Low molecular weight inhibitors of (p)ppGpp-dependent virulence factor production by Erwinia carotovora subsp. atroseptica

胡萝卜软腐欧文氏菌亚种 (p)ppGpp 依赖性毒力因子产生的低分子量抑制剂。

• 批准号: BB/G015171/1
• 负责人: Martin Welch
• 金额: $ 42.84万
• 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: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG015171%2F1
• 关键词: Low; molecular; weight; inhibitors; ppGpp

The potato is the World's fourth most important food crop, and the UK is the 11th largest producer, with each head of the UK population consuming an average of 94 kg of potatoes each year. The UK industry is worth around £4 billion p/a. Much of the prosperity and success of the industry has been brought about through the introduction of modern farming technologies, including the development of pesticides and other chemical interventions to control various diseases. However, to date, no effective pesticides have been developed to stop tubers from rotting, either in storage or in the ground, and this gap in the agricultural arsenal cumulatively leads to crop losses running to millions of pounds per year. One of the main causative agents of soft rot is the bacterial pathogen known as Erwinia carotovora subsp. atroseptica ('Eca' for short). The aim of the current study is to develop small molecule inhibitors that prevent this organism from causing disease in potato tubers. Eca infections can be spread from field to field through infected seed tubers, and once established, can rapidly decimate a crop. The substantial economic impact caused by Eca infections has been a powerful driver behind the recent successful effort to sequence the genome of this organism. These data have provided us with a wealth of information about the mechanism(s) of pathogenesis in Eca. The current project builds on these findings and also on some earlier observations made in the investigators lab, which showed that production of the enzymes that lead to soft rot by Eca is controlled by the nutritional status of the organism. Briefly, we have shown that if the production of an intracellular 'alarmone' called ppGpp is prevented by mutating the gene (relA) encoding the enzyme which makes this metabolite, the cell no longer produces the secreted enzymes that cause soft rot in the potato tuber. In the current work, we aim to try and mimic the phenotypic effect(s) of the relA mutation by developing low molecular weight compounds that block the synthesis or reception of ppGpp. This so-called 'chemical-genetic' approach could lead to the generation of new anti-rot agents. To begin with, we will make a mutant that is completely incapable of making or degrading ppGpp. This will require us to inactivate not only the relA gene, but also a second gene called spoT, which is responsible for generating small amounts of residual ppGpp in the relA strain. Next, using this 'ppGpp null' strain, we will employ microarray analysis to identify a set of genes that are exclusively/primarily controlled by ppGpp and not by other known regulatory inputs. These genes will be cloned and fused to an easily-assayable 'reporter gene' called GFP, allowing us to generate constructs that fluoresce in response to changes in the concentration of intracellular ppGpp. In parallel, we will identify additional components involved in the ppGpp signaling pathway in Eca, and develop high through-put assays to monitor the activity of these proteins. The combined in vivo (reporter constructs) and in vitro (biochemical assays) will be used to screen a library of structurally-diverse small molecules to see if any of these block any step in ppGpp signaling. This collection of small molecules has been designed to include both drug-like and natural product-like molecules, and samples a very large portion of the total structural diversity of 'chemical space'. Subsequent rounds of chemical modification will be aimed at improving the inhibitory activity of the 'hits' obtained in these assays. Ultimately, we aim to obtain one or a few so-called 'lead compounds' with good inhibitory activity, and these will be tested further for their ability to block virulence in tuber rot models.

马铃薯是世界上第四大粮食作物，英国是第11大生产国，英国每人每年平均消费94公斤马铃薯。英国工业价值约40亿英镑P/A。该行业的繁荣和成功在很大程度上是通过引进现代农业技术实现的，包括开发杀虫剂和其他化学干预措施来控制各种疾病。然而，到目前为止，还没有开发出有效的杀虫剂来阻止块茎在储存或地下腐烂，农业武器库中的这一差距累积起来导致每年数百万磅的作物损失。软腐病的主要病原体之一是称为胡萝卜软腐欧文氏菌胡萝卜软腐欧文氏菌胡萝卜atroseptica（简称'Eca'）。目前研究的目的是开发小分子抑制剂，防止这种生物体在马铃薯块茎中引起疾病。Eca感染可以通过受感染的种薯从一个田地传播到另一个田地，并且一旦建立，可以迅速毁灭作物。Eca感染所造成的巨大经济影响是最近成功测序这种生物体基因组的强大驱动力。这些数据为我们提供了丰富的信息的发病机制（S）在ECA。目前的项目建立在这些发现的基础上，也建立在研究人员实验室早期的一些观察结果的基础上，这些观察结果表明，导致Eca软腐的酶的产生受到生物体营养状况的控制。简单地说，我们已经表明，如果通过突变编码产生这种代谢物的酶的基因（relA）来阻止称为ppGpp的细胞内“alarmone”的产生，则细胞不再产生导致马铃薯块茎软腐的分泌酶。在目前的工作中，我们的目标是通过开发阻断ppGpp合成或接收的低分子量化合物来尝试和模拟relA突变的表型效应。这种所谓的“化学遗传”方法可能会导致新型防腐剂的产生。开始，我们将制造一个完全不能制造或降解ppGpp的突变体。这将要求我们不仅克隆relA基因，还克隆第二个基因spoT，它负责在relA菌株中产生少量残留的ppGpp。接下来，使用这种“ppGpp null”菌株，我们将采用微阵列分析来鉴定一组基因，这些基因完全/主要由ppGpp控制，而不是由其他已知的调控输入控制。这些基因将被克隆并融合到一个容易分析的“报告基因”，称为GFP，使我们能够产生荧光响应细胞内ppGpp浓度变化的结构。与此同时，我们将确定参与ppGpp信号通路在Eca的其他组件，并开发高通量检测来监测这些蛋白质的活性。组合的体内（报告构建体）和体外（生物化学测定）将用于筛选结构多样性小分子的文库，以查看这些小分子中是否有任何一种阻断ppGpp信号传导中的任何步骤。这种小分子的集合被设计为包括药物样和天然产物样分子，并对“化学空间”的总结构多样性的很大一部分进行采样。随后几轮的化学修饰将旨在提高这些测定中获得的“命中”的抑制活性。最终，我们的目标是获得一种或几种具有良好抑制活性的所谓的“先导化合物”，这些化合物将进一步测试其在块茎腐烂模型中阻断毒性的能力。

项目成果

Intra-species bacterial quorum sensing studied at single cell level in a double droplet trapping system.

在双液滴诱捕系统中，在单细胞水平上研究了种类的细菌群体传感。

• DOI: 10.3390/ijms140510570
• 发表时间: 2013-05-21
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Bai Y;Patil SN;Bowden SD;Poulter S;Pan J;Salmond GP;Welch M;Huck WT;Abell C
• 通讯作者: Abell C

A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

• DOI: 10.3389/fmicb.2015.01442
• 发表时间: 2015
• 期刊: Frontiers in microbiology
• 影响因子: 5.2
• 作者: Monson R;Smith DS;Matilla MA;Roberts K;Richardson E;Drew A;Williamson N;Ramsay J;Welch M;Salmond GP
• 通讯作者: Salmond GP

Robust routes for the synthesis of N-acylated-L-homoserine lactone (AHL) quorum sensing molecules with high levels of enantiomeric purity

• DOI: 10.1016/j.tetlet.2011.04.059
• 发表时间: 2011-06-29
• 期刊: TETRAHEDRON LETTERS
• 影响因子: 1.8
• 作者: Hodgkinson, James T.;Galloway, Warren R. J. D.;Spring, David R.
• 通讯作者: Spring, David R.