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

通过机会性病原体铜绿假单胞菌中的乙醛酸分流调节碳通量。

• 批准号: BB/M019411/1
• 负责人: Martin Welch
• 金额: $ 43.83万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM019411%2F1
• 关键词: Regulation; carbon; flux; through; glyoxylate

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.

过去二十年，人们越来越认识到——无论是在国家还是国际层面——我们迫切需要确定控制和管理细菌感染的新策略。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；对大多数类别抗生素的耐药性正在上升，与此同时，研发管线中出现的新抗生素越来越少。特别是，迫切需要针对所谓“革兰氏阴性”细菌的抗菌剂。这些细菌很难对抗，因为它们有两层膜状层将其内部与环境分开（这种双层使药物渗透变得困难），而且它们还经常表达几个“多药物外排泵”，顾名思义，这些泵在有机会产生任何效果之前将碰巧进入细胞的任何抗生素排出体外。一种特别可怕的革兰氏阴性“超级细菌”是机会性病原体，铜绿假单胞菌（以下简称“PA”）。这种细菌会引起毁灭性的感染，几天之内就会导致死亡。令人担忧的是，抗生素耐药性在 PA 人群中也很猖獗。 PA 在感染过程中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），使其能够将有毒蛋白质分子直接分泌到宿主细胞中，从而杀死它们。只需注入一个单分子毒素即可杀死宿主细胞，这使其成为 PA 武器库中最有效的毒力因子。 T3S 活性可以通过细菌和宿主细胞之间简单的物理接触来刺激。然而，最近的研究还表明，当细菌感觉到氧气耗尽时（许多感染部位的情况也是如此），T3S 也会被打开。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，由一种名为“乙醛酸分流”的生化途径产生。那么，什么是乙醛酸分流？在 PA 等细菌中，大多数类型的食物分子都可用于产生能量或产生生物质。然而，某些食品会出现问题，尤其是简单的分子，如乙酸盐或分解产生乙酸盐的分子（例如脂肪酸）。通常，细胞的中央代谢中心（“TCA 循环”）会吸收每个乙酸盐分子中的 2 个碳原子，并将其完全氧化，产生 2 个二氧化碳分子。红利是产生能量。然而，这也意味着进入 TCA 循环的所有碳都会以二氧化碳的形式损失——没有碳可以“固定”以纳入生物质中。为了规避这一点，细菌进化出了一种特殊的“分流器”来绕过循环中二氧化碳的释放步骤，从而“节省”碳原子并允许它们重新路由以产生生物质。如果没有乙醛酸分流，PA 就无法在许多食品上生长，并且乙醛酸分流缺陷的突变体无法在感染模型中引起疾病​​。尽管 T3S 减少和代谢不足都是可能的原因，但其原因仍不清楚。因此，乙醛酸分流酶被广泛认为是开发抗菌化合物的潜在靶标。问题在于，尽管乙醛酸分流在某些模式生物中已得到很好的表征，但 PA 中的 TCA 循环/乙醛酸分流分支点具有不同的结构，并且明显以非常不同的方式进行调节。事实上，尽管 PA 中的乙醛酸分流在控制适应性和毒力方面发挥着明显作用，但我们对如何通过 PA 中的乙醛酸分流来调节通量一无所知。拟议工作的目的是通过生成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分流破坏新陈代谢的最佳方法，并检查这对 T3S 的影响。

项目成果

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa.

• DOI: 10.3389/fmicb.2021.790742
• 发表时间: 2021
• 期刊: Frontiers in microbiology
• 影响因子: 5.2
• 作者: Abdelhamid Y;Wang M;Parkhill SL;Brear P;Chee X;Rahman T;Welch M
• 通讯作者: Welch M

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

• DOI: 10.1021/acs.jcim.2c00300
• 发表时间: 2022-05-23
• 期刊: JOURNAL OF CHEMICAL INFORMATION AND MODELING
• 影响因子: 5.6
• 作者: Wezen, Xavier Chee;Chandran, Aneesh;Eapen, Rohan Sakariah;Waters, Elaine;Bricio-Moreno, Laura;Tosi, Tommaso;Dolan, Stephen;Millership, Charlotte;Kadioglu, Aras;Grundling, Angelika;Itzhaki, Laura S.;Welch, Martin;Rahman, Taufiq
• 通讯作者: Rahman, Taufiq