Moyne Institute of Preventive Medicine

Dublin, Ireland

Moyne Institute of Preventive Medicine

Dublin, Ireland
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Kroger C.,Moyne Institute of Preventive Medicine | Dillon S.C.,Moyne Institute of Preventive Medicine | Cameron A.D.S.,Moyne Institute of Preventive Medicine | Papenfort K.,University of Würzburg | And 18 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2012

More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ70 (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ70-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.

Dillon S.C.,Moyne Institute of Preventive Medicine | Dorman C.J.,Moyne Institute of Preventive Medicine
Nature Reviews Microbiology | Year: 2010

Emerging models of the bacterial nucleoid show that nucleoid-associated proteins (NAPs) and transcription contribute in combination to the dynamic nature of nucleoid structure. NAPs and other DNA-binding proteins that display gene-silencing and anti-silencing activities are emerging as key antagonistic regulators of nucleoid structure. Furthermore, it is becoming clear that the boundary between NAPs and conventional transcriptional regulators is quite blurred and that NAPs facilitate the evolution of novel gene regulatory circuits. Here, NAP biology is considered from the standpoints of both gene regulation and nucleoid structure. © 2010 Macmillan Publishers Limited. All rights reserved.

Dillon S.C.,Moyne Institute of Preventive Medicine | Espinosa E.,University of Seville | Hokamp K.,Trinity College Dublin | Ussery D.W.,Technical University of Denmark | And 2 more authors.
Molecular Microbiology | Year: 2012

We report the first investigation of the binding of the Salmonella enterica LeuO LysR-type transcription regulator to its genomic targets in vivo. Chromatin-immunoprecipitation-on-chip identified 178 LeuO binding sites on the chromosome of S.enterica serovar Typhimurium strain SL1344. These sites were distributed across both the core and the horizontally acquired genome, and included housekeeping genes and genes known to contribute to virulence. Sixty-eight LeuO targets were co-bound by the global repressor protein, H-NS. Thus, while LeuO may function as an H-NS antagonist, these functions are unlikely to involve displacement of H-NS. RNA polymerase bound 173 of the 178 LeuO targets, consistent with LeuO being a transcription regulator. Thus, LeuO targets two classes of genes, those that are bound by H-NS and those that are not bound by H-NS. LeuO binding site analysis revealed a logo conforming to the TN11A motif common to LysR-type transcription factors. It differed in some details from a motif that we composed for Escherichia coli LeuO binding sites; 1263 and 1094 LeuO binding site locations were predicted in the S.Typhimurium SL1344 and E.coli MG1655 genomes respectively. Despite differences in motif composition, many LeuO target genes were common to both species. Thus, LeuO is likely to be a more important global regulator than previously suspected. © 2012 Blackwell Publishing Ltd.

Mara C.,Royal College of Surgeons in Ireland | Dempsey E.,Moyne Institute of Preventive Medicine | Bell A.,Moyne Institute of Preventive Medicine | Barlow J.W.,Royal College of Surgeons in Ireland
Bioorganic and Medicinal Chemistry Letters | Year: 2011

A series of phosphoramidate and phosphorothioamidate compounds based on the lead antitubulin herbicidal agents amiprophos methyl (APM) and butamifos were synthesised and evaluated for antimalarial activity. Of these compounds, phosphorothioamidates were more active than their oxo congeners and the nature of both aryl and amido substituents influenced the desired activity. The most active compound was 46, O-ethyl-O-(2-methyl-4-nitrophenyl)-N-cyclopentyl phosphorothioamidate, which was more effective than the lead compound. © 2011 Elsevier Ltd. All rights reserved.

Dillon S.C.,Moyne Institute of Preventive Medicine | Cameron A.D.S.,Moyne Institute of Preventive Medicine | Hokamp K.,Smurfit Institute | Lucchini S.,UK Institute of Food Research | And 2 more authors.
Molecular Microbiology | Year: 2010

The conjugative IncHI1 plasmid pSfR27 from Shigella flexneri 2a strain 2457T encodes the Sfh protein, a paralogue of the global transcriptional repressor H-NS. Sfh allows pSfR27 to be transmitted to new bacterial hosts with minimal impact on host fitness, providing a 'stealth' function whose molecular mechanism has yet to be determined. The impact of the Sfh protein on the Salmonella enterica serovar Typhimurium transcriptome was assessed and binding sites for Sfh in the Salmonella Typhimurium genome were identified by chromatin immunoprecipitation. Sfh did not bind uniquely to any sites. Instead, it bound to a subset of the larger H-NS regulatory network. Analysis of Sfh binding in the absence of H-NS revealed a greatly expanded population of Sfh binding sites that included the majority of H-NS target genes. Furthermore, the presence of plasmid pSfR27 caused a decrease in H-NS interactions with the S. Typhimurium chromosome, suggesting that the A + T-rich DNA of this large plasmid acts to titrate H-NS, removing it from chromosomal locations. It is proposed that Sfh acts as a molecular backup for H-NS and that it provides its 'stealth' function by replacing H-NS on the chromosome, thus minimizing disturbances to the H-NS-DNA binding pattern in cells that acquire pSfR27. © 2010 Blackwell Publishing Ltd.

Marin-Menendez A.,Moyne Institute of Preventive Medicine | Bell A.,Moyne Institute of Preventive Medicine
Protein Expression and Purification | Year: 2011

Malaria represents a global health, economic and social burden of enormous magnitude. Chemotherapy is at the moment a largely effective weapon against the disease, but the appearance of drug-resistant parasites is reducing the effectiveness of most drugs. Finding new drug-target candidates is one approach to the development of new drugs. The family of cyclophilins may represent a group of potential targets. They are involved in protein folding and regulation due to their peptidyl-prolyl cis-trans isomerase and/or chaperone activities. They also mediate the action of the immunosuppressive drug cyclosporin A, which additionally has strong antimalarial activity. In the genome database of the most lethal human malarial parasite Plasmodium falciparum, 11 genes apparently encoding cyclophilin or cyclophilin-like proteins were found, but most of these have not yet been characterized. Previously a pET vector conferring a C-terminal His6 tag was used for recombinant expression and purification of one member of the P. falciparum cyclophilin family in Escherichia coli. The approach here was to use an identical method to produce all of the other members of this family and thereby allow the most consistent functional comparisons. We were successful in generating all but three of the family, plus a single amino-acid mutant, in the same recombinant form as either full-length proteins or isolated cyclophilin-like domains. The recombinant proteins were assessed by thermal melt assay for correct folding and cyclosporin A binding. © 2011 Elsevier Inc. All rights reserved.

Corcoran C.P.,Moyne Institute of Preventive Medicine | Cameron A.D.S.,Moyne Institute of Preventive Medicine | Dorman C.J.,Moyne Institute of Preventive Medicine
Journal of Bacteriology | Year: 2010

The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Kroger C.,Moyne Institute of Preventive Medicine | Colgan A.,Moyne Institute of Preventive Medicine | Srikumar S.,Moyne Institute of Preventive Medicine | Handler K.,Moyne Institute of Preventive Medicine | And 8 more authors.
Cell Host and Microbe | Year: 2013

Bacterial transcriptional networks consist of hundreds of transcription factors and thousands of promoters. However, the true complexity of transcription in a bacterial pathogen and the effect of the environments encountered during infection remain to be established. We present a simplified approach for global promoter identification in bacteria using RNA-seq-based transcriptomic analyses of 22 distinct infection-relevant environmental conditions. Individual RNA samples were combined to identify most of the 3,838 Salmonella enterica serovar Typhimurium promoters in just two RNA-seq runs. Individual in vitro conditions stimulated characteristic transcriptional signatures, and the suite of 22 conditions induced transcription of 86% of all S. Typhimurium genes. We highlight the environmental conditions that induce the Salmonella pathogenicity islands and present a small RNA expression landscape of 280 sRNAs. This publicly available compendium of environmentally controlled expression of every transcriptional feature of S. Typhimurium constitutes a useful resource for the bacterial research community. © 2013 Elsevier Inc.

Boehm D.,Moyne Institute of Preventive Medicine | Bell A.,Moyne Institute of Preventive Medicine
Biotechnology Reports | Year: 2014

Most mammalian cell proliferation assays rely on manual or automated cell counting or the assessment of metabolic activity in colorimetric assays, with the former being either labor and time intensive or expensive and the latter being multistep procedures requiring the addition of several reagents. The proliferation of erythroid cells from hematopoietic stem cells and their differentiation into mature red blood cells is characterized by the accumulation of large amounts of hemoglobin. Hemoglobin concentrations are easily quantifiable using spectrophotometric methods due to the specific absorbance peak of the molecule's heme moiety between 400 and 420 nm. Erythroid proliferation can therefore be readily assessed using spectrophotometric measurement in this range. We have used this feature of erythroid cells to develop a simple erythroid proliferation assay that is minimally labor/time- and reagent-intensive and could easily be automated for use in high-throughput screening. Such an assay can be a valuable tool for investigations into hematological disorders where erythropoiesis is dysregulated, i.e., either inhibited or enhanced, into the development of anemia as a side-effect of primary diseases such as parasitic infections and into cyto-(particularly erythro-) toxicity of chemical agents or drugs. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND.

Leneghan D.,Moyne Institute of Preventive Medicine | Leneghan D.,University of Oxford | Bell A.,Moyne Institute of Preventive Medicine
Parasitology | Year: 2015

Immunophilins comprise two protein families, cyclophilins (CYPs) and FK506-binding proteins (FKBPs), and are the major receptors for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (tacrolimus), respectively. Most eukaryotic species have at least one immunophilin and some of them have been associated with pathogenesis of infectious or parasitic diseases or the action of antiparasitic drugs. The human malarial parasite Plasmodium falciparum has 13 immunophilin or immunophilin-like genes but the functions of their products are unknown. We set out to identify the parasite proteins that interact with the major CYPs, PfCYP19A and PfCYP19B, and the FKBP, PfFKBP35, using a combination of co-immunoprecipitation and yeast two-hybrid screening. We identified a cohort of putative interacting partners and further investigation of some of these revealed potentially novel roles in parasite biology. We demonstrated that (i) P. falciparum CYPs interacted with the heat shock protein 70, (ii) treatment of parasites with CYP ligands disrupted transport of the rhoptry-associated protein 1, and (iii) PfFKBP35 interacted with parasite histones in a way that might modulate gene expression. These findings begin to elucidate the functions of immunophilins in malaria. Furthermore, the known antimalarial effects of CsA, FK506 and non-immunosuppressive derivatives of these immunophilin ligands could be mediated through these partner proteins. © 2015 Cambridge University Press.

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