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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.