Antimicrobial targets of intracellular cyclic peptides

多药耐药感染的出现加上生物战的持续威胁 已经产生了发现新的抗微生物靶标的需要。该项目将利用一种新的基因 从功能上询问微生物基因组以寻找细胞存活所需的基因产物的策略。制做 基因序列和蛋白质重要性之间的联系曾经是至关重要的，因为30%到50% 的微生物ORF没有指定的生物学功能。E.线圈将成为肠道疾病的典范 感染，因为菌株被设计成具有非常高的转化效率。 这项建议的主要目标是检测基本的细菌基因，同时 获得稳定的、抑制其功能的环状肽(S)。本项目的具体目标是：(1) 确定单个毒肽的细胞靶点，(2)潜在抗菌剂的选择 其抑制所需的结构和化学决定因素的目标和分析，以及(3) 全基因组检测细胞存活所需的代谢功能。我们的努力成果将 为抗生素设计提供新的靶点和先导化合物。 研究计划将需要生产和选择大量的环肽序列 在大肠杆菌中。这些文库将使用多肽和蛋白质的分裂内含子环连获得 SICLOPPS)技术。实验方法将包括筛选杀菌剂/抑菌剂 )使用复制平板法和/或流式细胞术。每个已识别的有毒多肽都将被共转化 _在寄主基因组中发现的每一个ORF。受阻的代谢功能(S)将通过 靶蛋白的过度表达(S)。编码必需的、多肽抑制的基因组结构 功能将通过聚合酶链式反应恢复，并通过测序或与DNA杂交来揭示其身份 数组。在细菌物种中保存的、在人类中不存在的必要基因将被 被选中进行进一步研究。对于所选择的每个潜在目标，细胞内多肽库将是 重新筛选以检测分离的生物学功能的所有抑制剂。检索到的多肽家族将 对齐以确定抑制所需的保守氨基酸。

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.