Identification of nitrogen source and metabolism of Mycobacterium tuberculosis during intracellular replication.

结核分枝杆菌（Mtb）是结核病的病原，目前是全世界造成死亡的最具破坏性的传染性病原体，每年造成880万例结核病病例，导致140万人死亡。与人类免疫缺陷病毒（艾滋病毒）的合并感染，以及多重和广泛耐药（MDR和XDR）结核菌株的出现，使结核分枝杆菌再次成为世界各地的主要公共卫生威胁。具有抗结核活性的现有药物数量有限，以及根除感染所需的长期多药治疗方案是结核病治疗的根本问题。迫切需要抗结核分枝杆菌的新药。结核分枝杆菌细胞内代谢是开发新型抗结核药物的一个有吸引力的靶点；然而，大多数研究都集中在碳代谢上。氮也是结核分枝杆菌必需的营养物质，但很少有研究试图阐明病原菌在宿主体内生长时氮源的性质等基本问题。我们以前的研究已经确定氨基酸是Mtb在宿主细胞内生长时的假定氮来源。在这个项目中要研究的假设是Mtb从包括氨基酸在内的多种细胞内营养物质中获得氮。为了解决这些基本问题，我们将首先对结核分枝杆菌进行遗传分析，以确定参与吸收和同化一系列可能的氮源（如氨基酸）的基因。然后，我们将使结核分枝杆菌中的这些基因失活，并测量突变菌株（例如，缺乏摄取特定氨基酸的能力）在人类细胞中复制的能力。通过检查在摄取特定营养物质方面有特定缺陷的一系列突变体的细胞内复制效率，我们将确定哪些氮营养物质对细胞内复制是重要的。下一个问题是病原体如何吸收氮。为了解决这个问题，我们将直接分析用于吸收氮的氮代谢途径，使用我们在萨里大学率先采用的基于尖端系统的代谢组学技术。这将涉及开发全新的系统来同时测量病原体的氮代谢和碳代谢。最后，我们将建立一个Mtb氮碳代谢的计算机模型。该模型将用于整合项目中生成的数据，还将用于生成能够检验支撑项目的假设的预测。通过这种方式，我们将阐明氮代谢在这一重要病原体的生物学和毒力中的作用。这项研究还可能为开发抗结核药物提供新的靶点。

M. tuberculosis (Mtb), the etiological agent of TB, is presently the most devastating infectious agent of mortality worldwide, responsible for 8.8 million cases of TB each year resulting in 1.4 million deaths. Co-infection with the human immunodeficiency virus (HIV), along with the emergence of multi- and extensively- drug resistant (MDR and XDR) strains of TB, has reaffirmed Mtb as a primary public health threat throughout the world. The limited number of drugs available that have activity against Mtb, and the prolonged multi-drug regimen needed to eradicate the infection, are the fundamental problems of TB treatment. New drugs active against Mtb are urgently needed. Intracellular metabolism of Mtb is an attractive target for development of novel anti-tuberculosis drugs; however most studies have focussed on carbon metabolism. Nitrogen is also an essential nutrient of Mtb but few studies have attempted to elucidate fundamental questions such as the nature of the nitrogen source of the pathogen when it grows inside the host. Our previous studies have identified amino acids as putative sources of nitrogen for Mtb when growing inside host cells. The hypothesis to be investigated in this project is that Mtb obtains nitrogen from a diverse range of intracellular nutrients including amino acids.To address these fundamental questions we will first perform genetic analysis of Mtb to identify genes involved in uptake and assimilation of a range of possible nitrogen sources, such as amino acids. We will then inactivate these genes in Mtb and measure the ability of the mutated strains (lacking the ability, for example, to uptake a particular amino acid) to replicate in human cells. By examining the intracellular replication efficiency of a range of mutants that have specific defects in uptake of specific nutrients we will identify which nitrogen nutrients are important for intracellular replication. The next question is how the pathogen assimilates nitrogen. To tackle this question we in parallel we will directly analyse the nitrogen metabolic pathways used to assimilate nitrogen using cutting-edge systems-based metabolomics techniques that we have spearheaded at the University of Surrey. This will involve development of entirely novel systems to measure the nitrogen metabolism of the pathogen simultaneously with its carbon metabolism. Finally, we will develop a computer model of nitrogen and carbon metabolism in Mtb. The model will be used both to integrate data generated in the project but also to generate predictions that can test the hypothesis underpinning the project. In this way we will elucidate the role of nitrogen metabolism in the biology and virulence of this important pathogen. The study is also likely to generate novel targets for development of antituberculous drugs.