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FP7

SYBARIS Streszczenie raportu

Project ID: 242220
Źródło dofinansowania: FP7-HEALTH
Kraj: Germany

Final Report Summary - SYBARIS (Finding biomarkers of anti-microbial drug resistance via a systems biology analysis of fungal pathogen interactions with the human immune system)



Executive Summary:

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, reported increases of 4%, 8%, and 16% in antifungal resistant infections in the three years before 2008. 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 (Cornely OA, 2008, PMID: 18642109). 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 (Romani L., 2001, PMID: 11589476); 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.

The overarching objective of SYBARIS has been to orchestrate a consortium of partners from different disciplines, ranging from cell biology of the immune system to fungal disease clinicians and systems biology informaticians and data managers to deliver novel insights into mechanisms of antifungal drug resistance, fungi-host ecology and interactions and to produce strategies for new interventions.

For such an extraordinary task as we faced, it is important to realize that the European dimension has been key, because the problem of antimicrobial drug resistance is global, extremely complex and needs to be dealt with in a systematic, concerted effort, involving key actors joining forces towards a common solution strategy.

The SYBARIS consortium brought together leading clinical researchers, experts in fungal genetics and immunology and experts in data management and bioinformatics analysis to take a systems biology approach to the fungal challenge to human health. This generated new data on human patients, characterised fungal clinical isolates and enabled the investigation of mechanisms of fungal resistance to existing drugs. In addition to looking at fundamental questions of drug resistance in model organisms through core research constituent partners, we included an SME that is working to develop new treatments for people suffering from fungal allergies. The synergy between basic research, clinical fungal immunology and small biotech-based therapeutics development made our multidisciplinary consortium a real example of translational biomedical science.

Project Context and Objectives:

SYBARIS took a systems biology approach to study the response’s specificity 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 was 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 was assembled with cutting-edge laboratory instrumentation capacity, unique patient cohorts and access to major computational platforms and database resources.

SYBARIS had 5 principal objectives:

• To identify biomarkers of drug sensitivity and resistance in fungal microorganisms, producing a comprehensive knowledgebase of drug resistance genes for multiple safe and pathogenic species.
• To advance understanding of fundamental mechanisms of immunomodulation by antifungal drugs in the clinical context of host-pathogen interactions, developing high confidence models of drug action, resistance and toxicity.
• To identify high-precision genetic signatures of susceptibility and sensitivity to fungal infection in immunocompromised and normal patients.
• To identify potential novel candidate molecules for future antifungal drugs and develop them for clinical trials
• To set the groundwork for, and to serve as an example of a multidisciplinary systems biology approach to the study and treatment of infectious disease, bridging clinical immunology, human and fungi genetics and pharmacology with bioinformatics to answer fundamental questions of antimicrobial resistance.

The SYBARIS project was shaped as a three-year-and-a-half collaborative project developed along 7 workpackages conducted by 8 scientific partners.

SYBARIS attacked the issue of antifungal drug resistance via a three-pronged multidisciplinary approach: a molecular biology study of fungal pathogen interaction with the immune system (WP1 & WP3), multi-omics experiments profiling the transcriptional and translational activity under pathogenic stress against the landscape of genetic variation within the immunocompromised population (WP2), and a systems biology data analysis of the low- and high-throughput datasets accumulated in this project and available in the public domain (WP5). All produced data will be collected in a data management system (WP4) and statistical meta-analysis will be used to identify genes and pathways responsible for drug resistance and sensitivity. Project management and dissemination efforts are in WP6 and WP7, respectively, and include three major workshops on experiment design, data analysis methods and systems biology of fungal disease.

The consortium was coordinated by Misha Kapushesky (ostolop@ebi.ac.uk) and managed by Pascal Kahlem (pkahlem@ebi.ac.uk) at the European Bioinformatics Institute (UK). The Executive Committee included Misha Kapushesky (leader of WPs 4,5,6,7), Duccio Cavalieri (leader of WP3), Paul Bowyer (leader of WP1), and Ivo Gut (leader of WP2). The Executive Committee also gathered a panel of 4 scientists to form the Scientific Advisory Board, with expertise in the fields of systems biology, ethics, fungal disease and immunology: Prof. Ioannis Xenarios (Swiss Institute of Bioinformatics, Switzerland), Dr. Jane Kaye (Faculty of Law, University of Oxford, UK), Prof. Dr. Mihai G. Netea (Department of Medicine, Radboud University Nijmegen Medical Center, The Netherlands), Prof. Ken Smith (Department of Medicine, University of Cambridge, UK).

SYBARIS used 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. We performed a series of experiments where dendritic cells, macrophages and T lymphocytes from disease-free and immunocompromised individuals, with and without recurrent fungal disease, were challenged with pathogenic and non-pathogenic fungal agents. Whole genome analysis of gene expression together with deep sequencing allowed 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.

The genomes of environmental isolates of several pathogenic subspecies were sequenced and through comparative analysis we aimed at studying the evolution of drug resistance strategies in invasive and allergenic fungal disease. А comprehensive bar-coded S. cerevisiae gene deletion collection was treated with a panel of existing and potential antifungal drugs in order to construct models of drug resistance, and a similar strategy was employed against a library of several thousand antifungal drug-resistant or -sensitive mutants of Aspergillus. The resulting comprehensive models and candidate drug-sensitive genes were tested via specific gene knockout experiments in other fungal pathogenic species and selected targets further tested in a previously validated neutropenic mouse model. We applied 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.

Project Results:

The SYBARIS project met the criteria of the FP7 call 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.

We tackled the issues of anti-microbial drug resistance head-on via a multidisciplinary systems biology approach combining bacterial genetics, clinical and pharmacological research, integrating traditional wet-lab methods with those of functional genomics, proteomics, metabolomics and bioinformatics.

A cohort of patients presenting a panel of recurrent fungal infections and normal individuals were chosen for the collection of blood samples (WP1). At the same time we assembled a collection of fungi resistant to known anti-mycotics, isolated from patients and produced through in vitro high-throughput screens (WP3). A platform for recording exchanges of biological material within the consortium was created on the SYBARIS website, allowing to track the samples between the laboratories and their use in the experiments.

Peripheral Blood Monocyte Cells (PBMCs) produced from collected blood samples were shipped to the wet-lab partners for differentiation from monocytes to dendritic cells (DC), to be treated with S. cerevisiae cells, spores and pseudohyphae, C. albicans hyphae and cells, and Aspergillus spores and antigens/allergens. We assessed functional activity of fungus-exposed DC subsets, using high-throughput technologies, such as gene expression profiling, multiplex analysis of cytokine and chemokines, multiparameter flow cytometry and transcriptome analysis by deep sequencing. We characterized the genetic architecture in members of the cohort via whole genome sequencing and genotyping (WP2). These combined approaches allowed us to compare the different genetic backgrounds of the donors and could lead to an understanding of how a generally non-pathogen could be pathogenic in certain individuals. Challenge experiments were also performed in mice.

In addition to the examination of antifungal drug resistance in invasive fungal pathogens a complementary study of allergic fungal strains was undertaken including a comparative analysis of de novo sequenced genomes of several fungi species (WP5).

All produced data were collected in a data management system (WP4) and statistical meta-analysis was performed to identify genes and pathways responsible for drug resistance and sensitivity. Systems biology models incorporating the results of the Genome-Wide Association Study (GWAS) of fungal disease common variants were built to predict impacts of antifungal therapy on the immune system. The discovered biomarkers of antifungal drug resistance and disease susceptibility were deposited into an online knowledge base.

We set up a multigram-scale synthesis and bioassays of a recently developed new library of peptidomimetics that display strong in vitro and in vivo antifungal potency and are active on pharmacoresistant Candida albicans strains. These compounds were tested on the collection of approximately 100 strains of C. albicans and Aspergillus resistant to azoles and on S. cerevisiae in comparison to currently used antifungals such as amphotericin B, tamoxifen, voriconazole, fluconazole and others. The chemicals were tested in isolation or in the presence of patient’s monocyte-derived DCs (MoDCs), in order to assess whether any of the marker genes were affected. We assessed the indirect effect of the new molecules on the dendritic cells and if treated fungi were better recognized by the immune system.

Amongst the compounds found to have appropriate antifungal action and low toxicity, 3 were chosen as final outcomes of SYBARIS and prepared for further development and testing in clinical trials: the peptidomimetic 089 and the 2 morpholines (morpho3 and morpho4). We identified one possible way of action of 089, at least for the model S. cerevisiae.

The project is acknowledged in 41 scientific publications. All public deliverables, including the complete antifungal resistant strain collection, the biomarker knowledge base and predictive models are available on the SYBARIS project portal (http://www.sybaris-fp7.eu/workpackages) and disseminated through workshops, and at the end of the project, handed over to major well-established fungal research bodies. Collaboration with the members of the EU-funded project CommHERE (http://www.commhere.eu) also supports the public dissemination of the project outcomes.

The results have been 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 outline here major achievements of the project:

1.Development of a comprehensive library of drug-resistant/drug-sensitive fungal strains, with appropriate descriptions collected in a management system (D3.5 Collection of antifungal drug-resistant/sensitive fungal strains).

Strains from clinical isolate collections or laboratory-generated strains (WP3, WP4) were tested for drug resistance using standard MIC tests for azoles (Itraconazole, Posaconazole and Voriconazole) and amphotericin B. Results were recorded and used to construct a database of resistant isolates. 243 drug resistant isolates of Aspergillus fumigatus and 210 drug resistant Candida albicans were isolated from clinical sources. 1100 resistant isolates of A. fumigatus and 300 resistant C. albicans were generated in the laboratory as part of WP3.

The isolate collection developed in SYBARIS represents one of the largest and best characterised drug resistant fungal collections in the world. Isolates are freely available to other researchers and the searchable database of isolates is available at:

http://www.aspergillus.org.uk/

Additionally, we collected data on multiazole resistance in clinical and environmental fungal isolates on the following public webpage:

https://docs.google.com/spreadsheet/pub?key=0Agjk4o1KLiVUdHkwVXFQMDBhclFRS0FMZFJpNEVfVEE&single=true&gid=0&output=html

The following screenshot shows the results of the search for the strain Aspegillus fumigatus AF1. (Please see the attached pdf document)

2.Fungal transcriptome and de novo genome sequencing made available for multiple strains, enabling the comparative analysis of sequenced genomes helping to elucidate mechanisms of emergence of drug resistance (D4.6 Populated fungal strain collection presentation management system).

In the duration of the SYBARIS project, various Aspergillus, Candida, Saccharomyces and other fungal pathogen strains have been studied by whole genome sequencing, RNA-seq, Bar-seq, or transposon analysis. We have assembled and prepared files for submissions to major public data repositories:

- The European Nucleotide Archive (http://www.ebi.ac.uk/ena/)
- ArrayExpress (http://www.ebi.ac.uk/arrayexpress/)
- Ensembl Genomes (http://www.ensemblgenomes.org)

Sequenced and analysed fungal strains will be available publicly upon the publication of the corresponding scientific papers.

SYBARIS Fungal Collections Presentation Page on the Project Portal is available at:

http://www.sybaris-fp7.eu/fungal-strains-collections

3.Novel multi-omics datasets on human and mouse models of fungal disease deposited in public repositories and presented on the SYBARIS project portal.

In addition to the sequencing data (point 2 above), cytokine response was assayed on a series of DCs incubated in the presence of various strains of Aspergillus, Candida and Saccharomyces.

Data are available on the SYBARIS website and the direct link is:

https://docs.google.com/spreadsheet/pub?key=0Agjk4o1KLiVUdHpSN3Bxdkw1VkRuZDh2X1FJVTVHT0E&gid=4

DCs’ transcriptome in the presence of Saccharomyces cerevisiae, Candida, or C-lectin and TLR agonists are presented in the public deliverable D1.2.

Data were analysed using the DC-Atlas:

Cavalieri, Duccio, Damariz Rivero, LucaBeltrame, Sonja I Buschow, Enrica Calura, LisaRizzetto, Sandra Gessani, et al. (2010) DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells. Immunome Research 6: 10.

4.Knowledge-base of biomarkers and systems biology models of antifungal drug resistance and disease susceptibility developed and made available to the community via the SYBARIS project portal.

The deliverable D5.4 “Report on the GWAS of common variants of fungal disease” describes the preliminary study of 1731 genome variants associated with disease versus healthy groups: CCPA (n=116), ABPA (n=97), SAFS (n=50), healthy (n=280), non-atopic asthma (n=66) and atopic asthma (not fungally sensitised, n=167).

The prototype of integrated knowledge base of biomarkers and systems biology models of antifungal drug resistance and disease susceptibility was presented in the deliverable D4.4.

The deliverable D4.5 “Populated integrated knowledge base of biomarkers and systems biology models of antifungal drug resistance and disease susceptibility” submitted at M41 presented the final knowledge base populated with the curated set of biomarkers presented in the deliverable D5.1. We gathered basic information about each biomarker: the species, drugs tested, an abstract of the findings and the source, with space for citation information. Hundreds to thousands of experimental biomarkers have been identified, and population of this knowledge base is still on going.

As part of the work on the database of biomarkers a significant effort has been made at characterising the fungal strains involved. The strains from clinical isolate collections or laboratory-generated strains were tested for drug resistance using standard MIC tests for azoles and amphotericin. Results were recorded and reported in D3.5 and used to construct a database of resistant isolates. 243 drug resistant isolates of Aspergillus fumigatus and 210 drug resistant Candida albicans have been isolated from clinical sources. 1100 resistant isolates of A. fumigatus and 300 resistant C. albicans have been generated in the laboratory as part of WP3. The isolate collection developed in SYBARIS represents one of the largest and best characterised drug resistant fungal collections in the world. These isolates are freely available to other researchers and the searchable database of isolates is available at:

http://www.aspergillus.org.uk/indexhome.htm?/asd/asd_index.php~main

These datasets will be accessible at http://www.ebi.ac.uk/arrayexpress, the ArrayExpress database, and at http://www.ebi.ac.uk/ena, the European Nucleotide Archive. The curated biomarkers knowledgebase continue to be populated and remain available from the SYBARIS Consortium Pages at http://sybaris-fp7.eu. EMBL will sustain the SYBARIS website domain in the short-term, until the end of the corresponding funding, but all data will be transferred to public databases by then.

5.Development and study of several novel molecules with potential antifungal action via a systems biology study of fungal pathogen-host interactions (D3.4 Set of 3 or more molecules with potential antifungal action, tested on selected chemo-resistant fungal strains, ready to deploy in clinical trials).

We screened of the effects of three molecules (089, morpho3 and morpho5) with potential antifungal action on a set of chemo-resistant fungal strains. The molecules were previously selected from a wide library of newly synthesized small molecules as able to induce decrease of S. cerevisiae growth or to induce yeast death.

The molecules were tested on several fungal species, encompassing the yeast model Saccharomyces cerevisiae, the mould Aspergillus fumigatus and two Candida spp. Additionally, the toxicity of the molecules was assayed on a widely used human leukemic cell line (K562). In vivo assays are being carried out and the molecules evaluated for the development of novel antifungals.

6.Novel data analysis algorithms and models of immune response developed and published in peer-reviewed literature and via the SYBARIS project portal.

41 scientific publications acknowledging SYBARIS are cited on the website:

http://www.sybaris-fp7.eu/publications

It is of note that more than 10 publications are in preparation at the time of the project’s end. These publications will report on the latest findings of the project, including:

- the study of the involvement of fungi in Crohn’s disease and mouse models
- the association of colony morphology with genome features
- the challenge of DCs with fungal pathogens and analysis of the host-pathogen interactions
- study of non-CYP51A mediated azole resistance in Aspergillus fumigatus
- characterization of Cladosporium in allergic response
- the study of exome-sequencing derived variants from Aspergillosis patients
- assay studies of new candidate molecules efficacy on Aspergillus

7.A major antifungal disease workshop conducted with external paper submissions and presentations of SYBARIS results to the community.

In 2012 and 2013 the consortium organised public conferences, where several partners presented results:

- February 23, 2012: hosted by UMRS at Centre d’Immunologie de Marseille (France)
- March 13, 2013: hosted by UniFi at Fondazione Edmund Mach di San Michele all'Adige (Italy)

Results were also presented to the community by the partners when they participated individually to international conferences (see D7.6 Plan for the use and dissemination of foreground).

Additionally the partner UNIMAN participates to the regular Aspergillosis Patients Meetings in Manchester, to inform patients about the research progress in the area. These conferences are filmed and the videos are freely available online: http://www.ustream.tv/channel/aspergillosis-patients-meeting

8.The SYBARIS project portal developed and used to disseminate project deliverable documents and databases and to engage the community.

The SYBARIS portal was opened one month after the start of the project. It was used to present public information about the ongoing work, and also to share confidential information between the consortium partners.

The URL is: http://www.sybaris-fp7.eu/

Since all data obtained by the consortium are being uploaded on sustained public databases and the results will all be published in scientific journals, no unpublished data will remain on the SYBARIS website. We expect that the European Bioinformatics Institute, which hosts the SYBARIS website currently, will maintain an archived copy of it, migrating from the http://www.sybaris-fp7.eu URL to one under the http://www.ebi.ac.uk/ umbrella.

Potential Impact:

1.4.1. Potential impact

The project performed studies with the use of clinical samples. The study of host-pathogen interactions focused on assessment of resistance selection associated with the use of different antifungal agents. Such studies could be performed only in animals and human cell systems. However it should be noted that SYBARIS had access to all of the UK drug resistant fungal isolates identified in the last 10 years (via UNIMAN and collaborators). Once SYBARIS had identified mechanisms of drug resistance this collection could be directly interrogated using high-throughput sequencing to determine which drug resistance mechanisms were relevant in the clinical setting. Furthermore artificial mutants could be assessed for pathogenicity in animal models to further identify clinically relevant modes of drug resistance. These elements allowed SYBARIS to make a long-term impact into the study of the antifungals’ effect on the emergence of drug resistance.

The proposed research has been cutting-edge in terms of the knowledge that has been 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.

By working directly with major hospitals where a wide range of disease situations is encountered and by assembling an experimental cohort with significant numbers of normal, healthy individuals, we were able to add not only to an in-depth understanding of the pathogenic processes associated with infections disease but also to a general insight into human immunity. Research undertaken by SYBARIS was necessary to investigate the balance of pro-inflammatory and anti-inflammatory signalling as a prerequisite for successful host/pathogen interaction without the development of disease. The data derived from the clinical component in SYBARIS will guide research in infectious disease, vaccine development and fundamental research of the immune system for years to come.

The results follow as consequence of the combined strategy of looking comprehensively into antimicrobial (i.e. antifungal) resistance at the fundamental level, by characterizing genetic and genomic variability in large patient cohorts in terms of sensitivity to therapy and susceptibility to disease. To study the scale of the problem, we sought to identify its nature first and then we assessed it with a high-throughput sequencing approach using isolate collections already available at UNIMAN and collaborators (>10000 clinical isolates, >1000 azole-resistant isolates) and those collected within SYBARIS. In addition to producing candidate molecules for better treatment of multi-drug resistant fungal infections, with self-evidently relevant impact, the SYBARIS Knowledge Base has documented various factors affecting antifungal drug resistance – across a large panel of pathogens and individuals from both literature and experimental studies conducted in our consortium.

Given the results obtained by the consortium in three-and-a-half years, we believe that the SYBARIS initiative has energized antimicrobial drug research in infectious disease and immunology in Europe, providing direct, immediate impact in the clinical setting in terms of novel therapies and strategies for health.

The 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.

1.4.2.Main dissemination activities and exploitation of results

The SYBARIS consortium is strongly committed to liaising with the mycology and immunology communities, both the academia, industry and medicine, looking to ensure that our work remains relevant to current demands and challenges, that all possible synergies with other initiatives in the field are exploited, that the discovered biomarkers and models are adopted and that our contribution to the field has a long-lasting, sustainable effect.

Such a commitment requires a special effort to be directed towards building solid dissemination, exploitation and knowledge management strategies that support the creation of an environment of mutual trust and understanding beyond the consortium.

Reflecting the above, we have structured our work plan so that dissemination and exploitation activities form an integral part of the project. The entire WP4 was related to a variety of knowledge management tasks, and WP7 ensured the strategies for enactment of dissemination tasks for the project. Additionally, within the wet-lab workpackages (WP1-3) there were activities and reports to provide consultation and interaction amongst the partners through direct links and with the broader community via specially designed workshops open to participants from beyond the consortium. The global project leadership and management carried out in the framework of WP6 further ensured necessary interaction with specific initiatives, actors and the scientific community as a whole.

Dissemination activities:

There are several avenues of dissemination of materials produced in SYBARIS: (1) the project portal, (2) open workshops with invited speakers from outside the consortium, (3) scientific conferences and publications, and (4) the SYBARIS knowledge base. In total, more than 100 dissemination activities were undertaken throughout the project (deliverable D7.6).

(1)The SYBARIS project portal (www.sybaris-fp7.eu), developed from the first days of the project provides the scientific community with complete information about the project’s background, goals and progress (WP7). All public deliverables are accessible through this web site (http://www.sybaris-fp7.eu/workpackages). The portal links to the SYBARIS collection of drug-resistant fungal strains and to the developed biomarker knowledge base.
(2)Public conferences were held by the SYBARIS partners at the occasion of the AGMs in Marseille (February 2012) and in San Michele all’Adige (March 2013) (deliverable D7.5).

The partner UNIMAN participates to the regular Aspergillosis Patients Meetings in Manchester, to inform patients about the research progress in the area. These conferences are filmed and the videos are freely available online: http://www.ustream.tv/channel/aspergillosis-patients-meeting

(3)We engaged with the scientific community by attending several major conferences in fungal and immunological research, where we presented the work via talks or posters. 40 publications in high-impact journals are acknowledging SYBARIS funding since the start of the project (http://www.sybaris-fp7.eu/publications).
(4)The SYBARIS knowledge base of biomarkers of drug resistance and fungal disease susceptibility (WP4) is now a resource freely available to researchers in the fields of immunology and mycoses. It is still early to evaluate its impact, but this database has the potential to become a central point within the fungal research community.

Exploitation activities:

Several of SYBARIS deliverables lend themselves clearly to successful exploitation. Namely, these are the library of drug-resistant and drug-sensitive fungal strains, the newly derived low-toxicity antifungal compounds active against difficult to treat infections, and the biomarker knowledge base. Generally, we adhere to the policy of promoting open access to all products of the project (software source code, data, and others) via appropriate licensing.

The SYBARIS partner AlerGenetica is an SME based in Tenerife, Spain. The primary focus of our research is the development of fungal allergy vaccines. There is clearly evidence in the case of Aspergillus fumigatus that allergy to this fungus might be due to colonization of the patient. In addition, known allergens from the fungus Alternaria alternata comprise of intracellular proteins such as ribosome proteins again indicating a possible low-level infection.

As part of SYBARIS AlerGenetica in collaboration with Paul Bowyers´s group at UNIMAN attempted to express a number of allergens in the Pichia pastoris yeast expression system. We achieved the expression and secretion of recombinant proteins from P. pastoris transformant strains carrying an optimized gene sequence of potential allergen genes aspF2, aspF5 and aspF13 from A. fumigatus, and of altA5, altA6, altA7, altA8, altA10 and altA12 from A. alternata. We did not detect secretion of recombinant proteins in strains carrying the altA1 and altA4 optimized genes. The SYBARIS project allowed AlerGenetica to build the fungal molecular platform.

However, the secretion and purification of A. alternata major allergen: ALTA1 was not successful in either E. coli and in P. pastoris. Successful expression of ALTA1 was achieved by using the TNO (The Netherlands) under an MTA provided Aspergillus niger expression system.

Successful expression of ALTA1 allowed AlerGenetica to develop an animal model for A. alternata allergy. Based upon AlerGenetica´s results Professor H Kita, Head of Allergy, Mayo Clinic agreed to request NIH funding in 2013 for the A. alternata allergy vaccine work using his animal system.

Filamentous fungi such as A. niger have been successfully used to express recombinant proteins of biotechnological importance. However, expression levels of specific recombinant protein in A. niger vary considerably. In an attempt to understand rate the rate limiting steps to protein production in A. niger, AlerGenetica acquired four A. niger hyper-protein producers, these were sequenced in the SYBARIS project by CNAG and assembled by EBI. AlerGenetica intends to review the genome modifications of these sequenced strains with the public domain wild-type A. niger genome sequence to provide insights to mutations which facilitate over production of recombinant proteins. Several publication based upon this work are anticipated

In addition, AlerGenetica transformed A. niger with a plasmid containing the antifungal drug resistance gene for zeocin. Zeocin resistance is based upon (a prokaryotic) protein encoded by the zeocin resistance gene binding to the zeocin drug. Increased zeocin resistance is therefore correlated with increased production of the zeocin drug resistance protein. Previous unpublished studies have shown that either increased transcription of the zeocin or importantly host mutations which aid foreign protein secretion lead to increased zeocin resistance. Transformants with elevated drug resistance were isolated and have been genome sequenced by CNAG and assembled by EBI with the goal of identifying host mutations which lead to increased heterologous protein secretion.

The exposure AlerGenetica received with SYBARIS funded work, lead the German biotechnology company WeissBioTech funding research at AlerGenetica and forming a France based start-up company WeissBioTech SARL co-founded by WeissBioTech and two AlerGenetica co-founders to with the goal of developing new industrial biotechnology products.

Knowledge management activities:

The knowledge management agenda of SYBARIS aimed at facilitating knowledge transfer both internally among partners and to the external community, while also guiding new knowledge generation according to detected ‘gaps’.

‘Internal’ knowledge management activities had the objective of making the best use of knowledge and expertise existing in the consortium for undertaking the project itself. The need for this focus originates from the multidisciplinary nature of the consortium and the specificity of the field addressed. To deal with this in practice, frequent communication between partners via teleconferences and direct internal scientific meetings allowed the exchange of information and rapid transmission of know-how between consortium members as and when needed.

Internal knowledge-management and training was thus designed to bring about: a) mutual knowledge sharing of partner’s expertise, fostering better understanding and productive interactions amongst partners from different disciplines (particularly during the first year), and b) sharing knowledge on specific technologies/concepts/issues that was needed by other partners for them to carry out their work within the project (for instance the protocol standardization of derivation and culture of dendritic cells at university of Florence and at university of Manchester, which involved the exchange of post-docs between the 2 institutions). ‘External’ knowledge management, in contrast, dealt with the transmission of knowledge generated by the project to the outside world, via the dissemination activities mentioned above.

For both internal and external knowledge management undertakings, the SYBARIS project portal developed in WP7 served as reference. It served as one of the main platforms giving visibility to the project’s activities and providing free access to our results. It also incorporated a private area to support internal exchange of data, protocols and tools to support knowledge transfer among partners. Finally, it has been the major route by which we publicised the progress of the project and keep track of workshops, thereby raising awareness among prospective users and making the impact of the project in the community as high as possible.

List of Websites:

The project website has the address: www.sybaris-fp7.eu

Contractors involved:

European Molecular Biology Laboratory (EMBL-EBI): Misha Kapushesky
University of Florence (UniFi): Duccio Cavalieri
University of Manchester (UNIMAN): Paul Bowyer
University of Aveiro (UAVR): Manuel Santos
Centre National pour la Recherche Scientifique (CNRS): Philippe Pierre
Alergenetica (AG): Brian Miller
University of Perugia (UniPg): Luigina Romani
Parc Científic de Barcelona, Centro Nacional de Análisis Genómico, Spain (PCB): Ivo Gut

Coordinator contact details:

Consortium director: Misha Kapushesky
Consortium manager: Pascal Kahlem
EMBL- European Bioinformatics Institute
Wellcome Trust Genome Campus
Hinxton, Cambridge CB10 1SD
United Kingdom
Tel: +44 (0)1223 494 647
Email: ostolop@ebi.ac.uk


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