Metabolic Modeling of Invasive Bacteria and HeLa Cytosol

描述（由申请人提供）：NIAID优先病原体列表中所有三个类别的大多数细菌侵入人体细胞，并且对于许多细菌而言，唯一已知的感染生殖阶段发生在膜结合空泡中或直接发生在这些宿主细胞的胞质溶胶中。几乎所有的抗生素，以及所有口服的抗生素，都是通过阻断快速生长的细菌的某些代谢途径而不是破坏细菌细胞来起作用的。因此，鉴定抗生素作用的新靶点需要了解复制细胞内（IC）细菌的活性代谢。E.代谢的预测计算机模型。大肠杆菌（与福氏志贺菌（Shigella flexneri）有密切关系）已经使用基于约束的通量平衡分析（CFBA）构建。本文报道了快速生长的E.大肠杆菌细胞，并将这些信息用于建立CFBA模型。然而，真核细胞（基本上是IC细菌的生长培养基）的等效信息并不完整，将进行研究。真核细胞胞质溶胶的成分的知识是需要开发CFBA模型来分析细菌在IC复制阶段的代谢。S.福氏杆菌，一种B类病原体，将被用作：（具体目标1）生物传感器，以确定人源细胞的胞质溶胶的成分和（具体目标2）CFBA的这种创新应用的模式生物。S. Flexneri使其成为探测真核细胞胞质溶胶中细菌可接近化合物的良好选择：胞质溶胶中的复制（没有使分析复杂化的空泡膜）、大量摄取系统（以评估胞质溶胶中化合物对细菌的可用性）和检查异源摄取系统的遗传工具。CFBA将代谢网络（描述为化学计量的生物化学反应）转化为优化问题，该优化问题对生物体可以与其环境（在这种情况下是真核细胞质溶胶）交换的每个分子具有约束。将根据京都基因和基因组百科全书（KEGG）代谢数据库构建通用模型（以适应不同的细菌）。色葡萄flexneri CFBA模型将使用已发表的S.弗氏突变体。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剂，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.