Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination

Abstract

AvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-kappaB (NF-kappaB) pathway, IkappaBalpha and beta-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cell-free system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IkappaBalpha and beta-catenin in epithelial cells. AvrA deubiquitinates IkappaBalpha, which blocks its degradation and leads to the inhibition of NF-kappaB activation. Target genes of the NF-kappaB pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of beta-catenin. Target genes of the beta-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased beta-catenin further negatively regulates the NF-kappaB pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-kappaB and beta-catenin pathways.

Figure 1

AvrA inhibited the NF-κB pathway in cultured epithelial cell models. A: Western blot of IκBα in intestinal epithelia colonized by Salmonella nonvirulent PhoP^c, PhoP^cAvrA⁻ (AvrA⁻), or AvrA⁻ transcomplemented with wild-type AvrA (AvrA⁻/AvrA⁺) in vivo. Equal amounts of total cell lysate were sequentially immunoblotted with antibodies against IκBα and β-actin (loading control). B: Location of NF-κBp65 in HCT116 cells by immunofluorescence. Immunostaining of NF-κB in cells colonized with Salmonella PhoP^c or AvrA⁻. Cells were fixed, permeabilized, and stained with anti-p65 antibody, followed by A594 anti-goat secondary antibody and 4,6-diamidino-2-phenylindole (DAPI). Arrows indicate cell nuclei. Images shown are from a single experiment and are representative of three separate repeats.

Figure 2

AvrA inhibited the NF-κB pathway in a mouse model. A: Western blot of IκBα in mouse colonic epithelia cells colonized by PhoP^c, PhoP^c/AvrA⁻ (AvrA⁻), or AvrA⁻ transcomplemented with wild-type AvrA (AvrA⁻/AvrA⁺) in vivo. B: IL-6 levels in mouse serum 6 hours after bacterial infection. C: Real-time PCR of IL-6 mRNA in mouse colons. Data shown in B and C are mean ± SD for n = 3. Significance was at P ≤ 0.05.

Figure 3

AvrA is a deubiquitinase. A: AvrA deubiquitinated ubiquitinated IκBα. The purified GST-AvrA or GST-C186A (mutated at the key cysteine required for its activity) proteins were mixed with ubiquitinated IκB. The reaction was terminated at 30, 60, or 90 minutes. The ubiquitinated forms of IκB were detected by immunoblot using anti-IκB and anti-ubiquitin antibodies. Isopeptidase T is a deubiquitinase used as a positive control. B: Coomassie blue staining showed the total amount of AvrA or C186A in each reaction. C: AvrA reduced the ubiquitinated IκB protein. The purified GST-AvrA or GST-C186A (mutated at the key cysteine required for its activity) proteins were mixed with ubiquitinated IκB. The reaction was terminated in 30, 60, and 90 minutes. The ubiquitinated proteins were detected by immunoblot using anti-ubiquitin antibodies.

Figure 4

The Salmonella AvrA protein modulated β-catenin ubiquitination and activity. HeLa cells were incubated for 1 hour with PhoP^c, AvrA⁻, or PhoP^cAvrA⁻/AvrA⁺, washed, and incubated for the times indicated with the proteasome inhibitor MG262. Total cell lysates were analyzed for ubiquitinated β-catenin and β-actin (loading control) by immunoblot. Higher-molecular weight ubiquitinated β-catenin is indicated by arrows. Data shown are from a single experiment and are representative of three separate experiments. B: AvrA deubiquitinated ubiquitinated β-catenin. In the cell-free system, purified GST-AvrA or GST-C186A proteins were mixed with ubiquitinated β-catenin. The reaction was terminated in 60 minutes. The ubiquitinated forms of β-catenin were detected by immunoblot using anti-β-catenin antibody. The total amount of AvrA or C186A in each reaction was shown by immunoblot using anti-GST antibody. C: Location of β-catenin in HCT116 cells by immunofluorescence. Immunostaining of β-catenin in cells colonized with Salmonella PhoP^c, AvrA⁻, or AvrA⁻/AvrA⁺. Cells were fixed, permeabilized, and stained with anti-β-catenin antibody, followed by A488 anti-mouse secondary antibody and 4,6-diamidino-2-phenylindole (DAPI). Arrows indicate cell nuclei. Images shown are from a single experiment and are representative of three separate repeats. D: AvrA regulates activation of the β-catenin-TCF signaling pathway. Cells were transiently transfected with a TCF-responsive luciferase reporter plasmid containing either a wild-type TCF-binding site (pGL3-OT) or a defective TCF binding site (pGL3-OF). Cells were then colonized with PhoP^c or AvrA⁻ for 30 minutes, washed, and incubated in medium for 6 hours. TCF-dependent transcription was measured by luciferase activity. Data are the means ± SD of a single experiment assayed in triplicate and are representative of three separate experiments.

Figure 5

β-Catenin and its target gene expression are affected by AvrA in vivo. A: Western blot of β-catenin in intestinal epithelia colonized by Salmonella nonvirulent PhoP^c, PhoP^cAvrA⁻ (AvrA⁻), or AvrA⁻ transcomplemented with wild-type AvrA (AvrA⁻/AvrA⁺) in vivo. Equal amounts of total cell lysate were sequentially immunoblotted with antibodies against β-catenin and β-actin (loading control). Bands were quantified using NIH Image software (Bethesda, MD). B: Western blot of c-myc in intestinal epithelia colonized by Salmonella nonvirulent parental PhoP^c, PhoP^cAvrA⁻ (AvrA⁻), or AvrA⁻ transcomplemented with wild-type AvrA (PhoP^c AvrA⁻/AvrA⁺) in vivo. Bands were quantified using NIH Image software.

Figure 6

Murine large intestinal epithelial cell proliferation affected by AvrA expression. A: BrdU labeling of large intestine epithelial cells was performed 24 hours after infection with PhoP^c with or without AvrA. BrdU staining showed that the AvrA mutant strain induced dramatic decrease of epithelial cell proliferation, whereas PhoPc AvrA⁻/AvrA⁺ with AvrA overexpression increased cell proliferation. B: Proliferation index in intestinal epithelial cells. BrdU-positive cells per three high-powered fields were counted from both proximal and distal colons. n = 3 in each experimental group. *P < 0.0001.

Figure 7

Intestinal epithelial cell apoptosis is affected by AvrA expression. A: TUNEL staining on colonic epithelial cells was performed 24 hours after infection with PhoP^c, AvrA⁻, or AvrA⁻/AvrA⁺. AvrA⁻-infected mice had a dramatic increase in TUNEL-positive cells. Higher magnification of the box in A is AvrA⁻ showing apoptosis of the epithelial cells in AvrA⁻-infected mice. B: Apoptotic index in intestinal epithelial cells. TUNEL-positive cells (brown staining) versus nuclei (blue staining) were counted after being scanned in the ACIS system. Five fields were scanned and counted from colon. Apoptotic index, brown area/blue area. n = 3 in each experimental group.