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

土豆是世界第四大粮食作物，英国是第11大生产国，英国人均每年消费94公斤土豆。英国的农业产值约为40亿英镑/年。该产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，而农业武器库的这种缺口累积起来每年导致作物损失高达数百万英镑。软腐病的主要病原菌之一是被称为胡萝卜欧文氏亚种的细菌。阿托化症(简称ECA)。目前这项研究的目的是开发小分子抑制剂，以防止这种微生物在马铃薯块茎中致病。ECA感染可以通过受感染的种子块茎在田地之间传播，一旦确诊，就可以迅速摧毁作物。Eca感染造成的重大经济影响是最近成功地对这种生物的基因组进行测序的强大推动力。这些数据为我们了解食管瘤的发病机制(S)提供了丰富的信息。目前的项目建立在这些发现的基础上，也建立在调查人员实验室早先的一些观察结果的基础上，这些观察表明，导致ECA软腐的酶的产生受有机体的营养状态控制。简而言之，我们已经证明，如果通过突变编码产生这种代谢物的酶的基因(RelA)来阻止细胞内名为ppGpp的“警报”的产生，细胞就不再产生导致马铃薯块茎软腐的分泌酶。在目前的工作中，我们的目标是试图通过开发阻止ppGpp合成或接收的低分子化合物来模拟relA突变的表型效应(S)。这种所谓的“化学遗传”方法可能导致新的防腐剂的产生。首先，我们将制造一个完全不能制造或降解ppGpp的突变体。这不仅需要我们灭活relA基因，还需要灭活第二个基因Spot，它负责在relA菌株中产生少量残留的ppGpp。接下来，使用这个‘ppGpp空’菌株，我们将使用微阵列分析来鉴定一组由ppGpp独占/主要控制而不是由其他已知调控输入控制的基因。这些基因将被克隆并与一种名为GFP的易于检测的报告基因融合，使我们能够产生对细胞内ppGpp浓度变化做出反应的荧光构建体。同时，我们将确定ECA中ppGpp信号通路中涉及的其他成分，并开发高通量分析方法来监测这些蛋白质的活性。体内(记者构建)和体外(生化分析)相结合的方法将被用来筛选结构多样化的小分子文库，看看其中是否有任何一种阻止ppGpp信号的任何步骤。这组小分子被设计成既包括类药物分子，也包括天然产物类分子，并在“化学空间”的总结构多样性中采样了非常大的一部分。随后几轮的化学修饰将旨在提高在这些检测中获得的“HITS”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“先导化合物”，并将进一步测试它们在块茎腐烂模型中阻止毒力的能力。

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.