Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator
Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator
批准号:
10387865
负责人:
Christopher L Colbert
金额:
$19.99万
依托单位国家:
美国
项目类别:
财政年份:
2018
资助国家:
美国
项目状态:
已结题
起止时间:
2018-09-20 至 2023-08-31
关键词:
AffinityAntibiotic ResistanceAntibioticsBacteriaBacterial InfectionsBiological ModelsC-terminalCalorimetryCell surfaceCellsCellular AssayCenters for Disease Control and Prevention (U.S.)Circular Dichroism SpectroscopyComplexDevelopmentDimerizationDrug Delivery SystemsEnvironmentFutureGram-Negative BacteriaHealthHumanIronLeadLengthMembraneMetalsModernizationMolecular BiologyMolecular ConformationMulti-Drug ResistanceNutrientPrevalenceProcessReportingResearchRoentgen RaysRoleSiderophoresSigma FactorSignal TransductionStimulusStructureSystemTitrationsTranscriptional ActivationX-Ray Crystallographybiophysical techniquescombatdesigninterdisciplinary approachmetal chelatornext generationnovel therapeuticsperiplasmresistance mechanism
中文摘要
项目摘要/摘要
美国疾病控制与预防中心最近发布了一份报告,详细描述了美国的抗生素耐药性威胁。特别的
疾控中心报告中的重点是多重耐药的革兰氏阴性细菌(MDR-细菌)的流行增加。
(GNB),以及开发下一代抗生素来对抗它们的必要性。所有革兰氏阴性菌
依赖于一组同源但高度特异的外膜TonB依赖转运蛋白(TBDTs)来
从它们的环境中进口关键的营养物质,特别是铁等金属,它们被高亲和力结合在一起,
金属螯合化合物称为铁载体。最近的抗生素发展表明,
铁载体-抗生素结合物可以选择性地针对特定的细菌,这种传递
该机制克服了几个关键的抗生素耐药机制。这次交付的一个重要限制是
TbDts的低表达水平是该系统的一个重要特征。然而,这些TBDT的子集控制着它们自己的
通过细胞表面信号传递(CSS)过程上调自身的表达。长的-
本提案的术语目标是了解CSS调节过程并操纵TBDT表达
加强铁载体-抗生素联合治疗MDR-GNB感染。这样做的结果
该提案将有助于阐明由革兰氏阴性细菌Sigma调节因子形成的css的结构基础。作为一名
恶臭假单胞菌BN7/8假细菌转运系统模型系统,包括TBDT,
PupB是内膜σ调节因子PupR,细胞质σ因子是PupI。至
实现本提案的目标将追求以下三个具体目标:1)建立PupR
抗σ因子结构域二聚化影响PUPI的转录激活,2)鉴定结构
决定因素和描绘PupR:PupB周质相互作用对PupR稳定性的作用
周质C末端css结构域(CCSSD),以及3)决定全长PupB:PupR CCSSD的变化
在其同源铁载体的存在和不存在的情况下的复合体,伪BN7/8。
完成这些目标需要多学科方法,包括X射线结晶学、小角X射线
散射、分子生物学、细胞分析和生物物理技术,如等温滴定
量热法和圆二色谱;将使用。这项研究将提供关键的结构
有关σ调节因子的信息;解释它如何与内膜上的σ因子相互作用,以及作用程度
Tbdt和σ-调节子之间的周质构象变化导致蛋白降解
对控制转录激活很重要的降解。
英文摘要
PROJECT SUMMARY/ABSTRACT
The CDC recently released a report detailing antibiotic resistant threats in the US. Of particular
emphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR-
GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteria
rely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) to
import critical nutrients from their environment, especially metals like iron, which are bound by high-affinity,
metal chelating compounds called siderophores. Recent antibiotic developments have shown that
siderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this delivery
mechanism overcomes several key antibiotic resistance mechanisms. A significant limitation of this delivery
system is the low expression levels of the TBDTs. However, a subset of these TBDTs controls their own
expression through a cell-surface signaling (CSS) process that up-regulates their own expression. The long-
term objective of this proposal is to understand the CSS regulatory process and manipulate TBDT expression
to enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. The results of this
proposal will help elucidate the structural basis for CSS by a Gram-negative bacteria sigma-regulator. As a
model system the pseudobactin BN7/8 transport system from Psuedomonas putida that consists of the TBDT,
PupB, the inner membrane σ-regulator, PupR, and the cytoplasmic σ-factor, PupI, is being used. To
accomplish this proposal's objective the following three specific aims will be pursued: 1) establish that PupR
anti-σ-factor domain dimerization influences transcriptional activation by PupI, 2) identify the structural
determinants and delineate the role of the PupR:PupB periplasmic interactions on the stability of the PupR
periplasmic C-terminal CSS domain (CCSSD), and 3) determine changes in the full-length PupB:PupR CCSSD
complex in the presence and absence of its cognate siderophore, pseudobactin BN7/8. For the successful
completion of these aims a multidisciplinary approach; including X-ray crystallography, small-angle X-ray
scattering, molecular biology, cellular assays, and biophysical techniques such as isothermal titration
calorimetry and circular dichroism spectroscopy; will be employed. This research will provide critical structural
information about a σ-regulator; explain how it interacts with a σ-factor at the inner membrane, and the extent
to which periplasmic conformational changes between the TBDT and σ-regulator lead to proteolytic
degradation that is important for controlling transcriptional activation.
期刊论文(1)
专著(0)
科研奖励(0)
会议论文
Analytical ultracentrifuge with absorbance and interference optics.
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批准号:10177341
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项目类别:
-
资助金额:$38.44万
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财政年份:2021
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负责人:Christopher L Colbert
-
依托单位:
Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator
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批准号:9789675
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项目类别:
-
资助金额:$29.0万
-
财政年份:2018
-
负责人:Christopher L Colbert
-
依托单位:
Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator
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批准号:10004679
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项目类别:
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资助金额:$29.0万
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财政年份:2018
-
负责人:Christopher L Colbert
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依托单位:
Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator
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批准号:10240569
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项目类别:
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资助金额:$29.0万
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财政年份:2018
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负责人:Christopher L Colbert
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依托单位:
Mechanism of inner membrane sigma-regulator function in Gram-negative bacteria
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批准号:9316212
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项目类别:
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资助金额:$6.0万
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财政年份:2015
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负责人:Christopher L Colbert
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依托单位:
海外基金