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Spaciotemporal Regulation of Specific Penicillin Binding Protein (PBP) Function Determined by New Activity-Based Approaches

通过基于活性的新方法确定特定青霉素结合蛋白 (PBP) 功能的时空调节

基本信息

批准号：
9767233
负责人：
Erin Elizabeth Carlson
金额：
$ 43万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767233
关键词：
Address Anabolism Antibiotic Resistance Antibiotics Bacteria Bacterial Antibiotic Resistance Bacterial Infections Basic Science Biochemical Biological Biological Models Catalysis Cell Cycle Cell Cycle Stage Cell Wall Cell division Cells Cellular biology Chemicals Chemistry Clinical Complex Crystallization Crystallography Development Disease Enzymes Eubacterium Evolution Foundations Future Genetic Goals Grant Growth Health Homologous Gene Homologous Protein Human Individual Knowledge Lactones Lead Libraries Macromolecular Complexes Maps Methods Microbiology Microscopic Modeling Molecular Machines Molecular Structure Monobactams Organism Pathogenicity Penicillin-Binding Proteins Peptidoglycan Peptidyltransferase Process Protein Region Proteins Regulation Resolution Solid Streptococcus pneumoniae Superbug Techniques Testing Time Work base beta-Lactams combat crosslink design drug development experimental study genetic regulatory protein human pathogen imaging approach inhibitor/antagonist knowledge base knowledge of results molecular modeling mutant novel novel strategies pathogen pathogenic bacteria protein activation protein function scaffold structural biology success tool transpeptidation

项目摘要

Cell wall synthesis and remodeling are essential processes central to bacterial growth and division. The long-term goal of this basic-science proposal is to fill in major gaps in fundamental knowledge about the functions and regulation of the individual penicillin-binding proteins (PBPs) that synthesize peptidoglycan (PG) in bacterial cell walls. A major unmet challenge in mapping PBP activation and localization, as well as the relationships between the PBPs and their associated regulatory proteins, has been the inability to assess the functional state of the separate PBP homologs that are present in each bacterial species over the course of growth and division. We propose to generate new tools and approaches that will contribute to a comprehensive understanding of PBP function and regulation by pursuit of the following two Aims. Aim 1: Develop selective activity-based probes for each PBP homolog in a Streptococcus pneumoniae model utilizing both known and novel electrophilic scaffolds, in combination with protein crystallography and molecular modeling. Design of probes to target each PBP homolog in an organism requires the identification of scaffolds that selectively inhibit each enzyme. We have mapped the PBP selectivity of a library of β-lactams, providing a solid foundation for the development of activity-based probes; however, many PBPs are poorly inhibited by existing β-lactams. To address this additional challenge, we will employ molecular modeling and structural biology studies, as well as our newly identified PBP-selective β-lactone scaffold for the development of a suite of probes for the PBPs of S. pneumoniae, which is a powerful cell biology model to establish and validate the use of this new type of probe. Aim 2. Map the localization, timing, and regulation of the transpeptidase activity of specific PBPs by using activity-based probes and complementary approaches. We will use the specific activity-based probes from Aim 1 to answer fundamental questions about the function, localization, and interactions of the PBPs in a S. pneumoniae model; these questions cannot be answered with existing strategies. In this Aim, we will use cutting-edge high-resolution microscopic techniques to determine the spatial and temporal distributions of specific, functional PBPs at different stages of the S. pneumoniae cell cycle. In addition, we will determine how cellular amounts of active PBPs are altered in mutants defective in putative PBP regulators. Finally, we will determine the interactors of PBPs by using both activity-based and complementary approaches. This grant will answer fundamental questions about PBP function and spaciotemporal regulation in a “superbug” S. pneumoniae model and provide new tools and approaches for dissecting PG synthesis in other eubacteria, and thereby lead to a deeper understanding of this complex process in general. In addition, the new knowledge produced by this proposal will reveal novel targets and steps in PG synthesis that may be exploited as vulnerabilities for future drug development to combat the increasing emergence of antibiotic-resistant bacterial pathogens.

细胞壁的合成和重塑是细菌生长和分裂的重要过程。的