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Toward Cell Type-Specific In Vivo Dual RNA-Seq.
Frönicke L, Bronner DN, Byndloss MX, McLaughlin B, Bäumler AJ, Westermann AJ
(2018) Methods Enzymol 612: 505-522
MeSH Terms: Gene Library, RNA, Bacterial, Salmonella typhimurium, Sequence Analysis, RNA
Show Abstract · Added March 30, 2020
Dual RNA-seq has emerged as a genome-wide expression profiling technique, simultaneously measuring RNA transcript levels in a given host and its pathogen during an infection. Recently, the method was transferred from cell culture to in vivo models of bacterial infections; however, specific host cell-type resolution has not yet been achieved. Here we present a detailed protocol that describes the application of Dual RNA-seq to murine colonocytes isolated from mice infected with the enteric pathogen Salmonella Typhimurium. At day 5 after oral infection, the mice were humanely euthanized, their colons extracted, and colonocytes isolated and fixed. Upon antibody staining of cell type-specific surface markers, the fraction of Salmonella-invaded colonocytes was collected by fluorescence-activated cell sorting based on a fluorescent signal emitted by the internalized bacteria. Total RNA was extracted from cells enriched by this method, and ribosomal transcripts from host and microbial cells were removed prior to cDNA synthesis and library generation. We compared different protocols for library preparation and discuss their respective advantages and caveats when applied to minute RNA amounts that constitute an inherent challenge for in vivo transcriptomics. Our results introduce an ultralow input protocol that holds promise for cell type-specific in vivo Dual RNA-seq for charting gene expression of a bacterial pathogen within its respective in vivo niche, along with the consequent host response.
© 2018 Elsevier Inc. All rights reserved.
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In vitro safety pharmacology evaluation of 2-hydroxybenzylamine acetate.
Fuller JC, Pitchford LM, Morrison RD, Daniels JS, Flynn CR, Abumrad NN, Oates JA, Boutaud O, Rathmacher JA
(2018) Food Chem Toxicol 121: 541-548
MeSH Terms: Adult, Benzylamines, Blood Proteins, Cytochrome P-450 Enzyme System, ERG1 Potassium Channel, Erythrocytes, Female, Gene Expression Regulation, Enzymologic, Hepatocytes, Humans, Male, Middle Aged, Mutagenicity Tests, Salmonella typhimurium
Show Abstract · Added April 15, 2019
2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.
Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Genetic Ablation of Butyrate Utilization Attenuates Gastrointestinal Salmonella Disease.
Bronner DN, Faber F, Olsan EE, Byndloss MX, Sayed NA, Xu G, Yoo W, Kim D, Ryu S, Lebrilla CB, Bäumler AJ
(2018) Cell Host Microbe 23: 266-273.e4
MeSH Terms: Animals, Butyrates, Cell Line, Tumor, Clostridium, Colitis, Escherichia coli, Female, Humans, Intestines, Mice, Mice, Inbred CBA, Salmonella Food Poisoning, Salmonella typhi, Salmonella typhimurium, Type III Secretion Systems
Show Abstract · Added March 30, 2020
Salmonella enterica serovar (S.) Typhi is an extraintestinal pathogen that evolved from Salmonella serovars causing gastrointestinal disease. Compared with non-typhoidal Salmonella serovars, the genomes of typhoidal serovars contain various loss-of-function mutations. However, the contribution of these genetic differences to this shift in pathogen ecology remains unknown. We show that the ydiQRSTD operon, which is deleted in S. Typhi, enables S. Typhimurium to utilize microbiota-derived butyrate during gastrointestinal disease. Unexpectedly, genetic ablation of butyrate utilization reduces S. Typhimurium epithelial invasion and attenuates intestinal inflammation. Deletion of ydiD renders S. Typhimurium sensitive to butyrate-mediated repression of invasion gene expression. Combined with the gain of virulence-associated (Vi) capsular polysaccharide and loss of very-long O-antigen chains, two features characteristic of S. Typhi, genetic ablation of butyrate utilization abrogates S. Typhimurium-induced intestinal inflammation. Thus, the transition from a gastrointestinal to an extraintestinal pathogen involved discrete genetic changes, providing insights into pathogen evolution and emergence.
Copyright © 2018 Elsevier Inc. All rights reserved.
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Respiration of Microbiota-Derived 1,2-propanediol Drives Salmonella Expansion during Colitis.
Faber F, Thiennimitr P, Spiga L, Byndloss MX, Litvak Y, Lawhon S, Andrews-Polymenis HL, Winter SE, Bäumler AJ
(2017) PLoS Pathog 13: e1006129
MeSH Terms: Animals, Cell Respiration, Colitis, Disease Models, Animal, Host-Pathogen Interactions, Mice, Mice, Inbred C57BL, Polymerase Chain Reaction, Propylene Glycol, Salmonella Infections, Animal, Salmonella typhimurium, Virulence Factors
Show Abstract · Added March 30, 2020
Intestinal inflammation caused by Salmonella enterica serovar Typhimurium increases the availability of electron acceptors that fuel a respiratory growth of the pathogen in the intestinal lumen. Here we show that one of the carbon sources driving this respiratory expansion in the mouse model is 1,2-propanediol, a microbial fermentation product. 1,2-propanediol utilization required intestinal inflammation induced by virulence factors of the pathogen. S. Typhimurium used both aerobic and anaerobic respiration to consume 1,2-propanediol and expand in the murine large intestine. 1,2-propanediol-utilization did not confer a benefit in germ-free mice, but the pdu genes conferred a fitness advantage upon S. Typhimurium in mice mono-associated with Bacteroides fragilis or Bacteroides thetaiotaomicron. Collectively, our data suggest that intestinal inflammation enables S. Typhimurium to sidestep nutritional competition by respiring a microbiota-derived fermentation product.
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Iron acquisition pathways and colonization of the inflamed intestine by Salmonella enterica serovar Typhimurium.
Costa LF, Mol JP, Silva AP, Macêdo AA, Silva TM, Alves GE, Winter S, Winter MG, Velazquez EM, Byndloss MX, Bäumler AJ, Tsolis RM, Paixão TA, Santos RL
(2016) Int J Med Microbiol 306: 604-610
MeSH Terms: Animals, Bacterial Proteins, Cattle, Disease Models, Animal, Female, Gastroenteritis, Intestines, Iron, Male, Metabolic Networks and Pathways, Mice, Inbred C57BL, Salmonella Infections, Salmonella typhimurium, Virulence Factors
Show Abstract · Added March 30, 2020
Salmonella enterica serotype Typhimurium is able to expand in the lumen of the inflamed intestine through mechanisms that have not been fully resolved. Here we utilized streptomycin-pretreated mice and dextran sodium sulfate (DSS)-treated mice to investigate how pathways for S. Typhimurium iron acquisition contribute to pathogen expansion in the inflamed intestine. Competitive infection with an iron uptake-proficient S. Typhimurium strain and mutant strains lacking tonB feoB, feoB, tonB or iroN in streptomycin pretreated mice demonstrated that ferric iron uptake requiring IroN and TonB conferred a fitness advantage during growth in the inflamed intestine. However, the fitness advantage conferred by ferrous iron uptake mechanisms was independent of inflammation and was only apparent in models where the normal microbiota composition had been disrupted by antibiotic treatment.
Copyright © 2016 Elsevier GmbH. All rights reserved.
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Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.
Faber F, Tran L, Byndloss MX, Lopez CA, Velazquez EM, Kerrinnes T, Nuccio SP, Wangdi T, Fiehn O, Tsolis RM, Bäumler AJ
(2016) Nature 534: 697-9
MeSH Terms: Animals, Anti-Bacterial Agents, Carbohydrate Metabolism, Cecum, Female, Galactose, Gastroenteritis, Glucaric Acid, Glucose, Host-Pathogen Interactions, Intestinal Mucosa, Male, Mice, Nitric Oxide Synthase Type II, Operon, Oxidation-Reduction, Reactive Nitrogen Species, Salmonella typhimurium, Streptomycin, Sugar Acids
Show Abstract · Added March 30, 2020
Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.
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Salmonella Mitigates Oxidative Stress and Thrives in the Inflamed Gut by Evading Calprotectin-Mediated Manganese Sequestration.
Diaz-Ochoa VE, Lam D, Lee CS, Klaus S, Behnsen J, Liu JZ, Chim N, Nuccio SP, Rathi SG, Mastroianni JR, Edwards RA, Jacobo CM, Cerasi M, Battistoni A, Ouellette AJ, Goulding CW, Chazin WJ, Skaar EP, Raffatellu M
(2016) Cell Host Microbe 19: 814-25
MeSH Terms: Animals, Anti-Bacterial Agents, Antioxidants, Bacterial Proteins, Chelating Agents, Escherichia coli, Gastroenteritis, Intestinal Mucosa, Intestines, Leukocyte L1 Antigen Complex, Manganese, Mice, Mice, Inbred C57BL, Neutrophils, Oxidative Stress, Reactive Oxygen Species, Salmonella Infections, Salmonella typhimurium, Symbiosis, Zinc
Show Abstract · Added April 8, 2017
Neutrophils hinder bacterial growth by a variety of antimicrobial mechanisms, including the production of reactive oxygen species and the secretion of proteins that sequester nutrients essential to microbes. A major player in this process is calprotectin, a host protein that exerts antimicrobial activity by chelating zinc and manganese. Here we show that the intestinal pathogen Salmonella enterica serovar Typhimurium uses specialized metal transporters to evade calprotectin sequestration of manganese, allowing the bacteria to outcompete commensals and thrive in the inflamed gut. The pathogen's ability to acquire manganese in turn promotes function of SodA and KatN, enzymes that use the metal as a cofactor to detoxify reactive oxygen species. This manganese-dependent SodA activity allows the bacteria to evade neutrophil killing mediated by calprotectin and reactive oxygen species. Thus, manganese acquisition enables S. Typhimurium to overcome host antimicrobial defenses and support its competitive growth in the intestine.
Copyright © 2016 Elsevier Inc. All rights reserved.
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20 MeSH Terms
Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ⁵⁴ (RpoN) regulon of Salmonella Typhimurium LT2.
Samuels DJ, Frye JG, Porwollik S, McClelland M, Mrázek J, Hoover TR, Karls AC
(2013) BMC Genomics 14: 602
MeSH Terms: Binding Sites, Chromatin Immunoprecipitation, DNA-Binding Proteins, Gene Expression Regulation, Bacterial, Oligonucleotide Array Sequence Analysis, Open Reading Frames, Operon, Promoter Regions, Genetic, RNA Polymerase Sigma 54, Regulon, Salmonella typhimurium, Transcriptional Activation
Show Abstract · Added December 12, 2013
BACKGROUND - Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ⁵⁴ regulon in a bacterium is that the σ⁵⁴ RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ⁵⁴-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP-the AAA+ ATPase domain of DctD from Sinorhizobium meliloti-to activate transcription from all σ⁵⁴-dependent promoters for the characterization of the σ⁵⁴ regulon of Salmonella Typhimurium LT2.
RESULTS - The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ⁵⁴-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ⁵⁴-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ⁵⁴ binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ⁵⁴ binding sites suggest that many of these sites are capable of functioning as σ⁵⁴-dependent promoters.
CONCLUSION - Since the DctD AAA + ATPase domain proved effective in activating transcription from the diverse σ⁵⁴-dependent promoters of the S. Typhimurium LT2 σ⁵⁴ regulon under a single growth condition, this approach is likely to be valuable for examining σ⁵⁴ regulons in other bacterial species. The S. Typhimurium σ⁵⁴ regulon included a high number of intragenic σ⁵⁴ binding sites/promoters, suggesting that σ⁵⁴ may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.
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12 MeSH Terms
An HPLC-tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products.
Mullins EA, Rubinson EH, Pereira KN, Calcutt MW, Christov PP, Eichman BF
(2013) Methods 64: 59-66
MeSH Terms: Alkylation, Bacillus cereus, Bacterial Proteins, Chromatography, High Pressure Liquid, DNA, DNA Adducts, DNA Damage, DNA Repair, Humans, Saccharomyces cerevisiae Proteins, Salmonella typhi, Tandem Mass Spectrometry
Show Abstract · Added March 7, 2014
DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3 mA) and N7-methylguanine (7 mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described.
Copyright © 2013 Elsevier Inc. All rights reserved.
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12 MeSH Terms
Metabolic activation of polycyclic aromatic hydrocarbons and aryl and heterocyclic amines by human cytochromes P450 2A13 and 2A6.
Shimada T, Murayama N, Yamazaki H, Tanaka K, Takenaka S, Komori M, Kim D, Guengerich FP
(2013) Chem Res Toxicol 26: 529-37
MeSH Terms: Amines, Aryl Hydrocarbon Hydroxylases, Biotransformation, Carcinogens, Cytochrome P-450 CYP1B1, Cytochrome P-450 CYP2A6, Environmental Pollutants, Escherichia coli, Genes, Bacterial, Heterocyclic Compounds, Humans, Molecular Docking Simulation, Polycyclic Aromatic Hydrocarbons, Salmonella typhimurium
Show Abstract · Added March 7, 2014
Human cytochrome P450 (P450) 2A13 was found to interact with several polycyclic aromatic hydrocarbons (PAHs) to produce Type I binding spectra, including acenaphthene, acenaphthylene, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, and 1-nitropyrene. P450 2A6 also interacted with acenaphthene and acenaphthylene, but not with fluoranthene, fluoranthene-2,3-diol, or 1-nitropyrene. P450 1B1 is well-known to oxidize many carcinogenic PAHs, and we found that several PAHs (i.e., 7,12-dimethylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene-5,6-diol, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, 5-methylchrysene, benz[a]pyrene-4,5-diol, benzo[a]pyrene-7,8-diol, 1-nitropyrene, 2-aminoanthracene, 2-aminofluorene, and 2-acetylaminofluorene) interacted with P450 1B1, producing Reverse Type I binding spectra. Metabolic activation of PAHs and aryl- and heterocyclic amines to genotoxic products was examined in Salmonella typhimurium NM2009, and we found that P450 2A13 and 2A6 (as well as P450 1B1) were able to activate several of these procarcinogens. The former two enzymes were particularly active in catalyzing 2-aminofluorene and 2-aminoanthracene activation, and molecular docking simulations supported the results with these procarcinogens, in terms of binding in the active sites of P450 2A13 and 2A6. These results suggest that P450 2A enzymes, as well as P450 Family 1 enzymes including P450 1B1, are major enzymes involved in activating PAHs and aryl- and heterocyclic amines, as well as tobacco-related nitrosamines.
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14 MeSH Terms