Comment[ArrayExpressAccession] E-GEOD-43919 MAGE-TAB Version 1.1 Public Release Date 2013-06-15 Investigation Title Predicting and manipulating drug inactivation by the human gut microbiome Comment[Submitted Name] Predicting and manipulating drug inactivation by the human gut microbiome Experiment Description The trillions of microorganisms in the human gastrointestinal tract are an underexplored aspect of pharmacology. Despite numerous examples of microbial effects on drug efficacy and toxicity, there is often an incomplete understanding of the underlying mechanisms. Here, we dissect the inactivation of the commonly prescribed cardiac glycoside, digoxin, by Eggerthella lenta. Whole genome transcriptional profiling, comparative genomics, and culture-based assays revealed a cytochrome-encoding operon up-regulated by digoxin, absent in non-metabolizing E. lenta strains, and predictive of the efficiency of digoxin inactivation by the human gut microbiome. Digoxin inactivation was further enhanced by microbial interactions and inhibited by arginine. Pharmacokinetic studies using gnotobiotic mice revealed that increasing dietary protein reduces the in vivo metabolism of digoxin by E. lenta, with significant changes to drug concentration in the urine and serum. These results emphasize the importance of viewing pharmacology from the perspective of both our human and microbial genomes. RNA-Seq analysis of Eggerthella lenta cultured with or without digoxin. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Turnbaugh Haiser Turnbaugh Person First Name Peter Henry Peter Person Mid Initials James J J Person Email pturnbaugh@fas.harvard.edu Person Affiliation Harvard University Person Address Center for Systems Biology, Harvard University, 52 Oxford St Room 435.40, Cambridge, MA, USA Person Roles submitter Protocol Name P-GSE43919-4 P-GSE43919-1 P-GSE43919-3 P-GSE43919-2 Protocol Description Pre-processing of raw reads: binned according to barcode, trimmed ends to min score = 20 and minimum length = 20, required both pairs, concatenated each trimmed pair Mapped reads to E.lenta DSM2243 genome (GCA_000024265.1) using SSAHA (version 2; parameters: -best 1 -score 20 -solexa) The number of transcripts assigned to each gene was then tallied. Genome_build: ASM2426v1 Supplementary_files_format_and_content: Counts files were generated. Values are total reads assigned and fraction reads assigned (#reads/total reads). Digoxin was added at 10ug/ml final concentration Microbial cells were lysed by a bead beater (BioSpec Products), total RNA was extracted with phenol:chloroform:isoamyl alcohol (pH 4.5, 125:24:1, Ambion 9720), purified using MEGAClear columns (Ambion), and rRNA was depleted via subtractive hybridization (MICROBExpress, Ambion, in addition to custom depletion oligos). The presence of genomic DNA contamination was assessed by PCR with universal 16S rDNA primers. cDNA was synthesized using SuperScript II and random hexamers (Invitrogen), followed by second strand synthesis with RNaseH and E.coli DNA polymerase (New England Biolabs). Samples were prepared for sequencing with an Illumina GAII instrument after enzymatic fragmentation (NEBE6040L/M0348S). The size distribution of each library was quantified on an Agilent HS-DNA chip. Exponential and stationary phase growth in rich medium containing 0.25% or 1.25% arginine Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name GROWTH PHASE DRUG ARGININE Experimental Factor Type growth phase drug arginine Comment[SecondaryAccession] GSE43919 Comment[GEOReleaseDate] 2013-06-15 Comment[ArrayExpressSubmissionDate] 2013-01-30 Comment[GEOLastUpdateDate] 2013-06-15 Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP018311 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR653937-SRR653958 SDRF File E-GEOD-43919.sdrf.txt