Regulation of carbon flux through the glyoxylate shunt in the opportunistic pathogen, Pseudomonas aeruginosa.

在过去的二十年里，人们越来越认识到——在国家和国际层面上——我们迫切需要确定控制和管理细菌感染的新战略。由于抗生素的广泛使用和滥用，抗生素的“黄金时代”已经结束；对大多数种类抗生素的耐药性正在上升，与此同时，研发管道中出现的新抗生素越来越少。特别是迫切需要针对所谓的“革兰氏阴性”细菌的抗菌剂。这些细菌很难对付，因为它们有两层膜状层，将内部与环境分开（这层膜状层使药物难以渗透），而且它们还经常表现出几个“多药物外排泵”，顾名思义，这些外排泵在抗生素有机会发挥作用之前，将任何恰好进入细胞的抗生素排出体外。一个特别可怕的革兰氏阴性“超级细菌”是机会致病菌，铜绿假单胞菌（以下简称“PA”）。这种细菌会引起毁灭性的感染，几天内就会致人死亡。令人担忧的是，抗生素耐药性在PA种群中也很猖獗。PA在感染期间造成如此多的组织损伤的一个原因是，它有一种机制（称为“3型分泌”或T3S），允许它直接向宿主细胞分泌有毒蛋白质分子，从而杀死它们。只要注入一个毒素分子就能杀死宿主细胞，这使得它成为PA武器库中最有效的毒力因子。T3S的活性可以通过细菌和宿主细胞之间的简单物理接触来激发。然而，最近的研究也表明，当细菌感觉到氧气耗尽时，T3S也会被激活（就像许多感染部位的情况一样）。出乎意料的是，告诉细胞在缺氧情况下激活T3S的“信号”竟然是一种代谢信号，由一种叫做“乙醛酸分流”的生化途径产生。那么，什么是乙醛酸钠分流术？在像PA这样的细菌中，大多数类型的食物分子既可以用来产生能量，也可以用来产生生物质。然而，某些食品会出现问题，特别是像醋酸盐这样的简单分子或分解生成醋酸盐的分子（如脂肪酸）。正常情况下，细胞的中央代谢中枢（“TCA循环”）将每个醋酸酯分子中的2个碳原子完全氧化，产生2分子二氧化碳。红利是产生了能量。然而，这也意味着所有进入TCA循环的碳都以二氧化碳的形式损失了——没有碳可以“固定”地并入生物质。为了避免这种情况，细菌进化出了一种特殊的“分流”来绕过二氧化碳循环的进化步骤，从而“节省”了碳原子，并允许它们重新路由以产生生物量。没有乙醛酸分流，因此PA不能在许多食物上生长，并且在乙醛酸分流中有缺陷的突变体不能在感染模型中引起疾病。其原因尚不清楚，尽管T3S减少和代谢不足都可能是原因。因此，乙醛酸酯分流酶被广泛认为是开发抗菌化合物的潜在靶点。问题是，尽管在某些模式生物中已经很好地表征了乙醛酸酯分流，但PA中的TCA循环/乙醛酸酯分流分支点具有不同的结构，并且显然以非常不同的方式受到调节。事实上，尽管乙醛酸在控制适应性和毒力方面发挥着明显的作用，但对其如何通过PA中的乙醛酸分流调节通量尚不清楚。这项工作的目的是通过建立一个工作通量模型来解决这个问题，使我们能够探索通过乙醛酸酯分流器破坏代谢的最佳方法，并检查这对T3S的影响。

The last two decades have seen an increasing realisation - at both national and international level - that we urgently need to identify new strategies for controlling and managing bacterial infections. Due to widespread antibiotic use and abuse, the "golden age" of antibiotics is over; resistance to most classes of antibiotics is on the rise, and at the same time, fewer new antibiotics are emerging out of the R&D pipeline. In particular, antimicrobial agents that target the so-called "Gram-negative" bacteria are desperately needed. These bacteria are hard to fight because they have TWO membrane-like layers separating their interior from the environment (this double layer makes drug penetration difficult) and they also often express several "multi-drug efflux pumps" which, as their name suggests, export any antibiotics that do happen to get into the cell before they have a chance to have any effect. One particularly dreaded Gram-negative "superbug" is the opportunistic pathogen, Pseudomonas aeruginosa (hereafter, "PA"). This bacterium causes devastating infections that can kill in a matter of days. Worryingly, antibiotic resistance is also rampant in PA populations. One reason why PA causes so much tissue damage during infections is because it has a mechanism (called "Type 3 Secretion", or T3S) that allows it to secrete toxic protein molecules directly into host cells, thereby killing them. As little as a single molecule of injected toxin is all that is required to kill the host cell, making this the most potent virulence factor in the PA arsenal. T3S activity can be stimulated by simple physical contact between the bacterium and the host cell. However, recent work has also shown that T3S is also turned on when the bacterium senses that it is running out of oxygen (as is also the case at the site of many infections). Unexpectedly, the "signal" telling the cell to activate T3S in the absence of oxygen turned out to be a metabolic one, generated by a biochemical pathway called "the glyoxylate shunt". So, what is the glyoxylate shunt? In bacteria such as PA, most types of food molecule can be used to either generate energy or to generate biomass. However, a problem arises with certain foodstuffs - especially simple molecules like acetate or molecules which are broken down to yield acetate (e.g., fatty acids). Normally, the central metabolic hub of the cell (the "TCA cycle") takes the 2 carbon atoms in each acetate molecule and fully oxidizes these to yield 2 molecules of carbon dioxide. The dividend is that energy is produced. However, it also means that all the carbon that goes in to the TCA cycle is lost as CO2 - no carbon can become "fixed" for incorporation into biomass. To circumvent this, bacteria have evolved a special "shunt" to bypass the CO2 evolving steps of the cycle, thereby "saving" carbon atoms and allowing these to be re-routed to generate biomass. Without the glyoxylate shunt, PA therefore fails to grow on many foodstuffs, and mutants defective in the glyoxylate shunt are unable to cause disease in infection models. The reasons for this are still not clear, although diminished T3S and metabolic insufficiency are both probable contributors. Consequently, the enzymes of the glyoxylate shunt are widely accepted as potential targets for the development of antimicrobial compounds. The problem is that although the glyoxylate shunt has been well-characterized in certain model organisms, the TCA cycle/glyoxylate shunt branchpoint in PA has a different architecture and is clearly regulated in a very different manner. Indeed, nothing is known about how flux is regulated through the glyoxylate shunt in PA, in spite of its obvious role in controlling fitness and virulence. The aim of the proposed work is redress this issue by generating a working flux model, allowing us to explore the best ways(s) of disrupting metabolism through the glyoxylate shunt, and to examine the impact of this on T3S.