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

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.

The phytopathogen, Erwinia carotovora subsp. atroseptica (Eca) is the causative agent of soft-rot in potatoes. This soft rot is a major economic problem, and has a significant impact on the nearly billion-pound UK potato industry. There are currently no effective pesticides that specifically target Eca soft rot, and the current proposal aims to try and address/fill this need. We have previously shown that a relA mutant of Eca is substantially impaired in the production of PCWDEs, and that this effect is dominant to the other key regulatory input known to affect virulence, quorum sensing (QS). In this study, we will take these findings further by generating/characterizing a ppGpp null mutant (i.e., a relA spoT double mutant). Comparative microarray analysis will be used to define, on a global level, which genes are uniquely controlled by ppGpp, and which are affected by other regulatory inputs that operate during transition to the stationary phase. Reporter constructs will be made in these genes, and the resulting strains will be used to screen libraries of structurally-diverse drug-like and natural product-like molecules. We will also use biochemical approaches to identify novel binding partners for SpoT and RelA, and investigate whether ppGpp has additional (i.e., non-RNAP) binding partners in the cell. These biochemical approaches will be enabled by the generation of high affinity monoclonal antibodies against SpoT and RelA. A selection of high through-put in vitro assays will be developed that allow the effect(s) of small molecules on individual steps in the ppGpp production/reception/signal transduction pathway(s) to be measured. Hits obtained in these screens will be improved in subsequent rounds of focused chemical modification, ultimately leading (we anticipate) to one or a few lead compounds that offer potential as anti-rot agents. These leads will be characterized in detail to determine whether they have any potential as anti-soft rot agents.