Salmonella Enteritidis Effector AvrA Suppresses Autophagy by Reducing Beclin-1 Protein

Abstract

Autophagy is a cellular process to clear pathogens. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. However, major studies still focus on Salmonella enterica serovar Typhimurium. Here, we reported that AvrA, a S. Enteritidis effector, inhibited autophagy to promote bacterial survival in the host. We found that AvrA regulates the conversion of LC3 I into LC3 II and the enrichment of lysosomes. Beclin-1, a key molecular regulator of autophagy, was decreased after AvrA expressed strain colonization. In S.E-AvrA^--infected cells, we found the increases of protein levels of p-JNK and p-c-Jun and the transcription level of AP-1. AvrA-reduction of Beclin-1 protein expression is through the JNK pathway. The JNK inhibitor abolished the AvrA-reduced Beclin-1 protein expression. Moreover, we identified that the AvrA mutation C186A abolished its regulation of Beclin-1 expression. In addition AvrA protein was found interacted with Beclin-1. In organoids and infected mice, we explored the physiologically related effects and mechanism of AvrA in reducing Beclin-1 through the JNK pathway, thus attenuating autophagic responses. This finding not only indicates an important role of S. Enteritidis effector in reducing host protein as a strategy to suppress autophagy, but also suggests manipulating autophagy as a new strategy to treat infectious diseases.

Keywords: autophagy; effector; infection; organoids; paneth cells.

Figure 1

Salmonella Enteritidis AvrA inhibits autophagy in cell models. AvrA-regulated expression levels of autophagy related proteins. (A) The indicated HCT116 cell lines were infected with S.E WT, S.E-AvrA⁻ and S.E-AvrA⁺ strains (MOI:1:1, 1 h incubation before harvested, n = 4) as shown and analyzed for protein expression by immunoblotting. The immunoblotting of P62/LC3 was used to track the expression of P62 and the conversion of LC3 I into LC3 II for autophagic activity in the HCT116 cells after infection with the different S.E. The relative density of P62 (B) and LC3 II/LC3 I (C) was determined using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA). N = 4, **adjusted P < 0.01, ***adjusted P < 0.001 by ANOVA test. (D) HCT116 cell lines were incubated with 100 nM LysoTracker Deep Red Probe, then infected with S.E WT, S.E-AvrA- and S.E-AvrA+ strains (MOI:1:1, 1 h incubation before microscopic examination) to check the lysosomes staining. The immunofluorescence indicated that the HCT116 cells, pre-treated with LysoTracker, showed more lysosomes in the cells infected with the S.E-AvrA⁻ bacteria compared with the cells infected by the AvrA present strains. (E) Quantification of the number of lysotracker positive vesicles. The data are reported as the mean ± SE from three independent experiments, and a total of 100 cells per condition were analyzed. n = 100, **adjusted P < 0.01, ***adjusted P < 0.001 by ANOVA test. The numbers of associated Salmonella and internalized Salmonella in human epithelial cells colonized with wild-type Salmonella Enteritidis or AvrA mutant or AvrA-complemented strains. Human epithelial cells were grown on an insert, colonized with an equal number of the indicated bacteria for 30 min, after washed with HBSS, the number of associated Salmonella (Salmonella adhesion) was determined (F). Alternatively, cells were incubated in DMEM containing gentamicin (100 μg/ml) for 30 min, and the number of internalized intracellular Salmonella (Salmonella invasion) was then determined (G). Data are reported as the mean ± SE from six independent experiments, *adjusted P < 0.05, **adjusted P < 0.05 by ANOVA test.

Figure 2

AvrA changes Beclin-1 protein levels and interacts with Beclin-1 in HCT116 cells. AvrA changes Beclin-1 protein levels and interacts with Beclin-1 in HCT116 cells. (A) The Western blot shows the expression of Beclin-1 in the HCT116 cells after colonization with wild-type Salmonella Enteritidis or AvrA mutant or AvrA-complemented strains (MOI:1:1, 1 h incubation before harvested, n = 4). The relative density of Beclin-1 (B) was determined using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA). n = 4, *adjusted P < 0.05 by ANOVA test. (C) The Western blot shows the expression of Beclin-1 in the HCT116 cells after transfection with the AvrA wild-type and AvrA mutant plasmids (200 ng/μl, 24 h incubation, n = 5). The relative density of Beclin-1 (D) was determined using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA). n = 5, *adjusted P < 0.05, **adjusted P < 0.01 by ANOVA test. (E) The HCT116 cells were cotransfected with the AvrA WT plasmid (c-myc tag) and the Beclin-1 WT plasmid (HA-tag) (200 ng/μl, 24 h incubation, n = 4). At the indicated times, immunoprecipitation was performed with an anti-c-myc mouse monoclonal antibody. Pre-immune mouse IgG was used as a negative control. VPS34 was used as a positive control. The Western blot analyses of the pre-immunoprecipitation (Input) and immunoprecipitated samples (IP) were performed with an anti-c-myc mouse monoclonal antibody or with an anti-HA mouse monoclonal antibody. These results shown are representative of three independent experiments.

Figure 3

AvrA inhibits the JNK/c-jun/AP-1 signaling pathway to decrease Beclin-1 in HCT116 cells. AvrA inhibited the JNK pathway and decreased Beclin-1 in HCT116 cells, and the effect of AvrA on Beclin-1 expression was abolished by the JNK inhibitor SP600125. (A) The Western blot shows a change in the JNK pathway markers and Beclin-1 in the cells treated with the JNK inhibitor SP600125 (50 μM, 30 min) and those infected with the different S.E strains (MOI:1:1, 1 h incubation before harvested, n = 4), These results shown are representative of four independent experiments. (B) The luciferase reporter assay shows a change in AP-1 transcription in the cells treated with the JNK inhibitor SP600125 and in those infected with the different S.E strains (MOI:1:1, 1 h incubation before harvested, n = 6). *adjusted P < 0.05 by ANOVA test.

Figure 4

The AvrA C186A mutant plasmid abolishes the regulation of exogenous Beclin-1. The HCT116 cells were transfected with the indicated plasmids (200 ng/μl, 24 h incubation, n = 4). At the indicated times, the changes in the target proteins were measured. (A) The Western blot shows the expression of Beclin-1 in the HCT116 cells after transfection with the indicated plasmids. The relative density of Beclin-1 (B) was determined using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA). n=4, *P < 0.05 by student's t-test. (C) The Western blot shows the change in the JNK pathway markers and Beclin-1 in the HCT116 cells after transfection with the AvrA wild-type and AvrA mutant plasmids. These results shown are representative of four independent experiments.

Figure 5

Salmonella Enteritidis AvrA changes the levels of Beclin-1 in mouse intestinal organoids. Salmonella Enteritidis infection and invasion in the mouse organoids. (A) The Western blot shows the expression of Beclin-1, P62, LC3, and the JNK pathway markers in the organoids after infection with wild-type Salmonella Enteritidis or AvrA mutant or AvrA-complemented strains (MOI:1:1, 1.5 h incubation before harvested, n = 3). The relative density of Beclin-1 (B), P62 (C), and LC3 II/LC3 I (D) was determined using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA). n = 3, *adjusted P < 0.05, **adjusted P < 0.01, ^#unadjusted P < 0.05 by ANOVA test. (E) The representative images of the immunostaining of P62 in the organoids after infection with wild-type Salmonella Enteritidis or AvrA mutant or AvrA-complemented strains (MOI:1:1, 1.5 h incubation).

Figure 6

AvrA changes the levels of Beclin-1 and affects the function of Paneth cell granules of the ileal tissues in a mouse model. Wild-type Salmonella Enteritidis, AvrA mutant or AvrA-complemented strains were used to infect C57BL/6 mouse models (1.0 × 10⁸ CFU per mouse, i.g., n = 6). At 8 h post-infection, the ileal tissue was harvested for immunoblotting and immunofluorescence staining. (A) Western blot shows the expression of Beclin-1, P62, LC3, and the JNK pathway markers in the ileal tissue after infection with the different S.E strains. The relative density of Beclin-1 (B), P62 (C), and LC3 II/LC3 I (D) was determined using Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA). n = 5–6, *adjusted P < 0.05, **adjusted P < 0.01 by ANOVA test. (E) The representative images of the indirect immunofluorescence of the sections stained for lysozymes (42) in the ileal crypts of the C57BL/6 mice following infected with wild-type Salmonella Enteritidis or AvrA mutant strains. (F) The percentage of Paneth cells displaying a normal (D0) and abnormal (D1–D3) pattern of lysozyme expression. n = 5–6, *adjusted P < 0.05, **adjusted P < 0.01, ***adjusted P < 0.001 by ANOVA test.

Figure 7

Working model of AvrA inhibition of Beclin-1-dependent autophagy. Salmonella Enteritidis AvrA inhibits the autophagic response by decreasing Beclin-1 protein. At the molecular level, AvrA interacts with Beclin-1. This process occurs through the inhibition of the JNK signaling pathway. At the cellular level, AvrA affects the function of Paneth cell granules by inhibiting autophagy.