Figure 1.
GogB interacts with FBXO22 and Skp1 of the SCF E3 ubiquitin ligase.
A. Identification of GogB interacting partner in the host cell. GogB-HA delivered to HeLa cells by wild type Salmonella was immunoprecipitated and associated proteins were separated by SDS-PAGE. A silver-stained SDS-PAGE shows a ∼40 kDa band identified as F-box only protein (FBXO22) by mass spectrometry (arrow). B. Western blot analysis of the GogB-HA immunoprecipitation using antibodies against FBXO22 and actin. C. GogB interacts with the SCF ubiquitin ligase adapter protein Skp1 through the GogB C-terminus in a GST pull-down assay. GST-GogB and truncated GogB mutants encoding amino acid residues 1–253 (GogB-NT) and 254–497 (GogB-CT) were incubated with RAW 264.7 or HeLa cell lysates. Protein complexes were analyzed by Western blot using anti-Skp1 and anti-GST antibodies. GST beads alone mixed with lysates were used as control for non-specific binding. D. GogB immunoprecipitates a complex consisting of Skp1 and FBXO22 in host cells. GogB-HA or the truncation mutants were delivered to HeLa cells by the Salmonella T3SS and immunoprecipitated from infected cell lysates. Bound proteins were analyzed by Western blot using anti-Skp1 and FBXO22 antibodies. E. GogB interacts with FBXO22 in the absence of Skp1. HeLa cells were transfected with 80 pmol of either Skp1 siRNA or control siRNA. At 24 hr post-transfection, cells were infected with ΔgogB or ΔgogB complemented with pgogB-2HA. Cell lysates were mixed with anti-HA affinity beads after 20 hr post-infection and bound protein complexes were analyzed by Western blot using anti-Skp1, anti-FBXO22, and anti-HA antibodies. Anti-GAPDH was used as loading control for lysates.
Figure 2.
GogB contains an Fbox-like domain involved in Skp1 interaction.
A. Amino acid sequence alignment of the putative Fbox-like domain found at the C-terminus of GogB with the F-box consensus sequences (FBOX1 and FBOX2), the human F-box protein Skp2, and the secreted effector GALA1 from the plant pathogen Ralstonia solanacearum. The conserved leucine and proline residues are highlighted in red and similar amino acid residues are highlighted in blue boxes. B. Deletion of the GogB Fbox-like domain abrogates binding to Skp1 in vivo. HeLa cells were infected with ΔgogB or ΔgogB expressing GogB, GogB-NT1–253, GogB-CT254–497, GogB-Δ264–352, a mutant with a deletion from residues 264–352, and the mutants GogB LP-AA and GogB-CT LP-AA in which the conserved leucine and proline residues in the Fbox-like domain were mutated to alanine. At 20 hr post-infection, infected cells were lysed and lysates were incubated with anti-HA affinity beads and bound proteins were resolved by SDS-PAGE and analyzed by Western blot using anti-Skp1 and anti-HA antibodies.
Figure 3.
GogB decreases the host inflammatory response by inhibiting IκBα degradation and NFκB activation.
A. GogB inhibits IκBα degradation during infection. RAW264.7 cells were infected with wild type and ΔgogB Salmonella and lysed after 4, 8, and 20 h. Lysates were probed using anti-IκBα antibody. Actin was used as a loading control. B. Deletion of GogB or the Fbox-like domain on GogB increases ubiquitination of IκBα. RAW 264.7 cells were infected with wild type Salmonella, ΔgogB, or ΔgogB complemented with plasmid encoding GogB or GogB-Δ264–352 in the presence (+) or absence (−) of the proteasome inhibitor MG-132. At 4 hr post-infection, cells were lysed and normalized by protein content. Levels of ubiquitinated IκBα were determined by a co-immunoprecipitation assay using IgA magnetic beads coupled to anti-IκBα antibodies and mixed with the lysates. Western blot was done using HRP-conjugated anti-ubiquitin (FK2) and rabbit anti-IκBα. C. GogB-deficient Salmonella induce higher NFκB activation compared to wild type. RAW264.7 were transfected with a luciferase reporter and then infected with wild type Salmonella containing an empty pWSK129 vector, ΔgogB, and ΔgogB strains complemented with plasmids encoding full-length GogB, GogB-NT1–253, GogB-CT254–497, GogB LP270AA, GogB-CT254–497 LP47AA, and GogB-Δ265–352. At 20 hr post-infection, NFκB-driving luciferase activity was measured and normalized to β-galactosidase levels and CFUs enumerated from each strain after infection. Data are expressed as mean fold difference in NFκB levels compared to cells infected with wild-type SL1344/pWSK129 with standard errors from three independent experiments. D. Deletion of GogB increases IL1β activation in infected macrophages. RAW264.7 cells were infected with wild type Salmonella containing an empty pWSK129 vector, ΔgogB, or ΔgogB complemented with plasmids encoding GogB, GogB-NT, GogB-CT, GogB LP270AA, GogB-CT254–497 LP47AA, and GogB-Δ264–352. At 20 hr post-infection, IL1β levels were measured in culture supernatants of infected macrophages by ELISA and normalized to CFUs enumerated from each strain at 20 h post-infection. Data are expressed as mean IL1β concentration (pg/mL) with standard errors from three independent experiments. Asterisks denote significant difference between the indicated strains, P<0.01. E. NFκB activation in ΔgogB-infected epithelial cells is blocked in the absence of Skp1. HeLa cells co-transfected with 2 pmol Skp1 siRNA or negative control siRNA, and a luciferase gene reporter system. At 24 hr post-transfection, cells were infected with wild-type Salmonella, ΔgogB or the complemented mutant strains. NFκB activation was determined by reporter gene assay after 20 hr post-infection. Values are normalized to β-galactosidase levels and CFUs enumerated from each strain after infection. Data are expressed as mean fold difference in NFκB levels compared to cells infected with wild-type Salmonella containing an empty vector. Assays were done in triplicate in three independent experiments (n = 3, ± SEM). F. Representative Western blot analysis showing knockdown of Skp1 expression in HeLa cells transfected with Skp1 or control siRNA and NFκB reporter assay and infected with wild-type SL1344/pWSK129, ΔgogB or ΔgogB complemented strains. Lysates were normalized by protein content and analyzed by Western blot using anti-Skp1 and anti-GAPDH as loading control.
Figure 4.
Deletion of GogB does not affect replication in cells in vitro or acute infection of susceptible mice.
A. RAW 264.7 cells were infected with wild type Salmonella, ΔgogB, ΔgogB/pgogB-2HA, ΔgogB/pgogBNT-2HA and ΔgogB/pgogBCT-2HA. At 20 hr post-infection, infected cells were lysed and intracellular bacteria were enumerated. Fold replication represents the ratio of intracellular bacteria at 20 h and 2 h. Data are the means with standard error of three separate experiments. B. C57BL/6 mice were orally infected with a mixed inoculum of wild type Salmonella and the ΔgogB strain. The competitive index (CI) was determined in the spleen, liver and cecum after 3 days. Each data point represents one animal and horizontal bars indicate geometric means.
Figure 5.
Deletion of GogB promotes chronic colonization in Nramp+/+ mice.
Groups of 129/svImJ mice were orally infected with wild type Salmonella or the ΔgogB mutant. After 4, 7, 39, or 60 days of infection, viable bacteria were recovered from organs and enumerated. Each data point represents one animal and horizontal bars indicate geometric means. Asterisks denote significant differences between wild type and ΔgogB (P<0.05).
Figure 6.
Cecal colonization in ΔgogB-infected mice is accompanied by inflammation and tissue damage.
A. Hemotoxylin-eosin-stained cecal tissue from infected 129/svImJ mice at day 4. Images are representative of tissue from 5 mice per group, scored for pathology (B) as outlined in Methods. C. Hemotoxylin-eosin-stained cecal tissue from infected 129/svImJ mice at day 60. D. Tissue pathology at day 60 post-infection was quantified as described.
Figure 7.
Expression of inflammatory markers in the cecum and MLN of infected mice.
129svImJ mice were orally infected with wild type or ΔgogB Salmonella. At day 4 post-infection (A and B) and day 60 post-infection (C and D), RNA was extracted from the cecum and MLN and the relative mRNA levels of the inflammatory markers shown were determined by RT-PCR. Relative expression levels were normalized to 18S RNA and expressed as log-transformed mean fold change in expression with standard errors from 5 experiments. Asterisks denote significant difference from mice infected with wild type and ΔgogB Salmonella (P<0.05).