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. 2015 Jun;148(7):1405-1416.e3.
doi: 10.1053/j.gastro.2015.02.013. Epub 2015 Feb 18.

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters

Andrew T Schuster #  1 , Craig R Homer #  2 , Jacqueline R Kemp  1 , Kourtney P Nickerson  2 , Emily Deutschman  1 , Yeojung Kim  2 , Gail West  2 , Tammy Sadler  2 , Eleni Stylianou  2 , Dawid Krokowski  3 , Maria Hatzoglou  3 , Carol de la Motte  2 , Brian P Rubin  1   4 , Claudio Fiocchi  2 , Christine McDonald  2 , Michelle S Longworth  1
Affiliations

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters

Andrew T Schuster et al. Gastroenterology. 2015 Jun.

Abstract

Background & aims: Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium.

Methods: Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors.

Results: CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, the products of which heterodimerize to form an amino acid transporter in HT-29 cells after bacterial infection; levels of SLC7A5-SLC3A2 were increased in tissues from patients with UC compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5-SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3-deficient cells.

Conclusions: CAP-D3 down-regulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

Keywords: Condensin; Dysbiosis; Inflammatory Bowel Disease; Innate Immunity.

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Figures

Figure 1
Figure 1. CAP-D3 promotes bacterial autophagy in human colon epithelial cells
A) Immunoblot showing reduced expression of CAP-D3 in HT-29 cells treated with IPTG to induce expression CAP-D3 shRNAs in comparison to cells treated with PBS (mock) and cells expressing control (non-target) shRNA. CAP-D3 protein levels were normalized to tubulin and quantified in the bottom panel. Gentamycin protection assays of Salmonella infected HT-29 (B) or Caco-2 (C) cell lines at 1.5 hours post-infection, and of AIEC LF82 in HT-29 cells (D) demonstrate significant increases in bacteria in cells expressing CAP-D3 shRNAs. Mean±SD. E) Gentamycin protection assays performed in Salmonella-infected HT-29 cells expressing CAP-D3 shRNA or control shRNA show enhanced bacterial survival in CAP-D3 deficient cells beginning after 40 minutes. Bacterial survival graphed relative to CFU/well of control shRNA expressing cells at the earliest timepoint. Mean±SD. F) Immunoblots demonstrate lower ratios of LC3-II/LC3-I protein in Salmonella infected HT-29 cells expressing CAP-D3 shRNA. Protein levels normalized to actin, then quantified relative to uninfected controls. The experiment shown is representative of three independent immunoblots.
Figure 2
Figure 2. CAP-D3 protein levels are decreased in colonic epithelial cells from patients with active UC
A) Immunofluorescence analysis of CAP-D3 (green) and DAPI (blue) staining of colonic tissue from a control patient. B) Immunoblotting for CAP-D3 protein in epithelial cells isolated from resected colon tissue from control patients, patients with inactive CD, active CD, inactive UC, or active UC shows CAPD3 is significantly lower in patients with active UC. Protein levels normalized to villin, then quantified relative to a control sample. Mean±SEM.
Figure 3
Figure 3. Salmonella infection increases SLC7A5-SLC3A2 gene expression in CAP-D3 deficient cells
A) qRT-PCR for transcript levels of SLC7A5 and SLC3A2, in Salmonella-infected HT-29 cells normalized to GAPDH transcript levels demonstrate increases in CAP-D3 shRNA expressing cells. B) qRT-PCR for transcript levels of SLC3A2 and SLC7A5 in colonic epithelial cells isolated from resected patient tissues demonstrate increases in patients with active UC compared to controls and patients with inactive UC. Transcript levels were normalized to villin transcript levels and then to the transcript levels found in a single control patient sample.
Figure 4
Figure 4. SLC7A5 localizes to Salmonella-containing vacuoles early in infection and its transporter activity is essential for enhanced bacterial survival in CAP-D3 deficient cells
A-B) Immunofluorescence analysis of Salmonella (red) and SLC7A5 (green) and bacteria nuclei (DAPI-blue) in HT-29 cells demonstrates SLC7A5 localization to bacteria earlier (2 hours post-infection) in CAP-D3 deficient cells. C) Quantification of the percentage of SLC7A5+, LAMP1+, SLC7A5+/LAMP1+ Salmonella in HT-29 cell lines at various times during infection. D) The SLC7A5-SLC3A2 inhibitor, BCH, rescues the increased numbers of Salmonella in CAP-D3-deficient cells at 1.5h post-infection in gentamycin protection assays.
Figure 5
Figure 5. SLC7A5-dependent mTOR localization to Salmonella-containing vacuoles and mTORC1 activity are required for enhanced Salmonella survival in CAP-D3 deficient cells
A) Immunofluorescence analysis of LAMP1 (green) and mTOR (red) and bacterial nuclei (DAPI-blue) in HT-29 cells at 2 hours post-infection demonstrates increased colocalization in CAP-D3 deficient cells. B) Quantification of the percentage of mTOR+, LAMP1+, mTOR+/LAMP1+ Salmonella in HT-29 cell lines various times post-infection. C) Quantification as in (B) but in cells treated with BCH. D) The mTORC1 inhibitor, rapamycin, rescues the increased numbers of Salmonella in HT-29 cell lines at 1.5h post-infection in gentamycin protection assays.
Figure 6
Figure 6. Possible model of how CAP-D3 deficiency leads to enhanced Salmonella survival
A) In normal, colonic epithelial cells, Salmonella is endocytosed into an early endosome which becomes the early SCV (Salmonella-Containing Vacuole). The early endosome/SCV will acquire LAMP-1 (and other proteins) and become a Late Endosome/Lysosome/Intermediate SCV. The majority of Intermediate SCVs at this point will be targeted for autophagy since mTOR is not localized to the SCV and mTORC1 activity is low. A small number of Intermediate SCVs, however, will have begun to sequester existing SLC7a5 protein to their membranes and this could allow them to recruit mTOR, increase mTORC1 activity and block autophagy; this Late SCV would then escape destruction, and bacteria could replicate. In CAP-D3 deficient colonic epithelial cells, the burst of SLC7A5-SLC3A2 transcription occurring immediately after infection leads to increased levels of protein, allowing more Intermediate SCVs to be decorated with SLC7A5 earlier, thereby recruiting and activating mTORC1 more quickly and blocking autophagy sooner. This quicker version of events, leads to a larger number of SCVs escaping autophagy and enhances bacterial survival. This working model could also apply to other bacteria that invade epithelial cells and are typically cleared by autophagy, including AIEC LF82.
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