Antimicrobial targets of intracellular cyclic peptides

细胞内环肽的抗菌靶点

基本信息

批准号：
7214753
负责人：
Ernesto V Abel-Santos
金额：
$ 28.07万
依托单位：
UNIVERSITY OF NEVADA LAS VEGAS
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-01-01 至 2008-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7214753
关键词：
Amino Acid Sequence Amino Acids Animal Model Antiviral Agents Bacteria Bacterial Genes Bacterial Infections Bacterial Proteins Bacteriophages Binding Biological Biological Process Biological Warfare Catabolism Cell Survival Cell physiology Cells Chemicals Collection Complement Coupled Cyclic Peptides Cyclization DNA Data Detection Development Disease Engineering Ensure Enteral Environment Enzymes Escherichia coli Essential Genes Family Flow Cytometry Fluorogenic Substrate Food Poisoning Gene Targeting Genes Genetic Genome Genomic Library Genomics Goals Gram-Positive Bacteria Grant Growth Human Human Genome Immunomodulators In Vitro Individual Infection Lead Libraries Ligase Ligation Malignant Neoplasms Membrane Metabolic Methods Multi-Drug Resistance Numbers Open Reading Frames Organism Peptide Library Peptide Sequence Determination Peptides Pharmaceutical Preparations Polymerase Chain Reaction Population Procedures Production Proteins Proteolysis Rate Relative (related person)Research Screening procedure Sorting - Cell Movement Structure Techniques Technology Vertebral column antibiotic design antimicrobial antimicrobial drug bactericide cellular targeting chemical genetics in vivo inhibitor/antagonist intein interest member microbial microbial genome novel pathogenic Escherichia coli promoter protein aminoacid sequence protein function protein protein interaction

项目摘要

The emergence of multidrug resistant infections coupled with the continuous threat of biological warfare has created the need for the discovery of new antimicrobial targets. This project will utilize a novel genetic strategy to functionally interrogate microbial genomes for gene products required for cell viability. Making the connection between gene sequence and protein essentiality is ever so crucial since 30 to 50 percent of microbial ORFs have no assigned biological function. E. coil will serve as a paradigm for enteric infections, since strains have been engineered with very high transformation efficiencies. The main objective of this proposal is to detect essential bacterial genes, while simultaneously obtaining stable, cyclic peptides that inhibit their function(s). The specific aims of this project are: (1) Identification of the cellular targets for individual toxic peptides, (2) Selection of potential antimicrobial targets and analysis of the structural and chemical determinants required for their inhibition, and (3) Genome-wide detection of metabolic functions required for cell viability. The results of our efforts will provide new targets and lead compounds for antibiotic design. The research plan will require the production and selection of vast numbers of cyclic peptide sequences in E. coli. These libraries will be obtained using the Split Intein Circular Ligation Of Peptides and ProteinS SICLOPPS) technology. The experimental method will involve screening for bactericidal/bacteriostatic )eptides using replica plating and/or flow cytometry. Each identified toxic peptide will be co-transformed _vithevery ORF found in the host genome. The obstructed metabolic function(s) will be restored by over-expression of the targeted protein(s). The genomic constructs encoding essential, peptide-inhibited functions will be recovered by PCR and their identity revealed by sequencing or by hybridizing onto DNA arrays. Essential genes conserved across bacterial species and not represented in humans will be selected for further studies. For each potential target selected, the intracellular peptide library will be re-screened to detect all inhibitors of the isolated biological function. The family of retrieved peptides will be aligned to identify conserved amino acids required for inhibition.

多重耐药感染的出现加上生物战的持续威胁产生了发现新抗菌靶点的需求。该项目将利用一种新型基因功能性询问微生物基因组以获取细胞活力所需的基因产物的策略。制作基因序列和蛋白质必需性之间的联系至关重要，因为 30% 到 50% 的微生物 ORF 没有指定的生物学功能。大肠杆菌将作为肠溶的范例感染，因为菌株经过改造具有非常高的转化效率。该提案的主要目标是检测必需的细菌基因，同时获得抑制其功能的稳定的环肽。本项目的具体目标是：（1）识别单个有毒肽的细胞靶标，(2) 选择潜在的抗菌剂抑制其所需的目标和结构和化学决定因素的分析，以及（3）对细胞活力所需的代谢功能进行全基因组检测。我们努力的结果将为抗生素设计提供新的靶点和先导化合物。该研究计划将需要生产和选择大量环肽序列在大肠杆菌中。这些文库将使用肽和蛋白质的分裂内含肽环状连接获得 SICLOPPS）技术。实验方法将涉及筛选杀菌/抑菌剂）使用复制电镀和/或流式细胞术的肽。每个鉴定出的有毒肽都将被共转化 _在宿主基因组中发现的所有 ORF。受阻的代谢功能将通过以下方式恢复：目标蛋白的过度表达。编码必需的、肽抑制的基因组构建体功能将通过 PCR 恢复，并通过测序或与 DNA 杂交揭示其身份数组。跨细菌物种保守且在人类中不存在的重要基因将被选择继续深造。对于每个选定的潜在靶标，细胞内肽库将是重新筛选以检测分离的生物功能的所有抑制剂。回收的肽家族将进行比对以确定抑制所需的保守氨基酸。