SYBARIS is a systems biology study of the specificity of response of the cell-mediated immune system to fungal microorganisms in order to investigate the genetic basis of susceptibility to fungal disease and elucidate molecular mechanisms of drug resistance in fungal pathogens. An integrative approach, combining high-throughput and traditional wet-lab work with computational and bioinformatics methods, will be applied to identify biomarkers of resistance to currently available treatments and to develop novel putative drug target genes and pathways in different fungi. We will use Saccharomyces cerevisiae, a normally non-pathogenic yeast model organism, Candida albicans and Aspergillus fumigatus, two major recognized fungal pathogens as well as other Aspergillus spp. known to be multi-drug resistant and difficult to treat. This project meets the criteria of the call, the strategic objective of which is to confront the increasing emergence and spread of antimicrobial drug resistant pathogens in Europe by addressing a well-defined class of infectious disease caused by fungal pathogens, with significant morbidity and mortality in a large segment of the population, and a high economic cost due to resistance. The anticipated results are highly relevant to society in terms of reducing the burden of mortality and suffering in immunosuppressed patients and in terms of reducing medical costs associated with treating opportunistic fungal infections. The potential economic upside for novel broad-spectrum anti-infectives is very large. The worldwide market for antifungals is currently estimated at $4 billion US annually. We tackle the issues of anti-microbial drug resistance head on via a multidisciplinary systems biology study combining bacterial genetics, clinical and pharmacological research in a systems biology approach, integrating traditional wet-lab methods with those of functional genomics, proteomics, metabolomics and bioinformatics.[+] Read More
Incidence of fungal disease has risen dramatically in the past several decades, and this trend is exacerbated by the increased emergence and spread of antifungal drug resistant strains of fungal pathogens. University of Manchester, a major clinical centre in the Northwest of the UK and a partner in this consortium, reports increases of 4%, 8%, and 16% in antifungal resistant infections in the last three years. Invasive fungal infections are a major cause of mortality in immunocompromised patients, whose population is ever expanding through the aggressive use of cytotoxic chemotherapy, broad-spectrum antibiotics and underlying disease such as AIDS. In addition, filamentous fungi can produce a number of allergic diseases in immunocompetent patients, unlike common aeroallergens also colonizing human lung and other tissue to various degrees. Because fungal pathogens are eukaryotes and therefore share many of their biological processes with humans, most antifungal drugs are associated with severe toxicity. No standardized vaccines exist for preventing any of the fungal infections of humans, a situation attributed both to the complexity of the pathogens involved and to their sophisticated strategies for surviving in the host and evading immune responses. Evidence has emerged that the host immune response and antifungal therapy are the major determinants of the outcome of fungal disease and act in synergy; in fact, the newest antifungal agents are immunomodulators. Currently the number of antifungal agents is limited and often not well tolerated, showing important secondary effects. Additionally the number of fungal strains resistant to the most common antifungals is increasing dramatically. To achieve greatest impact in effective treatment of fungal disease we must therefore employ a multidisciplinary approach, combining the study of antifungal drug immunopharmacology, human and fungi genetics and clinical research to answer fundamental questions about the mechanisms of drug resistance in the pathogenic species.
An integrative approach, combining high-throughput and traditional wet-lab work with computational and bioinformatics methods, will be applied to identify biomarkers of resistance to currently available treatments and to develop novel putative drug target genes and pathways in different fungi. To this end a consortium of leaders in the fields of fungal pathology, immunology, functional genomics and proteomics technologies and bioinformatics is assembled with cutting-edge laboratory instrumentation capacity, unique patient cohorts and access to major computational platforms and database resources. We shall perform a series of experiments where dendritic cells, macrophages and T lymphocytes from disease-free and immunocompromised individuals, with and without recurrent fungal disease, are challenged with pathogenic and non-pathogenic fungal agents. Whole genome analysis of gene expression together with deep sequencing will allow us not only to identify the pathogen-specific gene responders but also to explore the genotype-phenotype association of susceptibility at the level of the individual patient.
We will sequence the genomes of environmental isolates of several pathogenic subspecies and through comparative analysis will gain a better understanding the evolution of drug resistance strategies in invasive and allergenic fungal disease. ? comprehensive bar-coded S. cerevisiae gene deletion collection will be treated with a panel of existing and potential antifungal drugs in order to construct models of drug resistance, and a similar strategy will be employed against a library of several thousand antifungal drug resistant or sensitive mutants of Aspergillus. The resulting comprehensive models and candidate drug sensitive genes will be tested via specific gene knockout experiments in other fungal pathogenic species and selected targets further tested in a previously validated neutropenic mouse model. We will apply our bioinformatics expertise for integrating data produced de novo with expert-curated relevant existing transcriptomic and proteomic knowledge, such as curated pathway and protein-protein interaction databases such as Reactome, WikiPathways, IntAct and others to create a systematic vision of the mechanisms of antifungal resistance and fungal disease susceptibility from an immunological perspective.
In the course of this project, we expect:
Systems biology techniques such gene expression profiling with microarrays, deep sequencing and modern approaches for gene regulatory pathway analysis will be applied to discriminate shared core response and pathogenicity-specific programs of gene expression. Mapping the information on existing pathways will enable us to reconstruct novel pathways, expand existing pathway knowledge and search for proteins acting as sensors and signal transducers developing new tools to reconstruct the hierarchy of the response.
Better understanding of the mechanisms that control the induction of different T-cell effector functions will enable the development of strategies to manipulate the immune system in the context of fungal infections. The expected results will be key to evaluate the downstream target genes induced by the pathogenic and non-pathogenic strains suggesting strategies and specific responses related to pathogenicity, and clarifying also if and how DCs discriminate pathogens from non-pathogens and enabling the discovery of new targets for antifungal therapy. Determining whether differences in the responses are advantageous to the pathogen, or to the host, is essential for understanding host-pathogen interactions. Precisely, determining how the molecular mechanisms underlying the plastic response of DCs to fungi and the functional consequences in terms of variegating immune responses and immune homeostasis in vivo will offer a new knowledge base for drug resistance genes that will be an invaluable resource for researchers and clinicians worldwide. These studies will be important to assess whether the commonly studied molecular components of these pathogens are sufficient to account for the live pathogen response, improve our understanding of immune system and provide targets for novel immunotherapies or for novel antifungals.
The proposed research is cutting-edge in terms of the knowledge that will be generated on the integrated molecular signatures from the innate immune system and the fungal pathogens that will be used for molecular engineering of future antifungal drugs. The anticipated results are highly relevant to society in terms of reducing the burden of mortality and suffering in immunosuppressed patients and in terms of reducing medical costs associated with treating opportunistic fungal infections.
As a consequence of the combined strategy of looking comprehensively into antifungal resistance at the fundamental level, by characterising genetic and genomic variability in large patient cohorts in terms of sensitivity to therapy and susceptibility to disease we will be able to appreciate the scale of the problem. In addition to producing candidate molecules for better treatment of multi-drug resistant fungal infections, the SYBARIS Knowledge Base will document exhaustively all factors affecting antifungal drug resistance across a large panel of pathogens and individuals, enabling further research to be undertaken with impact in areas of personalized pharmacogenomics.