Metabolic Modeling of Invasive Bacteria and HeLa Cytosol

入侵细菌和 HeLa 细胞溶质的代谢模型

• 批准号: 6809359
• 负责人: Anthony T Maurelli
• 金额: $ 22.46万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6809359
• 关键词: Escherichia coli; HeLa cells; Shigella; bacteria infection mechanism; bacterial genetics; biosensor device; cell line; cytoplasm; disease /disorder model; epithelium; flow cytometry; gene expression; genetic library; genome; host organism interaction; intracellular; mathematical model; microorganism culture; microorganism metabolism; microorganism reproduction; model design /development; mutant

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.

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都会侵入人类细胞，并且对于许多细菌来说，唯一已知的感染生殖阶段发生在膜结合液泡中或直接发生在这些宿主细胞的胞质中。几乎所有抗生素以及所有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破坏细菌细胞来发挥作用。因此，识别抗生素作用的新靶点需要了解复制细胞内（IC）细菌的活跃代谢。使用基于约束的通量平衡分析 (CFBA) 构建了大肠杆菌（福氏志贺氏菌的近亲）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被确定，并且该信息用于构建 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。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。 S. flexneri 是一种 B 类病原体，将用作：（具体目标 1）确定人源细胞胞浆成分的生物传感器，以及（具体目标 2）CFBA 创新应用的模型生物体。弗氏链球菌的几个属性使其成为探测真核细胞质中细菌可接触化合物的良好选择：在细胞质中复制（没有液泡膜使分析变得复杂），大量的摄取系统（以评估细菌对化合物的可用性）细胞质）和遗传工具来检查异源摄取系统。 CFBA 将代谢网络（描述为化学计量生化反应）转化为优化问题，并对生物体与其环境（在本例中为真核细胞质）交换的每个分子进行限制。通用模型（以适应不同的细菌）将根据京都基因和基因组百科全书（KEGG）代谢数据库构建。 S.flexneri CFBA 模型将从使用已发表的 S.flexneri 突变体的基因组和 IC 行为报告的参考模型衍生而来。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物质，并有助于指导新抗菌药物的搜索。