Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP

<p>Abstract</p> <p>Background</p> <p>When heterologous recombinant proteins are produced in <it>Escherichia coli</it>, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.</p> <p>Results</p> <p>We report that the <it>Brucella abortus </it>PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in <it>E. coli</it>, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.</p> <p>Conclusion</p> <p>The data reported here suggest that, in <it>E. coli</it>, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the <it>E. coli </it>cell to find its substrates.</p

Identification of the essential Brucella melitensis porin Omp2b as a suppressor of Bax-induced cell death in yeast in a genome-wide screening.

BACKGROUND: Inhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast. CONCLUSION/SIGNIFICANCE: We report here the first application to a bacterial genome-wide library of coding sequences of this "yeast-rescue" screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed

G1-arrested newborn cells are the predominant infectious form of the pathogen Brucella abortus

Several intracellular pathogens, such as Brucella abortus, display a biphasic infection process starting with a non-proliferative stage of unclear nature. Here, we study the cell cycle of B. abortus at the single-cell level, in culture and during infection of HeLa cells and macrophages. The localization of segregation and replication loci of the two bacterial chromosomes indicates that, immediately after being engulfed by host-cell endocytic vacuoles, most bacterial cells are newborn. These bacterial cells do not initiate DNA replication for the next 4 to 6 h, indicating a G1 arrest. Moreover, growth is completely stopped during that time, reflecting a global cell cycle block. Growth and DNA replication resume later, although bacteria still reside within endosomal-like compartments. We hypothesize that the predominance of G1-arrested bacteria in the infectious population, and the bacterial cell cycle arrest following internalization, may constitute a widespread strategy among intracellular pathogens to colonize new proliferation niches

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting &gt;100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved

Connecting the cell cycle and the pathogenicity of Brucella abortus: when a specific bacterial cell cycle arrest in G1 phase takes part in the colonization of mammalian cells

The bacterial cell cycle is a developmental process starting with newborn bacteria that progress to the predivisional status in which they eventually generate two daughter cells. Along their development, the newborns grow and replicate their genome that is in turn faithfully transmitted to the two siblings. The bacterial cell cycle has been extensively investigated in the alphaproteobacterium Caulobacter crescentus, which produces a small non-replicating (G1 phase) swarmer cell for the colonization of new niches in aquatic environments at each cell division. The pathogen Brucella abortus is also part of the alphaproteobacteria group but displays a singular cell cycle compared to C. crescentus. Opposite to C. crescentus, B. abortus growth mode is unipolar resulting in the asymmetric distribution of the cell wall material in the predivisionnal cell. This asymmetry is further exacerbated by an asymmetric division that generates a large cell composed of “old” cell wall material and a small cell made of a new envelope material. In addition, B. abortus also contains a genome divided into several replicons each encoding its own segregating apparatus. Among those replicons is included a chromid (a combination of chromosomal and plasmidic features) carrying a RepAB-based segregation system for which the localization has not been not assessed so far. During this PhD thesis, we characterized the mechanisms of unipolar growth and chromosomes segregation of B. abortus. Concerning the unipolar growth study, we localized the only monofunctional transpeptidase penicillin-binding protein (FtsI) identified in B. abortus that creates links between the different peptidoglycan strands at every growth zone. The localization of FtsI revealed that this protein is present at the growing pole during the unipolar elongation and at the constriction site during the division. These data suggest the presence of a unique growth machinery performing both the elongation (at the new pole) and the division (at the constriction site) in two different periods of the cell cycle. We therefore propose that B. abortus and other rhizobiales unipolar growth mode relies on a simple process involving only one machinery used after the loss of the genes involved in the formation of the core of the elongation machinery such as mreB or pbp2. We investigated the replication status of B. abortus by generating fluorescent reporters of the segregation markers (ParB and RepB) and the replication origins (ori) and terminators (ter). We showed that both chromosomes were oriented along the cell length axis and that oriI was strictly polarly localized while oriII was not anchored to the poles, similarly to plasmids. Moreover, we showed that the replication and segregation of chromosome I are initiated before the duplication of chromosome II origin. As B. abortus is a facultative intracellular pathogen characterized by a striking proliferation arrest during the first phase of its infectious process, we envisioned a potential link between its cell cycle and its infectivity. Importantly, our chromosomal reporters indicated that the newly generated non-replicating bacteria (newborns) are predominant during the non-proliferative stage of the infection. As the bacterial cell cycle appears to be blocked during this infectious period, we propose that the B. abortus newborns constitute the major cell type able to colonize host cells. These data demonstrate thus that only a subset of the Brucella population is dedicated to the prospection of new environmental niches similarly to the swarmer G1 blocked cells in C. crescentus. Overall, this work demonstrates the need to investigate bacterial cell cycle to improve our knowledge of bacterial pathogenicity, since G1 block may represent a general strategy of resistance form in stressful conditions encountered by the intracellular pathogens.(DOCSC03) -- FUNDP, 201

Un document hypertexte pour l'enseignement de l'anesthésie obstétricale

BREST-BU Médecine-Odontologie (290192102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Déterminants de l'accroissement nécessaire de la concentration en anesthésique local dans l'analgésie épidurale pour le travail obstétrical (étude prospective à propos de 235 parturientes)

BREST-BU Médecine-Odontologie (290192102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF