Chromosome-Associated Protein D3 Promotes Bacterial Clearance in Human Intestinal Epithelial Cells by Repressing Expression of Amino Acid Transporters

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 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, whose products heterodimerize to form an amino acid transporter in HT-29 cells following 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 downregulates 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.

Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells

“…We used an established cell culture assay to determine whether endogenous Condensin II subunits, CAP-D3 and CAP-H2, affect retrotransposition of the L1 retrotransposon. HT-29 colon epithelial adenocarcinoma cells expressing inducible Non-Target, CAP-D3, or CAP-H2 shRNAs [ 89 ] were transfected with a full-length, retrotransposition-competent L1 plasmid [ 22 , 90 , 91 ]. The L1 construct contains an indicator cassette in the 3′ end, which consists of an antisense copy of a neomycin phosphotransferase gene ( mneoI ) equipped with a heterologous promoter (Pr′) and polyadenylation signal.…”

“…In this context, GAIT has been shown to repress translation of select transcripts in response to IFN-γ [ 99 ]. Although HT-29 cells do not express IFN-γ ( S7 Fig and [ 89 ]), we tested whether a GAIT-like complex might act independently of IFN-γ to repress L1 RNA translation in coordination with CAP-D3. This idea was supported by the fact that the LC-MS/MS analysis identified L13a and GAPDH as putative CAP-D3 binding partners ( Fig 3 ).…”

Human Intestinal Enteroids as a Model System of Shigella Pathogenesis

“…7), possibly in response to Shigella-induced amino acid depletion. SLC7A5 has been implicated in the pathogenesis of other intracellular pathogens, such as Salmonella and Legionella (75)(76)(77)(78). Future studies will interrogate the role of amino acid transport in Shigella pathogenesis and how S. flexneri replication impacts host metabolism in human enteroids.…”

Role of Autophagy in the Maintenance of Intestinal Homeostasis

“…Defects in the restriction of bacterial replication have been linked to IBD, as proteins involved in autophagic bacterial clearance have decreased expression in epithelial cells from patients with active IBD. 30 Studies to date have used Salmonella as a model of infectious bacteria that are susceptible to autophagy. Mice with epithelial cell-specific deletion of Atg16l1 ( Atg16l1 ΔIEC ) or Atg5 (Atg5 ΔIEC ) showed significant defects in their ability to target Salmonella for autophagy, resulting in greater sensitivity to Salmonella infection and increased bacterial dissemination to other tissues in these mice, highlighting the importance of bacterial restriction in the epithelium.…”