Antimicrobial targets of intracellular cyclic peptides

细胞内环肽的抗菌靶点

• 批准号: 7327573
• 负责人: Ernesto V Abel-Santos
• 金额: $ 13.32万
• 依托单位: UNIVERSITY OF NEVADA LAS VEGAS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7327573
• 关键词: Antimicrobial; targets; intracellular; cyclic; peptides

DESCRIPTION (provided by applicant): 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. coli 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 peptides using replica plating and/or flow cytometry. Each identified toxic peptide will be co-transformed with every 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.

描述（由申请人提供）：多药耐药感染的出现，加上生物战的持续威胁，创造了发现新的抗菌靶点的需求。该项目将利用一种新的遗传策略对微生物基因组进行功能性询问，以获得细胞活力所需的基因产物。在基因序列和蛋白质的重要性之间建立联系是非常重要的，因为30%到50%的微生物orf没有指定的生物学功能。大肠杆菌将作为肠道感染的范例，因为菌株已经被设计成具有非常高的转化效率。本提案的主要目的是检测必需的细菌基因，同时获得抑制其功能的稳定的环状肽。该项目的具体目标是：(1)鉴定单个有毒肽的细胞靶点，(2)选择潜在的抗菌靶点并分析其抑制所需的结构和化学决定因素，以及(3)全基因组检测细胞活力所需的代谢功能。我们的研究结果将为抗生素设计提供新的靶点和先导化合物。该研究计划将需要在大肠杆菌中生产和选择大量的环肽序列。这些文库将使用多肽和蛋白质的分裂内环连接（SICLOPPS）技术获得。实验方法将包括使用复制电镀和/或流式细胞术筛选杀菌/抑菌肽。每个确定的毒性肽将与宿主基因组中发现的每个ORF共转化。受阻的代谢功能将通过靶蛋白的过表达得以恢复。编码必要的、抑制多肽功能的基因组结构将通过PCR恢复，并通过测序或杂交到DNA阵列上揭示其身份。在细菌物种中保守的、在人类中不存在的必要基因将被选择用于进一步的研究。对于选择的每个潜在靶点，将重新筛选细胞内肽库以检测分离的生物功能的所有抑制剂。检索的多肽家族将被对齐，以确定抑制所需的保守氨基酸。