Antimicrobial targets of intracellular cyclic peptides

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，导致需要发现新的抗菌靶标。该项目将利用一种新的遗传策略来功能性地询问微生物基因组中细胞活力所需的基因产物。在基因序列和蛋白质之间建立联系是非常重要的，因为30%到50%的微生物ORF没有指定的生物功能。E.大肠杆菌将作为肠道感染的范例，因为菌株已经被改造成具有非常高的转化效率。该提议的主要目标是检测细菌的必需基因，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）识别单个毒性肽的细胞靶点，（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。我们的研究成果将为抗生素的设计提供新的靶点和先导化合物。杆菌这些文库将使用肽和蛋白质的断裂内含肽环状连接（Split Intein Circular Ligation Of Peptides and ProteinS SICLOPPS）技术获得。实验方法将涉及使用复制平板和/或流式细胞术筛选杀菌/抑菌肽。每种鉴定的毒性肽将与宿主基因组中发现的每种ORF共转化。被阻塞的代谢功能将通过靶蛋白的过表达而恢复。编码必需的肽抑制功能的基因组构建体将通过PCR回收，并通过测序或通过与DNA阵列杂交来揭示其身份。将选择在细菌物种中保守且在人类中不存在的必需基因进行进一步研究。对于所选的每个潜在靶标，将重新筛选细胞内肽文库，以检测分离的生物功能的所有抑制剂。将比对回收的肽家族以鉴定抑制所需的保守氨基酸。

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.