Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.     

农药鱼藤酮对表达α-突触核蛋白细胞的作用

本研究利用农药鱼藤酮作用于神经细胞 ,观察鱼藤酮对神经细胞的损伤作用以及线粒体功能障碍与α- synuclein积聚之间的关系。方法 :本实验选用人神经母细胞瘤细胞株SH-SY5Y ,实验组为α-synuclein GFP基因转染的细胞 ,鱼藤酮处理的转基因细胞和非转基因细胞 ,对照组为未处理的SH-SY5Y细胞。通过RT-PCR检测α-synuclein基因转染细胞的基因表达情况。荧光显微镜观察细胞内α-synuclein GFP表达产物绿色荧光蛋白。MTT法检测各组细胞活性。DCF检测细胞氧应激。HE染色、免疫组化检测α-synuclein在细胞中的状态。电镜观察细胞超微结构的改变。结果 :RT-PCR显示转基因细胞α-synuclein基因的表达。荧光显微镜观察显示细胞浆内可见绿色荧光蛋白 ,绿色荧光分布不均匀 ,可见蛋白积聚。MTT检测结果显示 ,与对照组相比 ,鱼藤酮处理的细胞增殖速度明显减小 (P <0. 01 )。鱼藤酮的浓度为 75nmol/ L、1 0 0nmol/ L时 ,未转基因与转基因的细胞活性相比较 ,前者的细胞活性低于后者 (P <0 .0 5 )。HE染色 ,鱼藤酮处理的转基因细胞胞浆减少、转基因细胞胞浆内也可见自噬体。胞浆内有嗜酸性包涵体样结构。免疫组化可见鱼藤酮处理高表达α-synuclein细胞明显变成梭形并有很长的突起。电镜显示鱼藤酮处理的细胞线粒体肿胀 ,嵴断裂 ,胞浆内形成自噬体。DCF检测转基因细胞内存在明显的氧化应激 ,并随鱼藤酮处理加重。结论 :农药鱼藤酮对多巴胺能神经细胞有明显的损伤作用 ,转基因细胞显示对较高浓度的鱼藤酮损伤有一定的耐受作用。α-synuclein可引起神经细胞的氧化应激并随鱼藤酮处理加重。提示环境因素可能与α-synuclein表达相互作用使多巴胺能神经元氧化应激进行性加重 ,这可能是引起PD的主要原因。     

Metachromatic leukodystrophy in the habbanite Jews: high frequency in a genetic isolate and screening for heterozygotes.

A very high incidence of late infantile metachromatic leukodystrophy (MLD) (1/75 live births) was found in the Jewish Habbanite community which constitutes a genetic isolate of about 1,000-1,200 individuals. Screening in this population for aryl sulfatase A (ASA) levels in married adults revealed a carrier frequency for MLD of 17% and identified six couples of whom both partners were heterozygotes (6% of screened couples). In three pregnancies of these couples, prenatal diagnosis for the detection of ASA in the fetus was performed.     

Signal Transduction by BvgS Sensor Kinase: BINDING OF MODULATOR NICOTINATE AFFECTS THE CONFORMATION AND DYNAMICS OF THE ENTIRE PERIPLASMIC MOIETY*

The two-component sensory transduction system BvgAS controls the virulence regulon of the whooping-cough agent Bordetella pertussis. The periplasmic moiety of the homodimeric sensor kinase BvgS is composed of four bilobed Venus flytrap (VFT) perception domains followed by α helices that extend into the cytoplasmic membrane. In the virulent phase, the default state of B. pertussis, the cytoplasmic enzymatic moiety of BvgS acts as kinase by autophosphorylating and transferring the phosphoryl group to the response regulator BvgA. Under laboratory conditions, BvgS shifts to phosphatase activity in response to modulators, notably nicotinate ions. Here we characterized the effects of nicotinate and related modulators on the BvgS periplasmic moiety by using site-directed mutagenesis and in silico and biophysical approaches. Modulators bind with low affinity to BvgS in the VFT2 cavity. Electron paramagnetic resonance shows that their binding globally affects the conformation and dynamics of the periplasmic moiety. Specific amino acid substitutions designed to slacken interactions within and between the VFT lobes prevent BvgS from responding to nicotinate, showing that BvgS shifts from kinase to phosphatase activity in response to this modulator via a tense transition state that involves a large periplasmic structural block. We propose that this transition enables the transmembrane helices to adopt a distinct conformation that sets the cytoplasmic enzymatic moiety in the phosphatase mode. The bona fide, in vivo VFT ligands that remain to be identified are likely to trigger similar effects on the transmembrane and cytoplasmic moieties. This mechanism may be relevant to the other VFT-containing sensor kinases homologous to BvgS.     

Restoration of stratum corneum with nacre lipids

To discover potential new products for the atopic dermatitis treatment, lipids extracted from nacre from the oyster Pinctada margaritifera were tested on artificially dehydrated skin explants. Expression of filaggrin and transglutaminase 1 was investigated after treatment of dehydrated skin with P. margaritifera lipid extracts according to light microscopy after labelling with specific monoclonal antibodies. The lipids were extracted from the nacre with methanol/chloroform mixture at room temperature and the extract composition was determined according to TLC and densitometry measures. Relative to the dry nacre material, a yield of extraction in lipids of 0.54% (w/w) was determined. Fatty acids, triglycerides, cholesterol and ceramides were in low abundance. Then, application of lipid formulations on skin explants previously dehydrated gave after 3 h an overexpression of filaggrin and a decrease of transglutaminase expression as shown by light microscopy. Using immunofluorescence labelling, we showed that lipids extracted from the mother of pearl of P. margaritifera induced a reconstitution of the intercellular cement of the stratum corneum. The signaling properties of the nacre lipids could be used for a development of new active product treatment against the symptoms of the dermatitis.     

Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis

In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.