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Gestin B.,Joseph Fourier University | Valade E.,Institute Of Recherche Biomedicale Des Armees Crssa | Thibault F.,Institute Of Recherche Biomedicale Des Armees Crssa | Schneider D.,Joseph Fourier University | Maurin M.,Joseph Fourier University
Journal of Antimicrobial Chemotherapy | Year: 2010

Objectives: Francisella tularensis subsp. holarctica strains are classified as biovars I and II, which are susceptible and naturally resistant to the macrolide erythromycin, respectively. The present study was aimed at both selecting biovar I strains with increased levels of erythromycin resistance and characterizing the underlying genetic mechanisms. Methods: Serial cultures in the presence of increasingly high erythromycin concentrations were performed to select independent high-and intermediate-level erythromycin-resistant mutants from each of three different biovar I strains. The mutants were characterized for cross-resistance to several antibiotics, presence of mutations in the genes encoding the 23S rRNA and the L4 and L22 ribosomal proteins, and overexpression of efflux pumps. Results: Mutants displayed cross-resistance to all macrolide compounds tested but not to other classes of antibiotics. We found mutations in domain V of the 23S rRNA gene (G2057A, A2058G, A2058T and C2611T) and in the gene encoding L22, leading to either the G91D substitution or the M82K83R84 deletion. Analysis of mutants with intermediate resistance levels obtained over the course of the selection process revealed both a positive correlation between the number of mutated ribosomal operons and the resistance level, and an additional resistance mechanism in the early steps of selection. Conclusions: We showed that high-level resistance to macrolides can be easily obtained in vitro in F. tularensis subsp. holarctica biovar I strains, thereby suggesting that in vivo selection for resistance may explain reported failures of antibiotic treatment. Ketolides were the most effective macrolides tested, which may limit the risk of selection for resistance. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Ooi W.F.,Genome Institute of Singapore | Ong C.,DSO National Laboratories | Nandi T.,Genome Institute of Singapore | Kreisberg J.F.,Genome Institute of Singapore | And 16 more authors.
PLoS Genetics | Year: 2013

Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes - Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes - quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an "accidental pathogen", where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts. © 2013 Ooi et al.

Melzer M.,Blum Scientific Services | Melzer M.,Johannes Gutenberg University Mainz | Heidenreich A.,Blum Scientific Services | Dorandeu F.,Institute Of Recherche Biomedicale Des Armees Crssa | And 7 more authors.
Drug Testing and Analysis | Year: 2012

Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization - time of flight - mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD 50 dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose. Copyright © 2011 John Wiley & Sons, Ltd. Conjugates of DFPase and linear PEGs are characterizedusing ESI-ToF-MS and their in vitro and in vivo efficacy against toxic organophosphorus compounds is determined. A PEGylated variant of DFPase is shown to be able to protect rats from a 3x LD 50 challenge with soman. © 2011 John Wiley & Sons, Ltd..

Nachon F.,Institute Of Recherche Biomedicale Des Armees Crssa | Carletti E.,Institute Of Recherche Biomedicale Des Armees Crssa | Wandhammer M.,Institute Of Recherche Biomedicale Des Armees Crssa | Nicolet Y.,CNRS Institute of Pharmacology and Structural Biology | And 4 more authors.
Biochemical Journal | Year: 2011

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis-(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water. © The Authors Journal compilation © 2011 Biochemical Society.

Wandhammer M.,Institute Of Recherche Biomedicale Des Armees Crssa | Wandhammer M.,CNRS Laboratory of Design and Application of Bioactive Molecules | Carletti E.,Institute Of Recherche Biomedicale Des Armees Crssa | Van Der Schans M.,TNO | And 6 more authors.
Journal of Biological Chemistry | Year: 2011

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the PS adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VXR-(+) and VXS-(-) solutions lead to the formation of the PS and PR adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX R-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VXS-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with PR enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the PS enantiomer and ages. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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