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类的大多数细菌侵入人类细胞，并且对许多已知的唯一繁殖阶段的感染发生在膜结合液泡中或直接在这些宿主细胞的细胞质中。几乎所有的抗生素，以及所有的口服抗生素，都是通过阻断快速生长的细菌的某些代谢途径而不是通过破坏细菌细胞来起作用的。因此，确定抗生素作用的新靶点需要了解细胞内复制（IC）细菌的活性代谢。利用基于约束的通量平衡分析（CFBA）建立了大肠杆菌（与福氏志贺氏菌密切相关）代谢的预测计算机模型。快速生长的大肠杆菌细胞的化学成分已经被确定，这些信息被用于构建CFBA模型。然而，作为IC细菌生长介质的真核细胞的等效信息并不完整，有待进一步研究。要建立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。flexneri是一种B类病原体，将被用作：（Specific Aim 1）测定人源性细胞细胞质成分的生物传感器和（Specific Aim 2） CFBA创新应用的模式生物。flexneri的一些特性使其成为探索真核细胞质中细菌可接近的化合物的一个很好的选择：细胞质中的复制（没有使分析复杂化的空泡膜），大量的摄取系统（评估细胞质中化合物对细菌的可用性）和检测异源摄取系统的遗传工具。CFBA将代谢网络（描述为化学计量生物化学反应）转化为一个优化问题，该问题对生物体与环境（在本例中为真核细胞质）交换的每个分子都有限制。将从京都基因与基因组百科全书（KEGG）代谢数据库中构建一个通用模型（允许适应不同的细菌）。flexneri CFBA模型将基于参考模型，利用已发表的基因组报告和flexneri突变体的IC行为。模拟NIAID优先病原体的代谢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.