Metabolic Modeling of Invasive Bacteria and HeLa Cytosol

描述(由申请人提供)：NIAID优先病原体名单中的所有三类细菌中的大多数都入侵人类细胞，对许多人来说，已知的唯一感染繁殖阶段发生在这些宿主细胞的膜结合液泡中或直接发生在细胞质中。几乎所有的抗生素，以及所有口服的抗生素，都是通过阻止快速生长的细菌的某些新陈代谢途径来发挥作用，而不是通过扰乱细菌细胞。因此，识别抗生素作用的新靶点需要了解复制的细胞内(IC)细菌的活跃代谢。利用基于约束的通量平衡分析(CFBA)构建了大肠杆菌(与福氏志贺氏菌近缘)代谢的计算机预测模型。已经确定了快速生长的大肠杆菌细胞的化学成分，并将这些信息用于建立CFBA模型。然而，对于基本上是IC细菌生长介质的真核细胞来说，同样的信息并不完整，将进行调查。为了开发CFBA模型来分析细菌在IC复制阶段的代谢，需要了解真核细胞胞浆的成分。福氏志贺氏菌是一种B类病原体，将被用作：(特定目标1)测定人类来源细胞胞浆成分的生物传感器，以及(特定目标2)CFBA这一创新应用的模式生物。福氏志贺氏菌的几个特性使其成为探索真核细胞胞浆中细菌可接触化合物的良好选择：在胞浆中复制(没有空泡膜使分析复杂化)，大量摄取系统(评估胞浆中化合物对细菌的可用性)，以及检查异源摄取系统的遗传工具。CFBA将代谢网络(被描述为化学计量生物化学反应)转化为一个优化问题，该问题对有机体可以与其环境(在这种情况下是真核细胞胞浆)交换的每个分子都有限制。将从京都基因和基因组百科全书(KEGG)代谢数据库中构建一个通用模型(以允许适应不同的细菌)。福氏志贺氏菌CFBA模型将利用已发表的关于福氏志贺氏菌突变株基因组和IC行为的报告从参考模型中推导出来。对NIAID优先病原体的代谢进行建模将有助于我们了解大量潜在的生物恐怖因子，并有助于指导寻找新的抗菌药物。

DESCRIPTION (provided by applicant): Most of the bacteria in the NIAID Priority Pathogen list from all three Categories invade human cells and for many the only known reproductive stage of infection takes place in membrane-bound vacuoles or directly in the cytosol of these host cells. Almost all antibiotics, and all that are administered orally, act by blocking some metabolic pathway of rapidly growing bacteria rather than by disrupting the bacterial cell. Therefore, the identification of new targets for antibiotic action requires knowledge of the active metabolism of replicating intracellular (IC) bacteria. Predictive computer models of the metabolism of E. coli (a close relative to Shigella flexneri) have been constructed using Constraint-based Flux Balance Analysis (CFBA). The chemical constituents of rapidly growing E. coli cells have been determined, and this information was used in building the CFBA models. However, the equivalent information for eukaryotic cells, which is basically the growth medium for IC bacteria, is not as complete, and will be investigated. Knowledge of the constituents of eukaryotic cell cytosol is required to develop CFBA models to analyze the metabolism of bacteria during their IC replicative stage. S. flexneri, a Category B pathogen, will be used as: (Specific Aim 1) a biosensor to determine constituents of the cytosol of human-derived cells and (Specific Aim 2) the model organism for this innovative application of CFBA. Several attributes of S. flexneri make it a good choice to probe the eukaryotic cytosol for bacterially accessible compounds: replication in the cytosol (no vacuolar membrane to complicate the analysis), a large repertoire of uptake systems (to assess availability to bacteria of compounds in the cytosol) and genetic tools to examine heterologous uptake systems. CFBA translates a metabolic network (described as stoichiometric bio-chemical reactions) into an optimization problem with constraints on each molecule that the organism can exchange with its environment (in this case eukaryotic cytosol). A general model (to allow adaptation to different bacteria) will be constructed from the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic database. The S. flexneri CFBA model will be derived from the reference model using published reports of the genome and the IC behavior of S. flexneri mutants. Modeling the metabolism of NIAID Priority Pathogens will aid our understanding of a large number of potential bioterror agents and help direct the search for new antibacterial drugs.