Regulation of SsrA-mediated proteolysis of S. aureus

SsrA 介导的金黄色葡萄球菌蛋白水解的调节

基本信息

批准号：
9089861
负责人：
Ambrose Lin Yau Cheung
金额：
$ 24.3万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9089861
关键词：
ATP-Dependent Proteases Adult Affect Amino Acid Sequence Amino Acids Anterior nares Bacteria Borrelia burgdorferi C-terminal Candidate Disease Gene Carbon Clothing Communities DNA-Binding Proteins Data Diamide Environment Escherichia coli Exposure to Fluorescence Frequencies Future Genes Goals Growth Health Heating Homologous Gene Hospitals Human Hybrids Individual Infection Knowledge Lead Length Libraries Listeria Location Mediating Mediator of activation protein Messenger RNA Microbe Mind Molecular Chaperones Monitor Mutation Open Reading Frames Organism Oxidative Stress Parents Pathway interactions Pattern Peptide Hydrolases Plasmids Population Process Prokaryotic Cells Proteins Proteolysis Proteome Publishing Quality Control Reading Frames Regulation Regulation of Proteolysis Resistance Ribosomes Signal Transduction Site Source Staphylococcus aureus Starvation Stress Sulfhydryl Compounds Surface System Therapeutic Intervention Time Transcript Transcriptional Regulation Transfer RNA Translations Variant Venus biological adaptation to stress cell motility mutant novel pathogen protein degradation tmRNA transmission process

项目摘要

DESCRIPTION (provided by applicant): Proteolytic regulation of the SsrA degradation motif in the human pathogen Staphylococcus aureus occurs through a seemingly unique pathway as compared to other prokaryotes. In most cases, the ATP- dependent protease ClpP recognizes and degrades SsrA-tagged proteins following recognition by either the chaperone ClpX or ClpA. S. aureus instead uses the chaperone ClpC and the adaptor TrfA to assist ClpP in SsrA degradation. This novel mechanism is further enhanced by the observation that SsrA breakdown continues to occur during thiol or oxidative stress in strains lacking TrfA, suggesting an additional layer of proteolytic regulation beyond ClpPC/TrfA. To fully characterize the regulation of SsrA-tagged protein levels in S. aureus, our first aim is to uncover additional regulators and adaptors of the SsrA degradation motif in S. aureus and determine their interplay with the known SsrA regulator TrfA. A sequence-defined transposon mutant library of S. aureus USA300 LAC JE2 will be transformed with a plasmid capable of expressing the rapidly maturing Venus protein that is C-terminally tagged with SsrA. To cast the widest net in this search, fluorescence patterns from strains exposed to a variety of stress conditions (e.g. diamide, starvation, etc.) will be obtained. Most strains in these populations will demonstrate low levels o fluorescence due to their persistent degradation of SsrA- tagged Venus. However, a small number will have mutations that affect SsrA degradation, which when compared to patterns from strains lacking clpC or trfA, will allow initial categorization into various control pathways. Subsequent strains doubly disrupted for each candidate gene and trfA will be created to further guide this process, as well as downstream complementation with each identified gene to hone a hierarchy of SsrA regulation in S. aureus. The use of ClpPC/TrfA in S. aureus as mediators of SsrA proteolysis suggests that the established motifs in SsrA used by other microbes may not be valid for S. aureus. Therefore, the S. aureus SsrA sequence may contain novel degradation signals, and our second aim will be to identify novel motifs in the SsrA tag, and to evaluate the importance of various residues in these motifs. Individual and groups of the eleven amino acids of SsrA appended to Venus will be altered to identify critical regions for proteolytic regulation. The resulting changes will be evaluated by monitoring fluorescence over time and during exposure to various stresses. Ultimately, unraveling how S. aureus regulates SsrA-tagged proteins provides an opportunity to better understand how this bacteria adjusts its proteome to challenging environments, and can provide S. aureus- specific targets for treating infections.

描述（由申请人提供）：与其他原核生物相比，人类病原体金黄色葡萄球菌中 SsrA 降解基序的蛋白水解调节通过看似独特的途径发生。在大多数情况下，ATP 依赖性蛋白酶 ClpP 识别并降解 SsrA 标签蛋白。由伴侣 ClpX 或 ClpA 识别，而使用伴侣 ClpC 和金黄色葡萄球菌。接头 TrfA 协助 ClpP 参与 SsrA 降解，观察到缺乏 TrfA 的菌株在硫醇或氧化应激期间继续发生 SsrA 分解，这表明除了 ClpPC/TrfA 之外还有一层蛋白水解调节，从而进一步增强了这一新机制。金黄色葡萄球菌中 SsrA 标记蛋白水平的调节，我们的首要目标是发现 SsrA 降解基序的其他调节因子和接头金黄色葡萄球菌中，并确定它们与已知的 SsrA 调节剂 TrfA 的相互作用。金黄色葡萄球菌 USA300 LAC JE2 的序列定义转座子突变体库将用能够表达快速成熟的维纳斯蛋白的质粒进行转化，该蛋白的 C 端标记为SsrA。为了在这次搜索中撒下最广泛的网，暴露于各种应激条件（例如二酰胺、饥饿等）的菌株的荧光模式将被这些群体中的大多数菌株由于 SsrA 标记的 Venus 的持续降解而表现出低水平的荧光，但是，少数菌株会产生影响 SsrA 降解的突变，与缺乏 clpC 或 trfA 的菌株的模式相比，这种突变会影响 SsrA 的降解。允许初步分类为各种控制途径。将创建对每个候选基因和 trfA 进行双重破坏的后续菌株，以进一步指导该过程，以及与每个已识别基因的下游互补，以磨练金黄色葡萄球菌中 SsrA 调控的层次结构。 ClpPC/TrfA 在金黄色葡萄球菌中的使用。作为 SsrA 蛋白水解介质，表明其他微生物使用的 SsrA 中已建立的基序可能对金黄色葡萄球菌无效。金黄色葡萄球菌 SsrA 序列可能包含新的降解信号，我们的第二个目标是新识别 SsrA 标签中的基序，并评估这些基序中附加到金星的 SsrA 的 11 个氨基酸的个体和组的重要性。改变以确定蛋白水解调节的关键区域，将通过监测一段时间内的荧光和暴露于各种应激期间来评估所产生的变化，最终揭示金黄色葡萄球菌如何调节 SsrA 标记。蛋白质提供了一个机会，可以更好地了解这种细菌如何调整其蛋白质组以适应具有挑战性的环境，并可以提供金黄色葡萄球菌特异性靶点来治疗感染。