Developing a pipeline for antibiotic discovery – Ruth Airs – Plymouth Marine Laboratory
Project partners – Michiel Vos (University of Exeter)
Marine macroalgae exhibit stunning diversity, both in morphological form and the biochemical diversity of their metabolites. Seaweeds have been challenged throughout their evolution by microorganisms and have developed in a world of microbes. A large number of studies have demonstrated that extracts of many seaweed species are able to inhibit bacteria. Seaweeds are continually exposed to a large variety of potentially harmful microorganisms present in seawater but lack cell-based immune responses. It has therefore been hypothesized that they commonly exhibit strong antimicrobial activity to prevent fouling and disease. Although seaweeds offer a vast source of metabolic diversity, the discovery of novel antimicrobials can be laborious and time consuming, with de-replication necessary to avoid continual re-isolation of compounds that are already known. Using a well-characterized gram positive bacterium we have developed a high-resolution screen for antimicrobial activity of seaweed extracts based on statistical analysis of quantitative patterns of inhibition. A previous screen of a wide range of seaweeds, identified two species, Asparagopsis armata and Furcellaria lumbricalis, with promising antimicrobial activity. In order to characterize the biochemical interactions between
seaweed metabolites and bacteria, we here use an untargeted metabolomics approach to fingerprint metabolites. Using explicit comparison of closely related metabolomes with different or no antimicrobial activity, we will focus in on active compounds. Generation of non-active control extracts, chemically very similar to the active extract, is essential to the success of the metabolomics approach, as the chemical diversity of extracts is very high. Explicitly comparing related metabolic signatures with distinct antimicrobial spectra using a metabolomics MS pipeline greatly facilitates the discovery of active compounds, with novel modes of action. Compounds will be isolated and their interaction with bacterial cells characterised. The compounds may have application in several arenas, including agrochemical, animal health and cosmetics industries as well as in pharmaceuticals.
The discovery and characterisation of natural product derived small molecule compounds as novel anti‐leishmanials – Paul William Denny – Durham University
Project partners – Liam Evans (Hypha Discovery)
Single-celled protozoan parasites are a major cause of disease in humans and domestic animals. Insect vector borne protozoa of the Leishmania species cause leishmaniasis, a disease endemic in >80 countries – developing, industrialised and transitional. Leishmaniasis, which affects >12M people (350M at risk), is a zoonotic disease with multiple animal reservoirs. In addition to complicating infection control, this reservoir is a major veterinary concern with cats, horses and, most prominently, dogs affected. In common with human disease the treatment of animal infection is challenging, with a complete lack of well tolerated, easily administered, fully efficacious therapies or vaccines. A large market exists for veterinary antileishmanials, particularly in southern Europe and the Americas, to combat companion animal disease. Effective alternatives to the human drugs currently used to control, rather than cure, canine leishmaniaisis are urgently required to both improve treatment and to avoid the antimicrobial resistance arising in reservoir species being transferred to humans. With the almost complete failure of target-based drug screening and rational drug design to reveal new lead antiparasitics, attention has turned once again to the natural world – the original source of most of the currently used antimicrobials, including the 2015 Nobel prize winning antiparasitics (www.nobelprize.org). Funded by a BBSRC Impact Award (2013-14) Durham University and Hypha Discovery Ltd have, using Durham-based technology, conducted a preliminary screen of a library of fungal metabolites selected for their pharmacologically tractable nature. Through an iterative process of screening followed by the fractionation of hits to ever purer samples, we have identified a number of novel molecules with antiparasitic properties against Leishmania. In this Proof of Concept proposal we will determine the structural identity of these hits and, following triage for efficacy and chemical profile, evaluate them towards in vivo testing. This will be achieved by the development and use of a robust Leishmania infected cell system in a natural product screening programme, coupled with host cytotoxicity screening, and supported by genomic and bioinformatic approaches to identify the mode of action of the most promising of the antiparasitic natural compounds. At the conclusion of the project we will have identified novel, natural compound antiparasitic leads with the potential to be taken forward for development.
The role of Pseudomonas secondary metabolism in potato scab biocontrol – Jacob Malone – John Innes Centre
Project partners – Andrew Truman (JIC), Graham Tomalin (VCS Potatoes Ltd)
Potato scab is an important crop disease that affects potato quality and presents a significant economic burden to UK agriculture. Streptomyces scabies, the pathogenic bacterium that is the causal organism of scab, is ubiquitous and presents a threat in almost all soils. While scab can be controlled by properly managed irrigation, outbreaks still regularly occur in irrigated soil and alternative approaches to scab suppression are clearly required. One such alternative involves the exploitation of biocontrol agents: soil microorganisms that suppress or kill plant pathogens. Many soil-dwelling Pseudomonas species form beneficial relationships with plants, exhibiting potent antagonistic behaviour towards pathogenic microorganisms and boosting plant nutrition and health. Pseudomonas have been identified as the primary biocontrol organisms in numerous plant-microbe systems, suppressing pathogens with an array of secreted natural products (NPs) including siderophores and antibiotics. Furthermore, Pseudomonas are associated with potato scab suppression, with significantly increased Pseudomonas levels observed in irrigated fields, correlating with reduced levels of potato scab. In collaboration with VCS Potatoes, our analysis of S. scabies suppression by Pseudomonas has uncovered strong correlations between the ability of Pseudomonas isolates to suppress S. scabies growth, and the presence of multiple different (both characterised and novel) NP loci. This suggests that the production and secretion of these molecules is a common factor in Pseudomonas scab suppression. With this proposal, we intend to more fully define the relationship between S. scabies suppression and NP production. We will use a combination of molecular and environmental microbiology techniques to examine the effects of NP gene deletions on S. scabies suppression in laboratory assays, and virulence bioassays for plant infection. In parallel, novel NP molecules will be purified and their effects on S. scabies examined in the laboratory and in planta. Bioinformatic analysis of an existing library of soil Pseudomonas genomes will examine the relationship between NP gene clusters and S. scabies suppression, and the impact of irrigation on the distribution of these genes in the soil Pseudomonas population. This project will determine the importance of Pseudomonas natural products in the suppression of an economically important crop disease, and provide a strong basis for future work in this field.
Translational control of antibiotic production in an industrially important streptomycete – Colin P Smith – University of Brighton
Project partners – GlaxoSmithKline
The purpose of this proof of concept application is to establish whether translational control plays an important role in the production of antibiotics in an industrially important bacterium. This proposal builds on our recent exciting discovery that translational control plays a central role in the activation of antibiotic production by the model streptomycete, Streptomyces coelicolor. We will exploit the cutting edge technique of ribosome profiling to establish globally the translational efficiency of genes at different stages of growth of the industrial strain. This technique enables the accurate quantification of RNA molecules that are bound to ribosomes ‐ and are therefore being translated at that time point; this data can then be compared directly with more conventional transcriptome data (from RNA-seq) to provide a picture of the relative contribution of transcription and translation to the expression of each gene. This proposal is innovative in applying ribosome profiling to investigate antibiotic production and will involve close collaboration with GSK, who will provide know‐how for the cultivation of industrial strains and quantification of key secondary metabolites. Critically, they will also provide the two streptomycete strains that will be subjected to detailed analysis: the wild-type and an intermediate GSK production strain that exhibits similar growth properties while producing enhanced levels of secondary metabolites. Global transcription (transcriptome) and translation (translatome) will be monitored at different growth stages to establish whether translational control plays a role in production of its antibiotics and whether the production strain is altered in translational efficiency of key genes. If this POC study does serve to highlight translational control of key antibiotic production and/or regulatory genes then this will inform the development of a novel approach to engineering production of antibiotics; follow-on funding would be sought from the BBSRC/industry to identify the molecular players that govern this control. It is likely that this work will ultimately lead to novel generic approaches for the ‘translational engineering’ of actinomycetes in order to enhance production of established products, or to facilitate detection of ‘cryptic’ or ‘elusive’ products.