Antimicrobial targets of intracellular cyclic peptides

描述(申请人提供)：多重耐药感染的出现，加上生物战的持续威胁，创造了发现新的抗菌素靶标的必要性。该项目将利用一种新的遗传策略，从功能上询问微生物基因组，以获得细胞存活所需的基因产物。由于30%到50%的微生物开放阅读框没有指定的生物学功能，因此在基因序列和蛋白质重要性之间建立联系是至关重要的。大肠杆菌将成为肠道感染的范例，因为菌株已经被设计成具有非常高的转化效率。这项建议的主要目标是检测必要的细菌基因，同时获得稳定的、抑制其功能的环肽(S)。该项目的具体目标是：(1)确定单个有毒多肽的细胞靶标，(2)选择潜在的抗微生物靶标，并分析其抑制所需的结构和化学决定因素，以及(3)全基因组检测细胞存活所需的代谢功能。我们的努力结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SICLOPPS(SICLOPPS)技术获得。实验方法将包括使用复制平板法和/或流式细胞术筛选杀菌/抑菌肽。每个已识别的有毒多肽都将与宿主基因组中发现的每个ORF进行共转化。受阻的代谢功能(S)将通过靶蛋白(S)的过度表达而恢复。编码必需的多肽抑制功能的基因组结构将通过聚合酶链式反应恢复，并通过测序或杂交到DNA阵列来揭示它们的身份。将选择在细菌物种中保存的、在人类中不存在的必要基因进行进一步研究。对于每个选择的潜在靶点，将重新筛选细胞内多肽文库，以检测分离的生物功能的所有抑制剂。检索到的多肽家族将进行比对，以确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.