mM vanadate (V). Immobilized SptP and remaining proteins were eluted with reduced glutathione (G). Eluted fractions were analyzed by Coomassie blue-stained SDS-PAGE (upper panels) with molecular weight markers in kDa (left), and then immunoblotted (lower panels) using antibodies against VCP (arrowed). (B) Interaction between purified recombinant SptP and VCP. SptP^WT and SptP^D441A were immobilized on glutathione Sepharose beads and incubated with His-tagged VCP (VCP). Also shown are flowthrough containing unbound protein (F; lanes 1 and 2), washes (W; lanes 3 and 4), and proteins eluted with 1 M NaCl (N; lanes 5 and 6). Immobilized SptP and remaining VCP were eluted with reduced glutathione (G; lanes 7 and 8). Fractions were analyzed by Coomassie blue-stained SDS-PAGE. (C) Interaction between purified recombinant GST-GAP (GAP) or GST-PTP^D441A (PTP^D441A) SptP domains with VCP, performed as in (B).

Figure 3

SptP PTPase Activity toward VCP In Vitro (A) In vitro phosphorylation of VCP at tyrosine 805. Purified recombinant wild-type VCP (VCP^WT) and VCP with tyrosine 805 substituted for phenylalanine (VCP^Y805F) were phosphorylated in vitro (phos; −/+), and phosphorylation was assessed by immunoblotting with antibodies against phosphotyrosine (VCP^phos) and VCP. (B) SptP PTPase activity toward VCP. In vitro phosphorylated recombinant VCP, N-WASP, and aldolase (2 μM) were incubated with picomolar concentrations (top) of SptP^WT. Phosphorylation was assessed by immunoblotting samples with antibodies against phosphotyrosine (VCP^phos, N-WASP^phos, and aldolase^phos) and VCP, N-WASP, and aldolase as controls. (C) SptP^D441A PTPase activity toward VCP, performed as in (A).

Figure 4

VCP Promotion of S. Typhimurium Intracellular Replication (A) Subcellular localization of VCP and SptP^FLAG 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).

Figure 5

SptP-Dependent VCP Dephosphorylation during Infection (A) SptP interaction with VCP. HeLa cells were infected with S. Typhimurium ΔsptP expressing plasmid-encoded FLAG-tagged SptP variants (−, pSPTP, pR209A, pD441A). At 4 hr postinfection, cells were lysed with detergent, and SptP variants were immunoprecipitated from cell lysates with antibodies against SptP immobilized on protein G Sepharose. Eluted immunoprecipitates were assayed for presence of SptP and bound VCP and also supernatants for unbound VCP by immunoblotting with antibodies against FLAG (SptP) and VCP. (B) SptP-dependent dephosphorylation of VCP during S. Typhimurium cell infection. HeLa cells were individually infected with S. Typhimurium strains, described in (A), and wild-type (WT). At 4 hr postinfection, cells were lysed with detergent, and VCP was immunoprecipitated using antibodies against VCP immobilized on protein G Sepharose. Phosphorylation status of immunoprecipitated VCP was assessed by immunoblotting with antibodies against phosphotyrosine (VCP^phos) and VCP serving as a loading control (VCP). (C) SptP-dependent syntaxin5 interaction with VCP during cell infection. HeLa cells transiently transfected with pcDNA3.1-HA-STX5 were either noninfected or infected in parallel with wild-type S. Typhimurium, the ΔsptP (−) null mutant, or ΔsptP expressing plasmid-encoded SptP (pSPTP). HA-tagged syntaxin5 was immunoprecipitated from cell lysates with antibodies against HA immobilized on protein G Sepharose. Eluted immunoprecipitates were assayed for presence of syntaxin5 and bound VCP and also supernatants for unbound VCP by immunoblotting with antibodies against HA (syntaxin5) and VCP.

Figure 6

Mechanism for SptP Promotion of Intracellular Replication (A) SptP PTPase activity promotes intracellular replication. SptP-dependent dephosphorylation of VCP allows it to promote Sif formation and intracellular replication through the major adaptors p47 and Ufd1. SptP-dependent dephosphorylation of VCP enhances its association with pathway-specific coadaptors to determine downstream functions. (B) sptP null Salmonella are retarded for intracellular replication. Internalized ΔsptP Salmonella reside in SCVs, but VCP is not dephosphorylated. This prevents the association of VCP-p47 and VCP-Ufd1 complexes with pathway-specific coadaptor proteins and thus impairs specific VCP functions required for promoting Sif formation and intracellular replication.