Final Report Summary - ANT (Mining for new antimicrobials in ant associated actinomycetes) 1. Publishable SummaryThe quest for new antifungal and antibacterial compounds is urgent. Infectious diseases are a leading cause of death worldwide. In Europe, death rates from infectious diseases have reduced over the past century, but in the UK infectious diseases still account for over 10% of deaths1. Over the last decade this figure has doubled due to the emergence of multi-drug resistant strains. Invasive fungal infections are also increasing in frequency in all developed countries, and are often associated with otherwise successful surgical procedures. In 2013 a Nature editorial expounded the dangers posed by fungi2. Historically fungi are the word’s biggest killers having been responsible for over 70% of extinctions3. Fungi pose a grave threat to all five major crop staples, and there has been a tenfold increase in reports of fungal infections in animals as well as plants, worldwide, over the past 20 years. It has been suggested that this may be attributed to climate change3. In 2002, almost 10,000 patients in England were estimated to have acquired deep-organ fungal infections during otherwise successful treatment for a serious illness; nearly half of these died from their fungal disease4. Superficial fungal infections such as athlete’s foot and vaginal thrush are prevalent throughout the population and systemic fungal infections are becoming a serious complication for immunocompromised patients. New natural products are likely to provide a solution. A significant number of medicines in clinical use are derived from natural products. A vast proportion of these compounds are produced by soil-dwelling actinomycete bacteria. New, untapped ecological niches are likely to be the habitat for unique actinomycetes which have evolved to produce new palette of natural products, these provide a useful starting point for natural product discovery. In addition, over the past several years, the increased ease of genome scanning has revealed that common actinomycete genomes on average encode pathways for the production of 20 natural, with over 50% of these still undiscovered5. Many of these “cryptic” pathways encoding novel natural products need to be switched on (or unlocked) to force the organism to make their encoded secondary metabolites.The leaf-cutting ants can form a mutualism with Actinobacteria that supply antibiotics as weedkillers to defend their fungus gardens against infections by microbial pathogens, most notably fungal pathogens. This provides a potentially useful platform for searching for new antifungal compounds. Studies have shown that it is possible to isolate a variety of actinomycetes from the integument of leaf-cutting ants and that these microbes possess anti-microbial activity, especially anti-fungal activity. However, the identities of the antifungals produced by ant-associated actinomycetes remain largely unknown. Only several compounds have been identified so far including candicidin, nystatin-like compound6 and antimycin7. References:1. Health Protection Agency (2005) Health Protection in the 21st Century – Understanding the Burden of Disease; Preparing for the future, Part 4 Infectious diseases, available at: http://www.hpa.org.uk/publications/2005/burden_disease/.2. Jones N. Planetary disasters: It could happen one night. Nature, 2013, 493(7431): 154-156.3. Fisher MC, Henk DA, Briggs CJ, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature, 2012, 484(7393): 186-194.4. Health Protection Agency (2006) Fungal Diseases in the UK, available at: http://www.hpa.org.uk/webc/hpawebfile/hpaweb_c/1196942156347.5. Van Lanen SG and Shen B. Microbial genomics for the improvement of natural product discovery. Curr. Opin. Microbiol., 2006, 9(3): 252-260.6. Barke J, Seipke RF, Gruschow S, et al. A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus. BMC Biol., 2010, 8: 109.7. Seipke RF, Barke J, Brearley C, et al. A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus. PLoS One, 2011, 6(8): e22028.2. Project Objectives for the PeriodObjectives of this project are: 1) Gaining greater insight into the antifungal compounds produced in the ant-associated actinomycetes;2) Discovery of antifungal compounds by combined genome and bioactivity guided approach;3) Studying the synergistic effects of the isolated compounds with a combination of other natural products;4) Engineering the strains of compounds of particular interest to enable enhanced levels of compound production.Summary of Work Carried out and Results AcheivedTo achieve the objectives, we employed medium- to high-throughput screening techniques to explore the capability of the ant associated actinomycetes of producing antifungal compounds. By using bioassay guided fractionation, two classes of compounds with excellent anti-fungal activity were discovered in one of the Streptomyces strains. This is the first time that these antifungal compounds have been found in the ant/fungal/bacterial mutualism. These compounds have been fully chemically characterized.By genome mining, we found the potential gene clusters for the biosynthesis of these two families of compounds. We subsequently investigated the impacts of key genes in the clusters on the production of these antibiotics in order to confirm the biosynthetic pathway, increase the productivity, and explore the regulation of metabolite production within the organism. The activity of the combination of the two purified compounds against Candida and E. coli is currently being investigated to determine if there are any synergistic or antagonistic effects between the two compounds.ImpactFew compounds have been discovered so far from the ant/fungal/bacterial mutualisms; we are preparing to submit manuscripts reporting two further compounds that we have observed, for the first time, from this system. This brings about interesting ecological questions about the synergistic use of series of antifungal compounds in these systems. The potential for clinically applying similar synergistic antifungal treatments presents itself and we are carrying out further studies to gain better understanding of the way in which these combinations of compounds work. These studies could have impact on human health. The biogenesis of one of the antifungal compounds that we have isolated so far includes a series of unique enzyme catalysed reactions which we are further investigating. Additionally, this project is opening a further potential treasure trove of compounds. Sequencing studies that we have carried out on one of the ant associated microbes reveal a further 20 candidate biosynthetic clusters; analysis of these suggests that a number could potentially encode for the generation of previously unknown natural products; a cosmid library has been generated in order to work toward unlocking these cryptic clusters and establishing production of these compounds which could be of value to human health.