Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A Salmonella-encoded microRNA-like RNA facilitates bacterial invasion and intracellular replication via suppressing host cell inducible nitric oxide synthase

ABSTRACT: As the leading cause of food-borne illness in the world, Salmonella have evolved a sophisticated machinery to alter host cell function to promote virulence and survival.In this study, we compare production of non-coding RNAs between Salmonella-infected cells and mock infection cells. Using Solexa deep sequencing, we detected a panel of 19-24nt Salmonella-derived non-coding RNA fragments with considerable large copy numbers in human colonic epithelial HT-29 cells following Salmonella infection.The fragment with the highest copy number, Sal-1, was further validated by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blot. The generation of Sal-1 requires the infection of host cells by Salmonella, and the processing of the Sal-1 M-bM-^@M-^\primaryM-bM-^@M-^] or M-bM-^@M-^\precursorM-bM-^@M-^] to the mature Sal-1 in Salmonella-infected cells is Dicer-independent but Argonaute 2 (Ago2)-dependent. Functionally, Sal-1 suppresses the expression of colonic epithelial cell endogenous inducible nitric oxide synthase (iNOS) via targeting its open reading frame and thus reduces the bacterial resistance of host cells. Screening and identification of Salmonella-encoded microRNA-like RNA fragments

ORGANISM(S): Homo sapiens

SUBMITTER: Zhen Zhou

PROVIDER: E-GEOD-53586 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-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 CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients. Three RNA samples from 3 independent experiments including timepoints taken at 0, 0.5 and 7 hours post-infection were analyzed on a bioanalyzer for quality; one of the 0.5 hour post-infection samples was excluded at this time due to poor RNA purity. Directional, cDNA libraries made from cellular mRNAs were generated from the other 8 samples and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2000.

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A Salmonella-encoded microRNA-like RNA facilitates bacterial invasion and intracellular replication via suppressing host cell inducible nitric oxide synthase

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