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Layton A.N.,Institute for Animal Health | Hudson D.L.,Institute for Animal Health | Thompson A.,UK Institute of Food Research | Hinton J.C.D.,UK Institute of Food Research | And 5 more authors.
FEMS Microbiology Letters | Year: 2010

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. Intestinal colonization, induction of enteritis and systemic translocation by this bacterium requires type III protein secretion. Strategies that target this process have the potential to control infection, pathology and transmission. We defined the global transcriptional response of S. Typhimurium to INP0403, a member of a family of salicylidene acylhydrazides that inhibit type III secretion (T3S). INP0403 treatment was associated with reduced transcription of genes involved in T3S, but also increased transcription of genes associated with iron acquisition. We show that INP0403 restricts iron availability to Salmonella, and that inhibition of T3S system-1 by INP0403 is, at least in part, reversible by exogenous iron and independent of the iron response regulator Fur. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. 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.

Dempsey E.,Moyne Institute of Preventive Medicine | Prudencio M.,University of Lisbon | Fennell B.J.,Moyne Institute of Preventive Medicine | Fennell B.J.,Pfizer | And 3 more authors.
Molecular and Biochemical Parasitology | Year: 2013

Malarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules. Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these herbicides block schizogony in erythrocytic parasites of the most lethal human malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite tubulin. Here we show for the first time that the antimitotic herbicides also block the development of malarial parasites in the liver stage. Structure-based design of novel antimalarial agents binding to tubulin at the herbicide site, which presumably exists on (some) parasite and plant tubulins but not mammalian ones, can therefore constitute an important transmission blocking approach. The nature of this binding site is controversial, with three overlapping but non-identical locations on α-tubulin proposed in the literature. We tested the validity of the three sites by (i) using site-directed mutagenesis to introduce six amino acid changes designed to occlude them, (ii) producing the resulting tubulins recombinantly in Escherichia coli and (iii) measuring the affinity of the herbicides amiprophosmethyl and oryzalin for these proteins in comparison with wild-type tubulins by fluorescence quenching. The changes had little or no effect, with dissociation constants (Kd) no more than 1.3-fold (amiprophosmethyl) or 1.6-fold (oryzalin) higher than wild-type. We conclude that the herbicides impair Plasmodium liver stage as well as blood stage development but that the location of their binding site on malarial parasite tubulin remains to be proven. © 2013 Elsevier B.V.

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.

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.

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