Consistent activation of the β-catenin pathway by Salmonella type-three secretion effector protein AvrA in chronically infected intestine

Rong Lu, Xingyin Liu, Shaoping Wu, Yinglin Xia, Yong-guo Zhang, Elaine O. Petrof, Erika C. Claud, Jun Sun

Abstract

Salmonella infection is a common public health problem that can become chronic and increase the risk of cancer. Live, mutated Salmonella is used to target cancer cells. However, few studies have addressed chronic Salmonella infection in vivo. AvrA is a Salmonella type-three secretion effector that is multifunctional, inhibiting intestinal inflammation and enhancing proliferation. β-catenin is a key player in intestinal renewal, inflammation, and tumorigenesis. We hypothesize that in Salmonella-infected intestine, AvrA chronically activates the β-catenin pathway and increases cell proliferation, thus deregulating the intestinal responses to bacterial infection. We followed mice with Salmonella infection for 27 wk and investigated the physiological effects and role of AvrA on β-catenin in chronically infected intestine. We found that AvrA persistently regulated β-catenin posttranslational modifications, including phosphorylation and acetylation. Moreover, the upstream regulator Akt, transcription factors, T cell factors, nuclear β-catenin, and β-catenin target genes were enhanced in mice infected with Salmonella-expressing AvrA. AvrA has a chronic functional role in promoting intestinal renewal. In summary, we have uncovered an essential role of Salmonella AvrA in chronically activating β-catenin and impacting intestinal renewal in small intestine and colon. Our study emphasizes the importance of AvrA in chronic bacterial infection.

  • Wnt
  • β-catenin
  • acetylated β-catenin
  • phosphorylated β-catenin
  • T cell factor
  • AKT
  • proliferation
  • intestine
  • stem cells

salmonella outbreaks are a critical public health concern. In addition to Salmonellosis, the acute enteric infection, Salmonella colonization can become chronic (13) and can increase the risk of other gastrointestinal diseases, including chronic inflammation and cancer (12, 19). A recent population-based cohort study demonstrated an increased risk of inflammatory bowel diseases in individuals with Salmonella infections (12). Chronic Salmonella colonization of the intestine increases the risk of intestinal fibrosis in mice (13). Although the pathogenesis of acute Salmonella-induced diarrhea and inflammation has been extensively studied (38), the mechanism by which Salmonella bacteria regulate the host response in a chronic setting has not been fully explored.

Some Salmonella species are linked to carcinogenesis, whereas others appear promising in the diagnosis, prevention, and treatment of some cancers (28). Live, mutated noninvasive Salmonella species have been used as a vector to specifically target cancer cells (53). However, the chronic effects and molecular mechanisms of infection with nonpathogenic or mutated Salmonella in the host are largely unexplored.

Salmonella use a molecular “needle,” referred to as a type-three secretion system, to inject bacterial effector proteins into host cells. Stimulation of inflammation by effectors is crucial for Salmonella growth within the intestine (39). Effectors, such as SipA, SopE, and SopB, are known to activate inflammation in host cells (1–2, 8–9, 14, 17, 54). However, uncontrolled inflammation is harmful to the host and eventually damages the niche occupied by Salmonella during infection. Salmonella-secreted factor L (4, 20, 35), SspH1, SptP, and AvrA reverse the activation of signaling pathways induced by other Salmonella effectors (27, 29, 41, 5051). Studies on bacterial effectors and their ability to manipulate host signal transduction pathways may contribute to the understanding of the roles of these bacteria in human diseases.

Microbiologists used to believe that the bacterial effector proteins have a rapid and short-term effect in host cells. For example, wild-type Salmonella require only 80–200 s to inject bacterial effector protein SipE into the host cells (36, 52). Recently, increasing evidence indicates that Salmonella continue to synthesize effectors in colonized organs in the late stages of infection in mice (11, 24, 26). Our recent study (26) showed the chronic effects of Salmonella in vivo after 27 wk of infection. The chronic physiological effects of chronic Salmonella infection include altered body weight, intestinal pathology, and bacterial translocation in spleen, liver, and gallbladder (26). However, the molecular mechanism by which Salmonella proteins contribute to the chronic bacterial-host interactions remains unknown.

Our publications and others demonstrate that Salmonella AvrA is a multiple-function protein that plays a critical role in inhibiting inflammation, regulating epithelial apoptosis, and enhancing proliferation during bacterial infections in cell culture models and acutely infected mice (5, 18, 21, 48, 51). In the chronically Salmonella-infected mice, AvrA expression in Salmonella enhanced its invasion ability. Liver abscess and Salmonella translocation in the gallbladder were observed and may be associated with AvrA expression in Salmonella (26). The present study addressed roles of Salmonella AvrA on the host response in the chronically infected intestine.

β-catenin is a key regulator in proliferation and colon cancer (7, 15, 31, 34, 45, 55). Our previous studies have revealed that Salmonella AvrA activates the β-catenin pathway in cultured intestinal epithelial cell models and acutely infected mouse models (6, 23, 42, 51). In the present study, we address the following new and different questions: 1) is β-catenin signaling involved in chronic Salmonella infection? 2) is AvrA responsible for persistent activation of β-catenin signaling during chronic Salmonella infection? and 3) how does Salmonella AvrA regulate the host response in a chronic setting if the answers for questions 1 and 2 are “yes.” We hypothesize that AvrA has long-term effects in the Salmonella-infected intestine, including activating β-catenin and increasing cell proliferation, thus leading to the dysregulation of intestinal responses to chronic bacterial infections. We used the bacterial strain, PhoPC, for this chronic infection because previous work has indicated that PhoPC along with sufficient AvrA expression activates the β-catenin pathway in acute inflammation (42). PhoP-PhoQ is a two-component regulatory system that controls the expression of over 40 genes essential for S. typhimurium virulence and survival within macrophages. PhoPc is a PhoP-PhoQ constitutive mutation that increases the expression of PhoP-activated genes and represses the synthesis of 20 proteins encoded by PhoP-repressed genes (30). Wild-type Salmonella induced death of infected mice within a week (26). Therefore, we focused on mutated bacterial strains for these chronic studies. We used bacterial strains with wild-type or mutated AvrA, including parental PhoPc, PhoPc AvrA mutant (AvrA), and PhoPc AvrA with a complementary plasmid encoding AvrA (PhoPcAvrA/AvrA+) in vivo. We examined how AvrA regulates β-catenin at the posttranslational level. We also investigated transcriptional factor T cell factors (TCF), β-catenin target genes, and cell proliferation after chronic infection. Taken together, our study investigates the chronic effects of Salmonella AvrA in inducing intestinal dysfunction through β-catenin activation in vivo.

MATERIALS AND METHODS

Ethics statement.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Rochester Committee on Animal Resources (UCAR 2007-065). All efforts were made to minimize suffering.

Streptomycin-pretreated mouse model.

Animal experiments were performed by using specific pathogen-free female C57BL/6 mice (Taconic) that were 6–7 wk old, as previously described (6, 26). Water and food were withdrawn 4 h before oral gavage with 7.5 mg/mouse of streptomycin (100 μl of sterile solution). Afterward, animals were supplied with water and food ad libitum. Bacterial strains used in this study included Salmonella mutant strains PhoPc (30), PhoPc AvrA, and PhoPc AvrA/AvrA+. Nonagitated microaerophilic bacterial cultures were prepared as previously described (42). Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 h before the mice were infected with 1 × 106 CFU of S. typhimurium by oral gavage. At 1, 3, 6, 10, and 27 wk after Salmonella infection, tissue samples were collected. This represents a study that uses similar experimental groups as in a previous report that described a mouse model with persistent Salmonella infection (26). The present study used different animals and tissues. The previous report focuses on physiologic changes and bacterial effects in cecum, liver, gallbladder, and spleen, whereas in the present study we investigated the effects and molecular mechanisms of AvrA in Salmonella-infected small intestine and colon.

Detection of Salmonella in the intestine.

Intestinal Salmonella was detected by culturing fecal content at 37°C overnight on a BBL CHROMagar plate (BD Biosciences, San Jose, CA). Salmonella species appeared mauve (rose to purple) (26). Salmonella distribution in colon was also determined by immunostaining (49–50).

Microbial analysis.

After death, 1-ml samples from the cecal contents of colonized mice were taken and serially diluted in PBS. Total bacterial concentrations were determined by manual counting.

Real-time PCR measurement of Salmonella 16S rRNA.

DNA was extracted from colonic tissues after washing to remove residual feces. Primers specific to 18S rRNA were used as an endogenous control to normalize loading between samples. For the quantification of Salmonella, a pair of primers specific for Salmonella species with 16S rRNA was used to amplify this bacterial gene: 16srRNA, forward: 5′-TATAGCCCCATCGTGTAGTCAGAAC-3′, reverse: 5′-TGCGGCTGGATCACCTCCTT-3′; and 18S rRNA, forward: 5′-AGGGGAGAGCGGGTAAGAGA-3′, reverse: 5′-GGACAGGACTAGGCGGAACA-3′. The relative amount of 16S rRNA in each sample was estimated using the ΔΔCT method.

Salmonella-induced mouse cytokine secretion.

Mouse blood samples were collected 27 wk post-Salmonella infection by cardiac puncture and placed in tubes containing EDTA (10 mg/ml). Mouse cytokines were measured using mouse cytokine 10-Plex Panel kit (Invitrogen) according to the manufacturer's instructions, using the Luminex detection system (Perkin Elmer CS1000 Autoplex Analyzer) (26).

Immunoblotting.

Mouse colonic mucosa was collected by scraping both the proximal and distal regions of mouse colon as previously described (6). Mouse epithelial cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, and protease inhibitor cocktail). Immunoblot was performed with the following primary antibodies and visualized by enhanced chemiluminescence (43–44): anti-β-catenin (BD, Transduction Laboratories), anti-villin, anti-Frizzled 7 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GSK-3β (BD), anti-phosphor-GSK-3β (Ser9), anti-phospho-β-catenin (Ser552), anti-phospho-β-catenin (Ser33, 37, Thr41) and phosphor-Akt (T308) (Cell Signal, Beverly, MA), or anti-β-actin (Sigma-Aldrich, St. Louis, MO).

Coimmunoprecipitation assay.

Cells scraped from colonic tissues were rinsed twice in ice-cold HBSS and lysed in cold immunoprecipitation buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, and protease inhibitor cocktail (Boehringer Mannheim)]. Samples were immunoprecipitated with anti-lysine-acetylated antibody (Upstate, Lake Placid, NY) or anti-β-catenin antibody (Santa Cruz Biotechnology). Membranes were then stripped and reprobed with anti-β-catenin antibody and visualized using enhanced chemiluminescence. Protein input was normalized by the intestinal epithelial marker villin, as previously described (43).

Histological testing.

Tissues were processed by standard techniques (51). Sections (5 μm) were stained with hematoxylin and eosin. Histological score (0–4) to quantify the degree of gastrointestinal inflammation was determined according to the following criteria as described in a publication by Rath et al. (33): increased inflammatory cells, mucosal thickening, submucosal cell infiltration, destruction of architecture, and decreased of goblet cells. For immunostaining, antigens were retrieved by boiling the samples for 10 min in a 10-mM citrate solution (pH 6.0). The slides were stained as previously described (6). Blinded histologic inflammatory scores were performed using a validated scoring system (33) by a trained pathologist. 5-Bromo-2′-deoxyuridine (BrdU) staining and immunostaining were performed as previously described (51). Immunostaining and immunohistochemistry were performed with the following primary antibodies: anti-β-catenin, anti-phospho-β-catenin (Ser 552), anti-MMP-7, anti-phospho-c-myc, and anti-BrdU antibodies. Salmonella distribution in the intestine was determined by immunostaining using anti-Salmonella lipopolysaccharide antibody (Santa Cruz Biotechnology).

Quantitative real-time PCR analysis.

Total RNA was extracted from intestinal epithelial cells using Trizol reagent (Invitrogen, Carlsbad, CA). The cDNA reaction products were subjected to quantitative real-time PCR using the MyiQ single-color real-time PCR detection system and iQ SYBR green supermix (Bio-Rad, Hercules, CA) (51). All expression levels were normalized to the β-actin levels of the same sample. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All PCR reactions were performed in triplicate. PCR primers (Table 1) were designed using Lasergene (DNAStar, Madison, WI).

View this table:
Table 1.

Real-time PCR primers

Statistical analysis.

Data are expressed as means ± SD. All statistical tests were two-sided. The multiple comparisons of continuous variables were performed using ANOVA. The Tukey correction for multiple comparisons was used to adjust the significance level. P values < 0.05 after the correction for multiple comparisons were considered to be statistically significant. The nonparametric Kruskal-Wallis test was used for comparing the ordinal inflammation scores between groups. All statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, NC).

RESULTS

Intestinal inflammation is regulated by AvrA in vivo.

We established a chronic Salmonella colonization mouse model. After 3 wk of Salmonella infection, we found inflammation in the ileum and colon of mice infected with the AvrA strain. Less inflammation was found in the PhoPC group and PhoPC AvrA/AvrA+ group (Fig. 1A hematoxylin and eosin staining). After 3 wk of Salmonella infection, the overall inflammatory score in the ileum was significantly higher in the groups infected with the AvrA bacterial strain and the PhoPC AvrA/AvrA+ strain compared with the PhoPC strain. The overall inflammatory score in the colon was also significantly higher in the group infected with the AvrA bacterial strain compared with the PhoPC strain.

Fig. 1.

The physiological effect of AvrA on intestinal inflammation in vivo. A: hematoxylin and eosin staining of mouse ileum and colon after 3 wk of Salmonella infection. Experimental groups: control, normal wild-type mice; PhoPc with AvrA expression; PhoPc AvrA, AvrA mutant derived from PhoPc; and PhoPc AvrA/AvrA+, PhoPc AvrA with complemented plasmid encoding AvrA. B: inflammatory score in the intestine over 27 wk of Salmonella infection. Comparison was carried among the infected groups. **P < 0.01; n = 3 mice in each group. C: Salmonella in colon tissue was detected by 16S rRNA PCR after 27 wk of infection. Mice were infected with S. typhimurium PhoPc, PhoPc AvrA (AvrA gene mutant), or PhoPc AvrA/AvrA+; n = 3 mice in each group. D: Location of Salmonella in mouse colon using immunofluorescence staining. Green staining in the mouse colon showed Salmonella in the intestinal mucosa after 27 wk of infection.

The existence of Salmonella in the gut was monitored over 27 wk by culturing mouse feces overnight on a BBL CHROMagar plate, and Salmonella colonies appeared mauve (data not shown). Moreover, we used real-time PCR to amplify 16S rRNA to measure intestinal colonization by Salmonella. Figure 1C clearly shows the results of the bacterial 16S PCR assay which proves the persistence of Salmonella in the colon after 27 wk of infection. Persistent localized colonization of the colonic mucosa with Salmonella was also observed (bright green staining of Salmonella, Fig. 1D).

mRNA expression of genes associated with inflammation is altered by Salmonella infection.

Because chronic infection is associated with intestinal inflammation, we further investigated mRNA levels of the inflammatory cytokines ICAM, IL-1β, TNF-α, and MIP2. Real-time PCR showed elevated expression of inflammatory cytokines in Salmonella-infected colon after 3 or 27 wk of infection, particularly in mice infected with the AvrA mutant (Fig. 2A).

Fig. 2.

AvrA changes the expression of inflammatory cytokines in colon and serum. A: AvrA changes the expression of inflammatory cytokines at the mRNA level in colon infected with Salmonella. Total RNA from colon mucosa was extracted for real-time PCR 3 and 27 wk after infection. *P < 0.05, **P < 0.01. Each single experiment was assayed in triplicate. Data are presented as the means ± SD of n = 3 mice per group. B: serum cytokines in the mice infected with Salmonella for 27 wk. The cytokines in mouse serum were tested using mouse Cytokine 10-Plex Panel. *P < 0.05; **P < 0.01. The single experiment was assayed in triplicate. Data are the means ± SD of n = 4 mice in each group.

Three weeks after infection was defined as the chronic infection stage (25). At 3 wk, the AvrA infection group had significantly higher levels of ICAM compared with the control and PhoPC AvrA/AvrA+-infected groups. The AvrA infection group had significantly higher levels of MIP-2 compared with the control, PhoPC, and PhoPC AvrA/AvrA+ groups. The AvrA-infected group had significantly higher levels of IL-1β compared with the control, PhoPC, and PhoPC AvrA/AvrA+ groups. The AvrA infection group had significantly higher levels of TNF-α compared with the control, PhoPC, and PhoPC AvrA/AvrA+ groups.

After 27 wk of infection, the AvrA group still had significantly higher levels of MIP-2 and IL-1β expression compared with the control, PhoPC, and PhoPC AvrA/AvrA+ groups. These data indicate that in the absence of AvrA, mRNA levels of the inflammatory cytokine increased in vivo.

Serum cytokines in the Salmonella-infected mice at 27 wk postinfection.

We further investigated the profile of the cytokines in mouse serum at 27 wk postinfection (Fig. 2B). The AvrA group had significantly higher levels of IL-6 expression compared with the control, PhoPC, and AvrA/AvrA+ groups. The AvrA group also had significantly higher levels of IL-4 compared with the PhoPC group and higher INF-γ expressions compared with the control. We did not find significant differences in serum levels of IL-10, IL12, TNFα, and IL-1β in controls versus infected mice. There was no detectable colony-stimulating factor in any experimental groups (data not shown).

AvrA expression in Salmonella promotes intestinal epithelial cell proliferation.

Bacterial chronic infection plays a complex role in proliferation. We examined small intestinal epithelial cell proliferation at 1, 3, 6, 10, and 27 wk in Salmonella-infected mice using BrdU staining (Fig. 3A). BrdU incorporates into the newly synthesized DNA of proliferating cells. At week 1, compared with the PhoPC AvrA/AvrA+ group, the PhoPC AvrA group showed less intestinal proliferation (Fig. 3B, 1 wk). There were no significant findings in the small intestine among the control group without infection and infected groups at week 1. At week 3, the PhoPC AvrA/AvrA+ group had more proliferation than the control group, while the AvrA group showed less intestinal proliferation than the PhoPC AvrA/AvrA+ group. At week 6, the PhoPC and PhoPC AvrA/AvrA+ groups showed more intestinal proliferation than the control group, and the PhoPC AvrA group showed less intestinal proliferation than PhoPC and PhoPC AvrA/AvrA+ groups. At week 10, the PhoPC group showed significantly more intestinal proliferation compared with the control and AvrA groups, whereas the PhoPC AvrA/AvrA+ group showed more intestinal proliferation than the PhoPC AvrA group. At week 27, the AvrA group showed less intestinal proliferation than the control and PhoPC groups.

Fig. 3.

Effect of AvrA in intestinal proliferation over 27 wk of Salmonella infection in vivo. A: 5-bromo-2′-deoxyuridine (BrdU) staining of small intestine. B: number of proliferative cells in small intestine. Data are presented as the means ± SD of n = 5 mice per group. C: number of proliferative cells in the colon 27 wk after infection. Data are presented as the means ± SD of n = 5 mice per group. *P < 0.05; **P < 0.01.

The enhanced proliferation was also observed in mouse colon infected with the PhoPC and PhoPC AvrA/AvrA+ strains (Fig. 3C). At week 27, compared with the control, PhoPC AvrA, and PhoPC AvrA/AvrA+ groups, the PhoPC group showed more intestinal proliferation. The PhoPC AvrA/AvrA+ group showed more intestinal proliferation than the control and PhoPC AvrA groups. These data show physiological differences between the animal groups infected with Salmonella with or without AvrA expression.

AvrA modulates β-catenin posttranslational modification in a chronic Salmonella-infection mouse model.

β-catenin is known to control cell proliferation and regulate intestinal inflammation (7, 15, 31, 34, 45, 55). We hypothesized that chronic AvrA exposure also regulates the β-catenin signaling pathway, thus enhancing intestinal cell proliferation. First, we determined how AvrA modulates β-catenin at the posttranslational level: phosphorylation and acetylation.

There are several amino acid sites associated with β-catenin phosphorylation (pho-β-catenin) (46). Phosphorylated Ser 33/37 and Thr41 sites are associated with β-catenin degradation. We found that β-catenin phosphorylation at Ser33/37 and Thr41 was regulated by Salmonella infection and AvrA expression (Fig. 4A). Among the infected groups, at 6 wk, the AvrA group had the highest level of pho-β-catenin compared with the PhoPC and PhoPC AvrA/AvrA+ groups. At 10 wk, the AvrA group has statistically significantly higher expression compared with the control group. Finally, at 27 wk, compared with the PhoPC and control groups, the pho-β-catenin level in the AvrA group was significantly higher. Overall, densitometry data showed that the mice infected with the AvrA mutant strain had a significantly higher level of pho-β-catenin (Ser33/37, Thr41), compared with the mice infected with AvrA expressing Salmonella strains (Fig. 4A). The observed difference between the PhoPC and AvrA groups suggested that AvrA expression was able to suppress the phosphorylation of β-catenin at Ser 33/37 and Thr41. The effect of AvrA on β-catenin phosphorylation is maintained after chronic infection.

Fig. 4.

Salmonella AvrA modifies β-catenin posttranslationally in the colonic cells. A: phosphorylation of β-catenin (p-β-catenin Ser33/37 Thr41) in mouse intestinal mucosa after Salmonella infection in vivo. Relative band intensity of p-β-catenin in vivo. Data are presented as the means ± SD; n = 3 mice per group. *P < 0.05; **P < 0.01. B: bacterial effector protein AvrA modulates β-catenin acetylation in colonic epithelial cells. Acetylation of β-catenin in Salmonella-infected intestine after 6 and 10 wk in vivo. Please note that Salmonella AvrA increases β-catenin acetylation in the host cells. Colonic epithelial cells were harvested. Cell lysates were immunoprecipitated with anti-β-catenin or anti-acetyl-lysine antibody. Proteins were separated on SDS-PAGE gels for Western blot analysis. Protein input was normalized by the intestinal epithelial marker villin. Data are from a single experiment and are representative of n = 3 mice per group.

Acetylation of β-catenin regulates β-catenin transcriptional activity by modulating its affinity for TCF, thus being involved in regulating Wnt/β-catenin signaling and participating in proliferation (22, 47). Therefore, we also examined the acetylation pattern of β-catenin in the infected colon. In vivo immunoprecipitation data further showed that PhoPC infection consistently increased the acetylated form of β-catenin after 6 and 10 wk of infection (Fig. 4B).

β-catenin translocates to the nuclei of intestinal cells infected with PhoPc or PhoPc AvrA/AvrA+.

Based on the AvrA-associated change of acetylation of β-catenin in the infected colon, we further investigated the distribution of β-catenin in chronic Salmonella infection. In the intestines of uninfected mice, β-catenin was localized on the cell membrane and in the cytoplasm (Fig. 5A control). In contrast, we found that AvrA expression in Salmonella enhanced the nuclear β-catenin signal in the PhoPC and PhoPC AvrA/AvrA+-infected mouse colons, whereas less nuclear staining was identified in the AvrA group at 27 wk after infection (Fig. 5A). Statistical analysis showed that the numbers of nuclear β-catenin-positive cells per crypt in PhoPC AvrA/AvrA+ -infected mouse intestine were significantly higher than those in the control and AvrA groups (Fig. 5B).

Fig. 5.

AvrA changes the location and target genes of β-catenin in the colon after chronic Salmonella infection. A: location of β-catenin in the mouse large intestine after S. typhimurium infection. Some of β-catenin localizes in the cell membrane. Please note the enhanced nuclear staining (dark brown) in the mouse colon after S. typhimurium PhoPc and AvrA/AvrA+ infections. Left: β-catenin in the cell nuclei. The red box represents the higher magnification of β-catenin in colon. B: nuclear β-catenin-positive cells in intestinal crypts. Data are presented as the means ± SD; n = 3 mice per group. *P < 0.05; **P < 0.01. C: T cell factors-3 and -4 mRNA levels in the mouse large intestine after 6 and 27 wk of S. typhimurium infection; n = 3 mice per group. *P < 0.05; **P < 0.01. D: AvrA changes the β-catenin target genes at the protein level. Colonic epithelial cells were harvested. Total proteins were separated on SDS-PAGE gels for Western blot analysis. Data are presented as the means ± SD; n = 3 mice per group. *P < 0.05; **P < 0.01. E: location of cyclin D1, p-c-myc, and MMP7 in the infected colon. The red box represents the higher magnitude images of cyclin D1, p-c-myc, or MMP7 staining in colon.

Chronic Salmonella infection alters target genes of the β-catenin pathway.

Nuclear β-catenin is a transcription factor. Acetylation of β-catenin regulates β-catenin transcriptional activity by modulating its affinity for TCFs. We further identified significant changes in TCF3 and -4 over the 27-wk duration of infection (Fig. 5C). β-catenin is known to bind with TCFs, thus activating the promoters of target genes. Therefore, we investigated the expression of cyclin D1, p-c-myc, and mmp7 proteins encoded by genes that are normally upregulated by increased β-catenin transcriptional activity. At the protein level, there was a significant difference between animals infected with the Salmonella strains with AvrA and those infected with Salmonella strains lacking AvrA (Fig. 5D). At 6 and 27 wk postinfection, expression levels of cyclin D1, p-c-myc, and MMP7 were still significantly higher in the PhoPC and PhoPC AvrA/AvrA+ groups compared with the control and AvrA groups (Fig. 5D). For cyclin D1 at 6 wk, the increase was significant when the PhoPC-infected group was compared with the control, AvrA, and PhoPC AvrA/AvrA+ groups, respectively. There were no significant findings at 27 wk. For p-c-myc at 6 wk, there was significant alteration when the PhoPC infected group was compared with the PhoPC AvrA/AvrA+ and PhoPC AvrA groups, respectively. At 27 wk, the level of p-c-myc was significantly increased in PhoPC group when compared with the control and PhoPC AvrA groups, respectively. The P values were significant when the PhoPC AvrA/AvrA+ group was compared with the control and AvrA groups. For MMP7 at 27 wk, the PhoPC AvrA/AvrA+ group had the highest amount when it was compared with the control, PhoPC, and PhoPCAvrA groups, respectively. Western blot data indicated that c-myc, a target gene for β-catenin (15), was also increased in the PhoPC-infected mouse colon (data not shown).

By IHC analysis, we examined the location of cyclin D1 and phosphorylated c-myc (Fig. 5E). More cyclin D1 and nuclear staining of p-c-myc was found in the PhoPC-infected mouse intestine. Furthermore, we found that MMP7 staining was enhanced in the PhoPC and AvrA/AvrA+ groups, whereas less MMP7 staining was found in the AvrA-infected mice (Fig. 5E, MMP7).

Akt activation in the Salmonella-infected mice.

Akt is the upstream regulator of the β-catenin pathway. Our data showed that total Akt protein levels were increased in the PhoPC-and PhoPC AvrA/AvrA+-infected mice (Fig. 6A). Phosphorylated Akt was also increased, especially in the PhoPC AvrA/AvrA+ group (Fig. 6A). Akt phosphorylates β-catenin at Ser552 (50). The β-catenin (pho-β-catenin) phosphorylated at Ser552 was also increased in the PhoPC and PhoPC AvrA/AvrA+ groups' intestinal epithelial cells at 27 wk of infection.

Fig. 6.

Salmonella AvrA increases Akt/pho-β-catenin in vivo. A: protein expression of Akt, phospho-Akt, GSK-3β, and phospho-β-catenin in the intestine after infection 27 wk. B: localization of phospho-Akt in the intestine after 27 wk of infection. C: localization of p-β-catenin ser 552 in the intestine. The red box represents the higher magnitude images of p-β-catenin ser 552. D: nuclear p-β-catenin ser 552 in intestinal crypts. Data are presented as the means ± SD; n = 3 mice per group. *P < 0.05; **P < 0.01. E: working model of AvrA/β-catenin interaction in chronic intestinal Salmonella infection. Salmonella effector AvrA persistently regulates β-catenin posttranslational modification, including phosphorylation (P), ubiquitination (Ub), and acetylation (Ac). The hyperphosphorylation of Ser 33/37 and Thr41 by glycogen synthase kinase-3β (GSK3β) promotes the ubiquitylation and targeted destruction of β-catenin. Akt, the upstream regulator of β-catenin, phosphorylates this protein at Ser552 and contributes to the increased nuclear β-catenin signaling. Akt and β-catenin target genes were enhanced by Salmonella expressing AvrA. Hence, AvrA has a chronic function in promoting intestinal proliferation.

Moreover, we tested the location of phosphorylated-Akt (pho-Akt) (Fig. 6B). The red staining showed the activated form of Akt in the nuclei. It was undetectable in the normal intestine, but the nuclear staining of pho-Akt was very strong in the PhoPC AvrA/AvrA+-infected intestinal epithelial cells. The Pho-β-cat-Ser552 form is a marker of intestinal stem cells and has a nuclear distribution (16). In vivo IHC data showed that the PhoPC and PhoPC AvrA/AvrA+ infected mouse intestines had more nuclear staining of Pho-β-cat-Ser552 than the AvrA-infected or control mice (Fig. 6C). Statistical analysis confirmed a significant difference in the number of nuclear β-catenin-positive cells per crypt in the PhoPC-and PhoPC AvrA/AvrA+-infected mice compared with the control and AvrA groups (Fig. 6D).

DISCUSSION

In this present study, we have demonstrated the long-term effects and molecular mechanisms of the Salmonella effector protein AvrA in activating the β-catenin pathway in small intestine and colon. We found that Salmonella strains expressing AvrA consistently resulted in increased acetylation of β-catenin, enhanced TCF expression and β-catenin target genes, and increases in colonic cell proliferation in vivo. Our previously published studies mainly focused on acute infection and inflammation induced by Salmonella 4 h to 7 days after infection (23, 25, 42). We have demonstrated the acute effects of Salmonella and AvrA on the Wnt/β-catenin signaling pathway. We reported an established chronically Salmonella-infected model and showed physiologic changes in various organs, such as liver, gallbladder, spleen, and cecum (26). This present study focuses on biomarkers of β-catenin activation in mouse ileum and colon infected with Salmonella for 27 wk. It demonstrates that Salmonella strains expressing the enteric bacterial protein AvrA regulate the β-catenin signaling pathway in intestine over long-term chronic infection.

Our data show that the acetylation of the β-catenin is involved in chronic Salmonella-host interactions in the intestine. Acetylation of the β-catenin COOH terminus enhances its ability to activate TCF (37). Our data demonstrate that AvrA is able to increase the acetylation of the β-catenin and the expression level of TCFs 27 wk after Salmonella infection. β-catenin activity is modified by balancing acetylation, phosphorylation, and ubiquitination (10). It is possible that AvrA increases β-catenin acetylation, thus changing other modifications of β-catenin.

Phosphorylation of β-catenin is regulated by different upstream regulators and has different functions. Phosphorylated Ser 33/37 and Thr41 sites are associated with β-catenin degradation. This hyperphosphorylation of Ser 33/37 and Thr41 by glycogen synthase kinase-3β promotes the ubiquitylation and targeted destruction of β-catenin. In contrast, increased phosphorylation of β-catenin Ser552 is involved in inflammation-induced stem progenitor cell expansion (16). We found that AvrA mutation-infected colon had significantly higher phosphorylated Ser 33/37 Thr41 and AvrA that expression led to less phosphorylated Ser 33/37 Thr41. These differences in β-catenin postmodification may lead to stabilization of the total β-catenin and activation of the β-catenin signaling in the chronically infected intestine, which is consistent with our previous report in cell culture models (42). We also found that phospho-β-catenin at Ser552 was increased in colon epithelial cells of PhoPC and PhoPC AvrA/AvrA+-infected mice at 27 wk of infection. This finding is consistent with our previous study in the acute infected intestine that phospho-β-catenin at Ser552 was enhanced by AvrA (23). Enhanced phospho-β-catenin at Ser552 indicates simultaneous activation of phosphatidylinositol 3-kinase-Akt and Wnt signaling (50). Overall, knowledge gained from these studies indicates how bacterial AvrA activates β-catenin activity through posttranslational modifications.

Recently, we demonstrated that the Salmonella protein AvrA upregulates Wnt expression, the upstream regulators of the Wnt/β-catenin pathway (23, 26, 40). In the chronically infected mouse intestine, the Wnt receptor Frizzled 7 was also upregulated by AvrA (Fig. 6A). The Wnt and Akt pathways interact to control β-catenin nuclear localization and transcriptional activity in epithelial homoeostasis (16). It is known that AvrA influences eukaryotic cell pathways via proteins including NF-κB, JNK, and β-catenin (51, 56). Many proteins coordinate distinct signaling pathways in cells by having multiple functions. Bacterial effectors may possess multiple enzyme activities to modify different eukaryotic proteins and maximize the bacterial effector's ability to modulate cellular functions. The roles of AvrA in modulating the host signaling pathways are similar to those of a bacterial protein Salmonella-secreted factor L (4, 20, 35). As a multifunctional enzyme, AvrA blocks the host response to acute inflammation (18, 21, 51).

In the present study, we noticed that AvrA consistently influences the β-catenin pathway and impacts intestinal epithelial proliferation, rather than simply inhibiting acute inflammation. Although inflammation and proliferation may occur simultaneously in infection, we observed consistently increased proliferation in the intestine colonized with AvrA expressing bacteria postinfection 27 weeks, whereas significant inhibition of intestinal inflammation by AvrA occurred 3 wk after infection. Although we observe the changes in some serum cytokines at 27 wk postinfection, the systemic response may not directly reflect the local inflammation in intestine. Because infection is associated with fitness costs in the host, the increased epithelial cell renewal observed upon Salmonella infection may be a consequence of a host defense mechanism designed to replenish the damaged tissue in the gut (40). For persistent bacterial existence in the intestine, AvrA plays an essential role in regulating the long-term interactions between the host and Salmonella.

Live, mutated noninvasive Salmonella species have been used as a vector to specifically target cancer cells (53). Activation of the β-catenin pathway is associated with a mutant Salmonella PhoPc, which has been used as a nonpathogenic Salmonella for intestinal inflammation studies (3, 32). Our study demonstrating that Salmonella expressing AvrA persistently activates the β-catenin pathway raises concerns about using mutated Salmonella as a vector in cancer therapy. Further studies are needed to confirm the safety of these approaches for potential tumor therapy.

In summary, we have made specific advances and uncovered several novel aspects of Salmonella and the effector AvrA in this study: 1) Salmonella strains expressing AvrA consistently enhance acetylation of β-catenin, induce β-catenin nuclear translocation, and increase transcription factors TCFs and β-catenin target genes, 2) Akt and pho-Akt expression are enhanced in the intestine infected with AvrA expressing bacteria, and 3) Salmonella strains expressing AvrA persistently promote small intestinal and colonic epithelial proliferation in vivo over 10–27 wk. We speculate that chronic AvrA/β-catenin interaction occurs through posttranslational modification, thus promoting intestinal proliferation (Fig. 6E). Salmonella had chronic effects on modulating the eukaryotic signaling pathways related to proliferation, inflammation, and tumorigenesis. Our findings are significant because the mechanism of bacterial effectors in infection will be a prerequisite to the development of therapeutic protocols for digestive diseases and applications of nonpathogenic Salmonella as vectors in cancer therapy.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants KO1-DK-075386 and 1R03-DK-089010-01, American Cancer Society Grant RSG-09-075-01-MBC, and the IDEAL award from New York State's Empire State Stem Cell Board Grant N09G-279 (to J. Sun).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: R.L., X.L., S.W., Y.-g.Z., E.O.P., E.C.C., and J.S. conception and design of research; R.L., X.L., S.W., and Y.-g.Z. performed experiments; R.L., X.L., S.W., Y.X., Y.-g.Z., E.O.P., E.C.C., and J.S. analyzed data; R.L., S.W., Y.X., E.C.C., and J.S. interpreted results of experiments; R.L. and J.S. prepared figures; R.L., Y.X., and J.S. drafted manuscript; R.L., X.L., S.W., Y.X., Y.-g.Z., E.O.P., E.C.C., and J.S. edited and revised manuscript; R.L., X.L., S.W., Y.X., Y.-g.Z., E.O.P., E.C.C., and J.S. approved final version of manuscript.

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