X-ray structures of Clostridium perfringens sortase C with C-terminal cell wall sorting motif of LPST demonstrate role of subsite for substrate-binding and structural variations of catalytic site

2021 ◽  
Vol 554 ◽  
pp. 138-144
Author(s):  
Eiji Tamai ◽  
Hiroshi Sekiya ◽  
Hirofumi Nariya ◽  
Seiichi Katayama ◽  
Shigehiro Kamitori
Keyword(s):  
Cell Wall ◽  
Clostridium Perfringens ◽  
Substrate Binding ◽  
Catalytic Site ◽  
Terminal Cell ◽  
Structural Variations ◽  
X Ray ◽  
Sorting Motif

scholarly journals Synergistic Lethality of a Binary Inhibitor ofMycobacterium tuberculosisKasA

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Pradeep Kumar ◽  
Glenn C. Capodagli ◽  
Divya Awasthi ◽  
Riju Shrestha ◽  
Karishma Maharaja ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Substrate Binding ◽  
Content Type ◽  
X Ray ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Transcriptional Pattern

ABSTRACTWe report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) inMycobacterium tuberculosis. Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented thein vitroactivity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model ofM. tuberculosisinfection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCECell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors ofMycobacterium tuberculosisKasA—a key component for biosynthesis of the mycolic acid layer of the bacterium’s cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

scholarly journals Intracellular Siderophore Detection in an Egyptian, Cobalt-Treated F. solani Isolate Using SEM-EDX with Reference to its Tolerance

2017 ◽  
Vol 66 (2) ◽  
pp. 235-243 ◽  
Author(s):  
Farrag M. Rasha
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Fusarium Solani ◽  
X Ray ◽  
Cobalt Uptake ◽  
Molecular Studies ◽  
Control Regime ◽  
Co Concentrations ◽  
Scanning Electron

An Egyptian, plant pathogenic Fusarium solani isolate was grown on cobalt concentrations of 0, 50, 200, 500, 800, and 1000 ppm. The isolate survived concentrations up to 800 ppm, however failed to grow at 1000 ppm. Morphology and elemental analysis of the isolate under the investigated Co concentrations were studied using Scanning electron microscopy (SEM) and energy dispersive X-ray microanalysis (EDX). The isolate reserved its morphology up to a concentration of 200 ppm. Morphological distortions were dramatic at 500 and 800 ppm. EDX detected Co uptake through the hyphae, microconidia, macroconidia, and chlamydospores. Iron, calcium, and potassium were also detected. EDX results showed a linear relationship between Co% and Fe% up to a concentration of 500 ppm reflecting the possible ability of the isolate to synthesize intracellular siderophores storing iron and their release out of the vacuoles. The participation of such siderophores in conferring tolerance against cobalt is discussed. At 800 ppm, the % of Fe was greatly reduced with an accompanying increase in morphological distortions and absence of microconidia. Increasing the implicated cobalt concentrations resulted in increasing the percentages of the chelated cobalt reflecting the possible implication of the cell wall as well as extracellular siderophores in the uptake of cobalt. The current results recommend the absence of cobalt in any control regime taken to combat the investigated F. solani isolate and highlights the accomplishment of biochemical, ultrastructural, and molecular studies on such isolate to approve the production of siderophores and the role of cell wall in cobalt uptake.

scholarly journals Sortase B, a New Class of Sortase in Listeria monocytogenes

2004 ◽  
Vol 186 (7) ◽  
pp. 1972-1982 ◽  
Author(s):  
Hélène Bierne ◽  
Caroline Garandeau ◽  
M. Graciela Pucciarelli ◽  
Christophe Sabet ◽  
Salete Newton ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Virulence Factor ◽  
Cultured Cells ◽  
Surface Proteins ◽  
Gram Positive Bacteria ◽  
New Class ◽  
Sorting Motif

ABSTRACT Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The ΔsrtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.

scholarly journals Cell Wall Degradation in Irradiated Tomato Fruit

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 815B-815
Author(s):  
N. El-Assi ◽  
D.J. Huber ◽  
J.K. Brecht
Keyword(s):  
Cell Wall ◽  
Tomato Fruit ◽  
Selective Extraction ◽  
Specific Cell ◽  
Neutral Sugar ◽  
Cell Wall Degradation ◽  
X Ray ◽  
Green Fruit ◽  
Cell Wall Polymers

The irradiation of harvested fruit is typically accompanied by excessive tissue softening, a process that is not well understood. In this study, we examined the role of specific cell wall polymers and the extent of general cell wall degradation and softening in irradiated tomato fruit. `Sunny' tomato fruit at mature-green and pink stages were subjected to X-ray radiation at 0, 83, and 156 Krad. Immediate softening was noted for both maturation classes, although some postirradiation recovery was evident in green fruit. Pectic polymers of both mature-green and pink fruit exhibited depolymerization and altered neutral sugar profiles in response to irradiation. Pectins, either as components of total ethanol-insoluble solids (EIS), purified by selective extraction, or of commercial origin were similarly affected by irradiation. Cellulose preparations were unaffected by irradiation. The data demonstrate that the effect of irradiation on the cell wall exhibits specificity, can occur nonenzymatically, and does not require initiating adducts of cytosolic origin.

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

The role of silicon in forages: A quantitative X-Ray study

1987 ◽  
Vol 45 ◽  
pp. 416-417
Author(s):  
Janet H. Woodward ◽  
D. E. Akin
Keyword(s):  
Sodium Acetate ◽  
Dry Matter ◽  
Acetate Buffer ◽  
Plant Tissues ◽  
Sodium Acetate Buffer ◽  
X Ray ◽  
Dry Matter Digestibility ◽  
Percent Composition

Silicon (Si) is distributed throughout plant tissues, but its role in forages has not been clarified. Although Si has been suggested as an antiquality factor which limits the digestibility of structural carbohydrates, other research indicates that its presence in plants does not affect digestibility. We employed x-ray microanalysis to evaluate Si as an antiquality factor at specific sites of two cultivars of bermuda grass (Cynodon dactvlon (L.) Pers.). “Coastal” and “Tifton-78” were chosen for this study because previous work in our lab has shown that, although these two grasses are similar ultrastructurally, they differ in in vitro dry matter digestibility and in percent composition of Si.Two millimeter leaf sections of Tifton-7 8 (Tift-7 8) and Coastal (CBG) were incubated for 72 hr in 2.5% (w/v) cellulase in 0.05 M sodium acetate buffer, pH 5.0. For controls, sections were incubated in the sodium acetate buffer or were not treated.

Pathophysiological function of the mitochondria as a calcium reservoir: Quantitative x-ray microanalysis

1990 ◽  
Vol 48 (2) ◽  
pp. 164-165
Author(s):  
K. Teraoka ◽  
N. Kaneko ◽  
Y. Horikawa ◽  
T. Uchida ◽  
R. Matsuda ◽  
...  
Keyword(s):  
Bolus Injection ◽  
Contact Method ◽  
X Ray

The aim of this study was to elucidate the role of the mitochondria as a store of calcium(Ca) under the condition of pathophysiological Ca overload induced by a rise in extracellular Ca concentration and the administration of isoproterenol.Eight rats were employed, and hearts were perfused as in the Langendorff method with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2. Tow specimens were perfused with 2mM Ca for 30 min, and 2 were perfused with 5.5 mM Ca for 20 min. 4 specimens were perfused with 2 mM Ca for 5 min, and of these 4, 2 were infused with 10-7 mM/kg/min. isoproterenol for 5 min, and 2 were given a bolus injection of 3 x 10-7 mM isoproterenol. After rapid-cryofixation by the metal-mirror contact method with a Reichert-Jung KF80/MM80, and cryosectioning at -160 to -180° C with a Reichert-Jung Ultracut Fc-4E, ultrathin specimens (100nm) were free-ze-dreid for several hours at 10-5 Torr in the JEOL FD 7000, and mitochondrial Ca was determined by quantitative x-ray micranalysis (JEOL 1200EX, LINK AN 10000S).

Ionic homeostasis and subcellular element compartmentation

1990 ◽  
Vol 48 (2) ◽  
pp. 150-151
Author(s):  
Ann LeFurgey ◽  
Peter Ingram ◽  
J.J. Blum ◽  
M.C. Carney ◽  
L.A. Hawkey ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Ionic Homeostasis ◽  
X Ray ◽  
Functional Studies ◽  
Cell Compartments ◽  
X Ray Imaging ◽  
Resolution Electron ◽  
Multiple Cell ◽  
Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

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