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SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases.

Mazurkiewicz P, Thomas J, Thompson JA, Liu M, Arbibe L, Sansonetti P, Holden DW - Mol. Microbiol. (2008)

Bottom Line: SpvC is encoded by the Salmonella virulence plasmid.SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems.Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK.

ABSTRACT
SpvC is encoded by the Salmonella virulence plasmid. We have investigated the biochemical function of SpvC and the mechanism by which it is secreted by bacteria and translocated into infected macrophages. We constructed a strain carrying a deletion in spvC and showed that the strain is attenuated for systemic virulence in mice. SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems. Cell biological and genetic experiments showed that translocation of the protein into the cytosol of macrophages by intracellular bacteria is dependent on the SPI-2 T3SS. Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif. A Salmonella strain expressing spvC from a plasmid downregulated cytokine release from infected cells.

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Effect of Salmonella on cytokine release by infected cells. ELISA was used to measure amounts of IL-8 released by infected HeLa cells (A) and TNF-α released by J774 macrophages (B) into the growth media at the indicated time points. Similar infection efficiencies was confirmed by microscopy in every experiment. P-values were calculated for cytokine levels from cells infected with the ΔspvCpspvC strain compared with cells infected with WT bacteria were significantly different (> 0.05) at all time points tested for A, and at 14, 18 and 21 h for B.
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fig07: Effect of Salmonella on cytokine release by infected cells. ELISA was used to measure amounts of IL-8 released by infected HeLa cells (A) and TNF-α released by J774 macrophages (B) into the growth media at the indicated time points. Similar infection efficiencies was confirmed by microscopy in every experiment. P-values were calculated for cytokine levels from cells infected with the ΔspvCpspvC strain compared with cells infected with WT bacteria were significantly different (> 0.05) at all time points tested for A, and at 14, 18 and 21 h for B.

Mentions: The action of OspF on MAPKs affects histone modification and chromatin accessibility of NF-κB at the IL-8 promoter, and negatively regulates neutrophil recruitment in infected tissues (Arbibe et al., 2007). We therefore measured secretion of IL-8 from HeLa cells at different times after infection with bacterial strains. Infection with wild-type bacteria induced IL-8 production but no increased production could be detected in cells infected with the ΔspvC mutant (Fig. 7A). However, cells infected with the ΔspvCpspvC strain showed significantly less IL-8 production compared with the wild-type strain at all time points tested (P ≤ 0.05 for all time points) (Fig. 7A). As TNF-α production following stimulation of TLR-4 by LPS is MAPK-dependent (van der Bruggen et al., 1999), we also examined production of this cytokine by murine J774 macrophages. Time points for sampling were chosen to coincide with detectable SpvC translocation into host cells. Again, no significant differences were detected between cells infected with the wild-type or spvC mutant, but cells overexpressing SpvC produced less TNF-α at 14, 18 and 21 h post uptake (Fig. 7B). We also infected BALB/c mice with either the wild-type, spvC mutant or ΔspvCpspvC strains. At 4, 24 and 72 h post inoculation, mouse sera were recovered and analysed for IL-6, IL-10, MCP-1, INF-γ, TNF-α and IL12p70 by ELISA. No statistically significant differences were detected between mice infected with these strains (data not shown).


SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases.

Mazurkiewicz P, Thomas J, Thompson JA, Liu M, Arbibe L, Sansonetti P, Holden DW - Mol. Microbiol. (2008)

Effect of Salmonella on cytokine release by infected cells. ELISA was used to measure amounts of IL-8 released by infected HeLa cells (A) and TNF-α released by J774 macrophages (B) into the growth media at the indicated time points. Similar infection efficiencies was confirmed by microscopy in every experiment. P-values were calculated for cytokine levels from cells infected with the ΔspvCpspvC strain compared with cells infected with WT bacteria were significantly different (> 0.05) at all time points tested for A, and at 14, 18 and 21 h for B.
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Related In: Results  -  Collection

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fig07: Effect of Salmonella on cytokine release by infected cells. ELISA was used to measure amounts of IL-8 released by infected HeLa cells (A) and TNF-α released by J774 macrophages (B) into the growth media at the indicated time points. Similar infection efficiencies was confirmed by microscopy in every experiment. P-values were calculated for cytokine levels from cells infected with the ΔspvCpspvC strain compared with cells infected with WT bacteria were significantly different (> 0.05) at all time points tested for A, and at 14, 18 and 21 h for B.
Mentions: The action of OspF on MAPKs affects histone modification and chromatin accessibility of NF-κB at the IL-8 promoter, and negatively regulates neutrophil recruitment in infected tissues (Arbibe et al., 2007). We therefore measured secretion of IL-8 from HeLa cells at different times after infection with bacterial strains. Infection with wild-type bacteria induced IL-8 production but no increased production could be detected in cells infected with the ΔspvC mutant (Fig. 7A). However, cells infected with the ΔspvCpspvC strain showed significantly less IL-8 production compared with the wild-type strain at all time points tested (P ≤ 0.05 for all time points) (Fig. 7A). As TNF-α production following stimulation of TLR-4 by LPS is MAPK-dependent (van der Bruggen et al., 1999), we also examined production of this cytokine by murine J774 macrophages. Time points for sampling were chosen to coincide with detectable SpvC translocation into host cells. Again, no significant differences were detected between cells infected with the wild-type or spvC mutant, but cells overexpressing SpvC produced less TNF-α at 14, 18 and 21 h post uptake (Fig. 7B). We also infected BALB/c mice with either the wild-type, spvC mutant or ΔspvCpspvC strains. At 4, 24 and 72 h post inoculation, mouse sera were recovered and analysed for IL-6, IL-10, MCP-1, INF-γ, TNF-α and IL12p70 by ELISA. No statistically significant differences were detected between mice infected with these strains (data not shown).

Bottom Line: SpvC is encoded by the Salmonella virulence plasmid.SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems.Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK.

ABSTRACT
SpvC is encoded by the Salmonella virulence plasmid. We have investigated the biochemical function of SpvC and the mechanism by which it is secreted by bacteria and translocated into infected macrophages. We constructed a strain carrying a deletion in spvC and showed that the strain is attenuated for systemic virulence in mice. SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems. Cell biological and genetic experiments showed that translocation of the protein into the cytosol of macrophages by intracellular bacteria is dependent on the SPI-2 T3SS. Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif. A Salmonella strain expressing spvC from a plasmid downregulated cytokine release from infected cells.

Show MeSH
Related in: MedlinePlus