The palindromic DNA-bound USP/EcR nuclear receptor adopts an asymmetric organization with allosteric domain positioning

International audienceNuclear receptors (NRs) regulate gene expression through DNA- and ligand-binding and thus represent crucial therapeutic targets. The ultraspiracle protein/ecdysone receptor (USP/EcR) complex binds to half-sites with a one base pair spaced inverted repeat (IR1), a palindromic DNA response element (RE) reminiscent of IRs observed for vertebrate steroid hormone receptors. Here we present the cryo electron microscopy structure of the USP/EcR complex bound to an IR1 RE which provides the first description of a full IR-bound NR complex. The structure reveals that even though the DNA is almost symmetric, the complex adopts a highly asymmetric architecture in which the ligand-binding domains (LBDs) are positioned 5' off-centred. Additional interactions of the USP LBD with the 5'-flanking sequence trigger transcription activity as monitored by transfection assays. The comparison with DR-bound NR complexes suggests that DNA is the major allosteric driver in inversely positioning the LBDs, which serve as the main binding-site for transcriptional regulators

The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10

Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulatio

Both the <i>esx-5</i> and <i>ppe10</i> mutants have reduced hemolytic activity and show reduced ubiquitin co-localization upon infection of host cells.

<p>A) Contact-dependent hemolysis of red blood cells (RBCs) by <i>M</i>. <i>marinum</i>. <i>M</i>. marinum E11 wild-type cells and isogenic transposon mutants disrupted in <i>espG</i>_<i>5</i>, <i>eccCb</i>_<i>1</i> and <i>ppe10</i> and the complemented <i>ppe10</i> mutant were grown in the presence or absence of Tween-80. Subsequently, washed cells were used for the hemolysis assay. B) Quantification of differentiated THP-1 cells that were infected with <i>M</i>. <i>marinum</i>. Only cells gated as positive for co-localization of the bacteria with ubiquitin were chosen for further analysis (full data and statistics in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s006" target="_blank">S6 Fig</a>). The <i>ppe10</i>::<i>tn</i> mutant showed similar levels of ubiquitin co-localization as <i>eccCb</i>_<i>1</i>::<i>tn</i> indicating that this mutant has no cytosolic access in the early stages of infection. C) Images obtained by imaging flow cytometry of wild-type <i>M</i>. <i>marinum</i> and <i>ppe10</i>::<i>tn</i> three hours after infection of differentiated THP-1 cells. Bacteria express <i>mCherry</i> (Red), while ubiquitin is visualized by FK-2 antibody against poly-ubiquitin (Green). D) Super-resolution confocal microscopy images of wild-type <i>M</i>. <i>marinum</i> and <i>ppe10</i>::<i>tn</i> illustrating differential bacterial clustering and ubiquitin labeling of bacteria. Bacteria express <i>mCherry</i> (Red), while ubiquitin is visualized by FK-2 antibody against poly-ubiquitin (Green). A 3-dimensional view of these images can be found in supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s009" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s010" target="_blank">S2</a> Movies.</p

Surface labelling of capsular protein EspE and capsular glycans is dependent on ESX-5.

<p>A) Parental strains of wild-type <i>M</i>. <i>marinum</i> E11, or isogenic mutant strains <i>espG</i>_<i>5</i>::<i>tn</i>, <i>eccCb</i>_<i>1</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i> expressing pSMT3::<i>mCherry</i>, were grown in the presence or absence of Tween-80 and labeled with the α-EspE antibody and a FITC labeled secondary antibody. Subsequently, cells were analyzed by flow cytometry analysis. High levels of surface expression of EspE could be detected in the absence of Tween-80 in both wild-type <i>M</i>. <i>marinum</i> and the <i>espG</i>_<i>5</i>::<i>tn</i> mutant strain, while <i>ppe10</i>::<i>tn</i> showed an intermediate EspE surface labelling. When grown in the presence of Tween-80, surface labeling of EspE was almost completely lost and reduced to similar levels of the ESX-1 mutant strain <i>eccCb</i>_<i>1</i>::<i>tn</i>. B) Immuno-electron microscopy analysis of <i>M</i>. <i>tuberculosis</i> CDC1551, the <i>esx-5</i> mutant Mtb-<i>eccC</i>_<i>5</i>::<i>tn</i> and the mutant complemented with an integrative plasmid containing the complete <i>esx-5</i> region; pMV-<i>esx-5</i>. Cells were grown in liquid culture with 0.05% Tween-80 (lower row) or without Tween-80 (upper row) and labeled with a monoclonal antibody directed against PIM₆-LAM capsular glycolipids and a gold-labeled secondary antibody. C) Gold labeling of the different Mtb strains depicted in B was quantified by counting the number of gold particles per cell after growth in the presence or absence of Tween-80 (error bars indicate the standard deviation). D) The capsule morphology of wild-type <i>M</i>. <i>marinum</i> and <i>espG</i>_<i>5</i>::<i>tn</i> was analyzed after plunge freezing of bacteria grown in the absence of Tween-80 by cryo-electron microscopy. The black bar indicates the mycobacterial capsular layer. E) Reduced hydrophobicity of the <i>espG</i>_<i>5</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i> strains, measured as the OD of the aqueous phase after 1 OD of bacteria was incubated with 0.5% (v/v) Xylene in PBS for two hours. Statistical differences were calculated by GraphPad Prism software using one-way ANOVA and (Dunnett’s) multiple comparisons against a single control. ** = <i>p</i><0.01, n.s = not significant.</p

The <i>M</i>. <i>marinum ppe10</i>::tn mutant is attenuated in zebrafish embryo infections.

<p>A) <i>M</i>. <i>marinum</i> E11 wild type strain and the isogenic mutants <i>espG</i>_<i>5</i>::<i>tn</i>, <i>eccCb</i>_<i>1</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i>, as well as the complemented <i>ppe10</i>::<i>tn</i> strain (PPE10-C), were pre-cultured in liquid medium containing Tween-80. 50–100 CFUs of bacteria were injected in the bloodstream of zebrafish embryos at 28 hours post fertilization. Embryos were homogenized five days post infection and plated to establish the number of CFU per embryo. Three independent experiments of six embryos per group were performed and the data were pooled. B) Visualization of <i>M</i>. <i>marinum</i> infection of zebrafish embryos. Wild-type <i>M</i>. <i>marinum</i> or <i>ppe10</i>::<i>tn</i> containing the plasmid pSMT3::<i>mCherry</i> was injected in the bloodstream of zebrafish embryos as described above. After 5 days of infection the embryos were examined by brightfield (top) or fluorescence (bottom) microscopy. C) Quantification of fluorescence in infected zebrafish embryos. <i>M</i>. <i>marinum</i> wild type (closed symbols) and the <i>ppe10</i>::<i>tn</i> (open symbols) mutants expressing <i>mCherry</i> were injected in the bloodstream of zebrafish embryos 28 hours post fertilization. Images were acquired by fluorescence microscopy at day 5 (blue) and 7 (red) post infection and were analyzed for fluorescent intensity by dedicated software [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.ref043" target="_blank">43</a>]. Differences on day 5 and day 7 were analyzed by GraphPad Prism software using Mann-Whitney two-tailed test. * = <i>p</i><0.05, ** = <i>p</i><0.01, n.s. = not significant.</p

The beam and detector of the NA62 experiment at CERN

NA62 is a fixed-target experiment at the CERN SPS dedicated to measurements of rare kaon decays. Such measurements, like the branching fraction of the K(+) → π(+) ν bar nu decay, have the potential to bring significant insights into new physics processes when comparison is made with precise theoretical predictions. For this purpose, innovative techniques have been developed, in particular, in the domain of low-mass tracking devices. Detector construction spanned several years from 2009 to 2014. The collaboration started detector commissioning in 2014 and will collect data until the end of 2018. The beam line and detector components are described together with their early performance obtained from 2014 and 2015 data.NA62 is a fixed-target experiment at the CERN SPS dedicated to measurements of rare kaon decays. Such measurements, like the branching fraction of the $K^{+} \rightarrow \pi^{+} \nu \bar\nu$ decay, have the potential to bring significant insights into new physics processes when comparison is made with precise theoretical predictions. For this purpose, innovative techniques have been developed, in particular, in the domain of low-mass tracking devices. Detector construction spanned several years from 2009 to 2014. The collaboration started detector commissioning in 2014 and will collect data until the end of 2018. The beam line and detector components are described together with their early performance obtained from 2014 and 2015 data