Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Mar 28:14:1268243.
doi: 10.3389/fcimb.2024.1268243. eCollection 2024.

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli

Alison Da Silva  1 , Guillaume Dalmasso  1 , Anaïs Larabi  1 , My Hanh Thi Hoang  1   2 , Elisabeth Billard  1 , Nicolas Barnich #  1 , Hang Thi Thu Nguyen #  1
Affiliations

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli

Alison Da Silva et al. Front Cell Infect Microbiol. .

Abstract

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation.

Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used.

Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Keywords: Crohn’s disease; NDP52; adherent-invasive E. coli (AIEC); autophagy; p62.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer NR declared a past co-authorship with the author NB to the handling editor.

Figures

Figure 1
Figure 1
NDP52 and p62 are implicated in the control of AIEC LF82 intracellular number. (A–C). T84 cells were transfected with 50 nM of control siRNA or siRNA against NDP52, p62 or Optineurin (A), or TAX1BP1, NBR1, or Optineurin (B), or with both siRNA against NDP52 and p62 or siRNA against NDP52 or p62 separately (C). 48 h after transfection, the cells were infected with AIEC LF82 strain at a MOI of 10 for 3 h. The cells were then washed and incubated with the infection media containing 100 μg/ml gentamicin for 1, 7 or 21 h, which corresponded to 4, 10 or 24 h post-infection on the graph respectively. The cells were washed, lysed and plated on LB agar plate to determine the colony-forming units of LF82. p62 KO HeLa (D) or NDP52 KO HeLa (E) cells and their corresponding control cells were infected with the AIEC LF82 strain as in A-C, and the colony-forming units of LF82 were determined. Results are presented as means ± SEM from 3 independent experiments. Different points on the graph presented replicates from 3 independent experiments. Statistical analyses were performed using one-way Anova test followed by a post-test Bonferroni correction. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
Figure 2
Figure 2
Depletion of NDP52 or p62 leads to enhanced intracellular number of clinical AIEC strains in HeLa cells. P62 KO HeLa (A) or NDP52 KO HeLa (B) cells and their corresponding control cells were infected with the K12 C600 strain, or different clinical AIEC strains (LF82, CEA501S or CEA614S) at a MOI of 10 for 3 h. The cells were then washed and incubated with the infection media containing 100 μg/ml gentamicin for 1 h. The cells were washed, lysed and plated on LB agar plate to determine the bacterial colony-forming units. Results are presented as means ± SEM from 3 independent experiments. Different points on the graph presented replicates from 3 independent experiments. Statistical analyses were performed using one-way Anova test followed by a post-test Bonferroni correction. **P ≤ 0.01; ***P ≤ 0.001.
Figure 3
Figure 3
NDP52 and p62 directly colocalize with AIEC LF82 bacteria and the autophagic LC3 protein in HeLa cells. (A) HeLa cells were infected with AIEC LF82-GFP at a MOI of 100 for 3 h, washed and incubated with 100 μg/ml gentamicin for 3 h. Immunofluorescent labeling to detect p62 (blue) and LC3 (red) was performed. (B) HeLa-GFP-LC3 cells were infected with AIEC LF82-mCherry at a MOI of 100 for 3 h, washed and incubated with 100 μg/ml gentamicin for 3 (h). Immunofluorescent labeling to detect NDP52 (magenta) was performed. Observation was performed on a Zeiss LSM 800 Airyscan confocal microscope. Each experiment was repeated 3 times, and 2 replicates (2 coverslips) were prepared for each experiment. For each coverslip, 20 images were taken. Bars: 2 μm.
Figure 4
Figure 4
Depletion of NDP52 or p62 leads to increased AIEC LF82-induced pro-inflammatory cytokine production in HeLa cells. p62 KO (A, B) or NDP52 KO (C, D) HeLa cells and their corresponding control cells were infected with AIEC LF82 strain at a MOI of 10 for 3 h. The cells were then washed and incubated with the infection media containing 100 μg/ml gentamicin for 1, 7 or 21 h, which corresponded to 4, 10 or 24 h post-infection on the graphs respectively. Cell culture supernatants were collected at 4, 10 and 24 h post-infection, and the amount of secreted IL-6 (A, C) and IL-8 (B, D) were analyzed by ELISA. Results are presented as means ± SEM from 3 independent experiments. Different points on the graph presented replicates from 3 independent experiments. Statistical analyses were performed using one-way Anova test followed by a post-test Bonferroni correction. *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
Figure 5
Figure 5
The CD-associated NDP52Val248Ala variant does not impact AIEC LF82 intracellular number in HeLa cells. (A) Western blot analysis of NDP52 expression in NDP52 KO HeLa cells transfected with different quantities (250, 500, 750 or 1000 ng/well) of a construct that expresses wild-type NDP52 or the mutated NDP52Val248Ala or the empty plasmid or with different volumes of vehicle (corresponding to the volumes of plasmid added). HeLa cells from ATCC and the corresponding control cells of NDP52 KO cells were used in parallel. (B, C) NDP52 KO HeLa cells expressing NDP52-GFP or NDP52Val248Ala-GFP were infected with AIEC LF82-mCherry at a MOI of 100 for 3 h. The cells were then washed and incubated with the infection media containing 100 μg/ml gentamicin for 3 h. (B) The percentage of mCherry-positive cells (LF82-infected cells) among GFP-positive cells (transfected cells) was determined by flow cytometry. (C) Immunofluorescent labeling to detect LC3 (magenta) was performed. Observation was performed on a Zeiss LSM 800 Airyscan confocal microscope. Each experiment was repeated 3 times, and 2 replicates (2 coverslips) were prepared for each experiment. For each coverslip, 20 images were taken. Bars: 2 μm. (D) NDP52 KO HeLa cells transfected with the construct expressing wild-type NDP52 or NDP52Val248Ala protein were infected with AIEC LF82 at a MOI of 100 for 3 h. The cells were then washed and incubated with the infection media containing 100 μg/ml gentamicin for 1, 7 or 21 h, which corresponded to 4, 10 or 24 h post-infection on the graphs, respectively. The cells were washed, lysed and plated on LB agar plates to determine the bacterial colony-forming units (CFUs). Results are presented as means ± SEM from 3 independent experiments.
Figure 6
Figure 6
The CD-associated NDP52Val248Ala variant does not impact AIEC LF82-induced NF-κB activation and pro-inflammatory cytokine production in HeLa cells. NDP52 KO HeLa cells were transfected with a construct that expresses wild-type NDP52 or the mutated NDP52Val248Ala (A–C), and were infected with AIEC LF82 at a MOI of 10 for 3 h. The cells were then washed and incubated with the infection media containing 100 μg/ml gentamicin for 1, 7 or 21 h (which corresponded to 4, 10 or 24 h post-infection on the graphs respectively) (A, B) or 3 h (C). (A, B) Cell culture supernatants were collected at 4, 10 and 24 h post-infection, and the amounts of secreted IL-6 and IL-8 were analyzed by ELISA. Results are presented as means ± SEM from 3 independent experiments. Different points on the graph represented replicates from 3 independent experiments. (C) Western blot analysis for phospho-IκB levels. The immunoblot is representative of three independent experiments..
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Appleyard R. K. (1954). Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from escherichia coli K12. Genetics 39, 440–452. doi: 10.1093/genetics/39.4.440 - DOI - PMC - PubMed
    1. Bjørkøy G., Lamark T., Brech A., Outzen H., Perander M., Øvervatn A., et al. . (2005). p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 171, 603–614. doi: 10.1083/jcb.200507002 - DOI - PMC - PubMed
    1. Bretin A., Carrière J., Dalmasso G., Bergougnoux A., B’chir W., Maurin A.-C., et al. . (2016). Activation of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway triggers autophagy response to Crohn disease-associated adherent-invasive Escherichia coli infection. Autophagy 12, 770–783. doi: 10.1080/15548627.2016.1156823 - DOI - PMC - PubMed
    1. Bretin A., Lucas C., Larabi A., Dalmasso G., Billard E., Barnich N., et al. . (2018). AIEC infection triggers modification of gut microbiota composition in genetically predisposed mice, contributing to intestinal inflammation. Sci. Rep. 8, 12301. doi: 10.1038/s41598-018-30055-y - DOI - PMC - PubMed
    1. Buisson A., Douadi C., Ouchchane L., Goutte M., Hugot J.-P., Dubois A., et al. . (2019). Macrophages inability to mediate adherent-invasive E. coli replication is linked to autophagy in crohn’s disease patients. Cells 8, 1394. doi: 10.3390/cells8111394 - DOI - PMC - PubMed

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the “Ministère de l'Enseignement Supérieur et de la Recherche”, Inserm (Institut national de la santé et de la recherche médicale; UMR1071), INRAE (Institut national de recherche en agriculture, alimentation et environnement; USC 1382), the Agence Nationale de la Recherche of the French government through the program “Investissements d’Avenir” I-SITE CAP 20-25 CLERMONT (Projects HAPPYCROHN, RUNNINGUT and EXOCROHN to HN) and the ANR (French National Research Agency; project AAPG2021_RESTOGUT to HN and project AAPG2022_TOPIC to NB and HN).
-