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.

在过去的二十年中，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策略。由于广泛的抗生素使用和滥用，抗生素的“黄金时代”已经结束。对大多数类别的抗生素的耐药性正在上升，同时，从研发管道中出现了更少的新抗生素。尤其是针对所谓的“革兰氏阴性”细菌的抗菌剂。这些细菌很难战斗，因为它们具有两个类似膜的层，它们将其内部与环境分开（这种双层使药物穿透很困难），而且它们也经常表达几种“多药外排泵”，顾名思义，这些抗生素会导出任何确实会遇到任何效果的抗生素。一个特别令人恐惧的革兰氏阴性“超级咬合”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，可能会在几天内杀死。令人担忧的是，PA种群中的抗生素耐药性也猖ramp。 PA在感染过程中造成如此多的组织损伤的原因之一是因为它具有一种机制（称为“ 3型分泌”或T3S），该机制使其可以将有毒蛋白质分子直接分泌到宿主细胞中，从而杀死它们。杀死宿主细胞所需的只需要单个注射毒素的分子，这使其成为PA砷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的简单物理接触来刺激。但是，最近的工作还表明，当细菌感知其氧气不足时，T3S也被打开（在许多感染部位也是如此）。出乎意料的是，“信号”告诉细胞在没有氧气的情况下激活T3s是一种代谢途径，该代谢途径被称为“甘酰基分流器”。那么，什么是甘氧基分流器？在细菌（例如PA）中，大多数类型的食物分子可用于产生能量或产生生物量。然而，某些食物的问题 - 尤其是简单的分子，例如乙酸盐或分子，这些分子被分解以产生醋酸盐（例如脂肪酸）。通常，细胞的中央代谢枢纽（“ TCA循环”）在每个乙酸盐分子中取2个碳原子，并将其完全氧化以产生2个分子的二氧化碳。股息是产生能量。但是，这也意味着所有进入TCA周期的碳都会丢失，因为CO2 - 没有碳可以“固定”以掺入生物质中。为了解决这个问题，细菌已经进化出一种特殊的“分流”，以绕过二氧化碳的发展步骤，从而“保存”碳原子，并允许将它们重新铺设以产生生物质。因此，如果没有乙二醇分流，PA就无法在许多食物上生长，而在乙二醇分流中有缺陷的突变体在感染模型中无法引起疾病。尽管T3S和代谢功能不全均可能是可能的贡献者，但原因仍然不清楚。因此，乙二基分流的酶被广泛接受为抗菌化合物发展的潜在靶标。问题在于，尽管在某些模型生物体中已充分表征乙二醇分流，但PA中的TCA循环/甘酰胺分流点具有不同的结构，并且以截然不同的方式进行了调节。实际上，尽管如何通过PA中的甘酰胺分流器调节通量，尽管它在控制适应性和毒力中具有明显的作用，但尚无了解。拟议工作的目的是通过产生工作通量模型来纠正这个问题，从而使我们能够通过乙氧基分流器探索破坏新陈代谢的最佳方法，并研究该问题对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