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：
8951755
负责人：
Ambrose Lin Yau Cheung
金额：
$ 20.25万
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 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8951755
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 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 public health relevance 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（由适用提供）：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发生在看似独特的途径中。在大多数情况下，依赖ATP的蛋白质CLPP在伴侣CLPX或CLPA识别后识别并降解了SSRA标记的蛋白质。 S.金黄色葡萄球菌使用伴侣CLPC和适配器TRFA来协助CLPP进行SSRA降解。通过观察到缺乏TRFA的菌株，SSRA分解继续发生的观察结果进一步增强了这种新型机制，这表明除了CLPPC/TRFA之外，还表明了额外的蛋白水解调节层。为了充分表征金黄色葡萄球菌中SSRA标记的蛋白水平的调节，我们的第一个目的是发现金黄色葡萄球菌中SSRA降解基序的其他调节剂和适配器，并确定其与已知SSRA调节剂TRFA的相互作用。金黄色葡萄球菌USE300 LAC JE2的序列定义的转座子突变库将使用能够表达用SSRA标记为C末端标记的快速成熟的金星蛋白的质粒转化。为了在此搜索中施放最宽的网络，将获得暴露于各种应力条件（例如，杜敏，饥饿等）的荧光模式。这些人群中的大多数菌株由于SSRA标记的金星的持续降解而表现出低水平的荧光。但是，少数数字将具有影响SSRA降解的突变，而与缺乏CLPC或TRFA的菌株的模式相比，该突变将使初始类别成为各种控制途径。随后的每个候选基因和TRFA的菌株将被创建以进一步指导这一过程，以及下游完成，每个鉴定的基因都可以磨练S. Aureus中SSRA调节的层次结构。在金黄色葡萄球菌中使用CLPPC/TRFA作为SSRA蛋白水解的介体表明，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。因此，金黄色葡萄球菌SSRA序列可能包含新的降解信号，我们的第二个目的是识别SSRA标签中的新基序，并评估这些基序中各种残差的重要性。附加到维纳斯的SSRA的11个氨基酸的个体和组将被改变，以识别蛋白水解调节的关键区域。随着时间的推移和暴露于各种应力期间，将通过监测荧光来评估所得的变化。最终，阐明金黄色葡萄球菌如何调节SSRA标记的蛋白质提供了一个机会，可以更好地了解该细菌如何调整其蛋白质组以挑战环境，并可以为治疗感染提供特定于金黄色葡萄球菌。