title: Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Schuster
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Craig R
authors.initials: CR
authors.lastname: Homer
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Jacqueline R
authors.initials: JR
authors.lastname: Kemp
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Kourtney P
authors.initials: KP
authors.lastname: Nickerson
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Emily
authors.initials: E
authors.lastname: Deutschman
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Yeojung
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Gail
authors.initials: G
authors.lastname: West
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Tammy
authors.initials: T
authors.lastname: Sadler
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Eleni
authors.initials: E
authors.lastname: Stylianou
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio.
authors.forename: Dawid
authors.initials: D
authors.lastname: Krokowski
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio.
authors.forename: Maria
authors.initials: M
authors.lastname: Hatzoglou
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Carol
authors.initials: C
authors.lastname: de la Motte
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.|Department of Anatomic Pathology, Taussig Cancer Institute, Cleveland, Ohio.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Rubin
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Claudio
authors.initials: C
authors.lastname: Fiocchi
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Christine
authors.initials: C
authors.lastname: McDonald
authors.affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Longworth
mesh_terms: D000251:Adenosine Triphosphatases; D001343:Autophagy; D018938:Caco-2 Cells; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003093:Colitis, Ulcerative / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D003424:Crohn Disease / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D029721:Drosophila Proteins; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology*; D004926:Escherichia coli / Q000276:immunology / Q000502:physiology*; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D019073:HT29 Cells; D006801:Humans; D007113:Immunity, Innate; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology*; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D050296:Microbial Viability; D046912:Multiprotein Complexes / Q000378:metabolism; D034622:RNA Interference; D012475:Salmonella / Q000276:immunology / Q000502:physiology*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027283:Large Neutral Amino Acid-Transporter 1; D046912:Multiprotein Complexes; C000599979:NCAPD3 protein, human; C545989:SLC3A2 protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; C529732:CAP-D3 protein, Drosophila
keywords: Condensin; Dysbiosis; Inflammatory Bowel Disease; Innate Immunity