Maynooth University Department of Biology

March 2021...ELISA and bioreagents for COVID-19 antibody detection including SARS-CoV-2 recombinant Spike protein, recombinant baculovirus encoding-Spike gene, recombinant E. coli clone expressing SARS-CoV-2 N protein, purified N protein and CoV-2 ELISA materials are now available.

New immunoreagents for diagnosis of COVID-19 associated fungal/aspergillosis infection are also available for evaluation or licence. We also invite contact from industry with challenging molecular biotechnology projects.

Contact: sean.doyle@mu.ie

To date, Doyle's molecular and applied proteomics work has resulted in over 100 peer-reviewed publications, over 5000 citations (h-index: 40), patented fungal diagnostic technology (EP2709659, awarded 2018), commercialised products and his work accounts for over 6% of Maynooth University output on Pubmed from 1997-2018 (10% from 2015-2016 inclusive). It is directed towards understanding microbial pathogenicity, antimicrobial resistance and identifying new biotechnological, therapeutic and diagnostic targets. See https://scholar.google.com/citations?user=DJhTVdEAAAAJ&hl=en

Doyle also secured competitive funding (€89,000) for Lightcycler 480 instrumentation which was offered, and loaned free-of-charge, to the National Virus Reference Laboratory (ultimately Enfer Scientific) from March - July 2020 to underpin essential and large-scale National molecular testing of patients for SARS-CoV-2, the causative agent of COVID-19. The impact of this, and equivalent contributions by other Irish Universities, on the establishment of control over what was an exponentially evolving pandemic, cannot be underestimated.

He was also instrumental in securing competitive SFI funding for Departmental quantitative mass spectrometry (MS) facilities (€566,540) which has enabled multiple new projects within Biology at MU- and is also available to internal & external users. All MS-related publications (85), equating to 1 per month since commissioning, to April 2021:

Doyle has also successfully supervised 28 PhD and 10 MSc student to completion, and currently supervises 3 PhD student projects.

His group also has a number of academic and industrial collaborations and group members have been pro-active in the promotion of the public understanding of science through participation in such activities as the Maynooth University Chain Reaction Lecture Series and the National Science Week. Research work in the laboratory is funded by The Irish Research Council (IRC) and MU.

Research area 1. Aspergillus fumigatus: He studies the biology, diagnosis and molecular genetics of the human pathogenic fungus, Aspergillus fumigatus - a major cause of infection in immunocompromised individuals. His group has identified a number of potential virulence factors (proteins) in the organism, which may contribute both to its pathogenicity and emerging resistance to conventional chemotherapies. He was the first to identify and characterise a novel self-protection mechanism against gliotoxin in Aspergillus fumigatus. His group also identified a number of non-ribosomal peptide synthetases and glutathione s-transferases in A. fumigatus. Interestingly, non-ribosomal peptide synthetases in fungi represent a novel source of bioactive metabolites and his published work is at the cutting-edge of this internationally competitive area. The primary research methods involved in this work comprise gene deletion studies, proteomics, NMR and LC-MS. Collaborators: Professor Gary Jones (Leeds-Beckett University, UK, Dr David Fitzpatrick, Dr Ozgur Bayram, Professor Hubertus Haas (Innsbruck, Austria), Professor Gustavo Goldman (Sao Paulo, Brazil), Dr Siobhan McClean (UCD) & Dr Maire Callaghan (ITT Dublin).

Research area 2 Immunodiagnostics: Using a well-established recombinant protein expression systems (E. coli and baculovirus) Doyle's group has expressed and continues to express A. fumigatus proteins in order to assess their diagnostic potential. In addition, he has developed novel immunoassays to detect fungal metabolites as disease indicators. In June 2020, he was awarded funding to develop COVID-19 ELISA and lateral flow-based immunodiagnostics. This work is in collaboration with Professor John P. Dalton, NUIG. Proteomic studies of a number of organisms, using Departmental protein MS facilities, are underway and peer-reviewed publications are emerging from these efforts.

Research area 3. Armillaria mellea: In the past, this plant pathogenic fungus has proven recalcitrant to detailed molecular analysis. However, Doyle with Dr David Fitzpatrick has undertaken genome sequencing of this organism and has established a robust proteomics pipeline to identify novel biocatalysts (enzymes) with biotechnological, pharmaceutical and biofuel applications. This work involves conventional individual protein mass spectrometry and shotgun proteomics. The primary research methods involved in this work comprise genome sequencing, bioinformatics, proteomics, HPLC and small molecule LC-MS. Collaborators: Dr David Fitzpatrick and Dr Rebecca Owens (Maynooth University).

Research equipment infrastructure available for Industry-Academia Collaborations: The Department of Biology possesses a range of modern molecular and proteomic instrumentation which is used for both PhD and Post-Doctoral Research projects. This equipment, much of which has been funded by competitive awards from the HEA, HRB, SFI and Enterprise Ireland, is also available for use by companies on a commercial basis. For further details see:https://www.maynoothuniversity.ie/biology/instrumentation-facilities

Professor Sean Doyle- Applied and Discovery Research (2021)

1. Bioconjugation strategy development and process transfer to manufacturing of: (i) anti-human IgG-HRP conjugate (Hepatitis C virus serodiagnostic systems; Wellcome Diagnostics, UK), (ii) multiple IgG(anti-GST)-HRP conjugates (Human, canine, porcine and murine GST detection systems; Biotrin) (Rees et al., 1995), (iii) biotinylated recombinant VP2 capsids (FDA approved Parvovirus B19 IgM detection system, Biotrin) (Doyle et al., 2000), (iv) chemical modification of haemoglobin (Haptoglobin detection; Tridelta Development Limited; Eckersall et al., 1999) and (v) Phosphorylcholine-BSA-HRP conjugation (C-Reactive Protein detection; Tridelta Development Limited; Deegan et al., 2003).

2. Bioconjugation strategies for immunization and chemoenzymatic research applications: (i) Fumagillin and helvolic acid bioconjugation for immunogen synthesis (Fox et al., 2004), (ii) synthesis of fluorescent and biotinylated coenzyme A derivatives as substrates for novel 4’-phosphopantetheinyl transferase activity determination by microwell assay (Stack et al., 2009), (iii) fluorescent derivatization of gliotoxin to enhance detection by RP-HPLC and MALDI-MS (Davis et al., 2011), (iv) siderophore (fusarinine C (FsC) and triacetylfusarinine C (TAFC)) conjugation to proteins as immunogens (EP patent awarded), (v) fluorescent alkylation of antioxidant ergothioneine (Gallagher et al., 2012), (vi) siderophore (ornibactin) conjugation to proteins as an immunogen (Whelan et al., 2015), (vii) fluorescent derivatization of FsC for fungal uptake studies (Moloney et al., 2016) and (viii) simultaneous chemical acetylation and activation of FsC for immunogen synthesis (Moloney et al., 2018).

3. Proteogenomic-mediated discovery of novel biological systems: Since 2004, Doyle has pioneered an integrated proteogenomic approach to new systems discovery in fungi, which has led to the first identification of: (i) oxidoreductase mediated self-resistance mechanisms against redox-active peptides, (ii) negative regulatory system for redox-active metabolite production, (iii) new biosynthetic functionality of detoxification enzymes, (iv) integration of primary and secondary metabolism via methylation systems, (v) a novel metal biochelator, (vi) detailed proteogenomic investigation of plant pathogen Armillaria mellea and (vii) discovery of supra-antioxidant, ergothioneine, presence and biosynthesis in Aspergillus fumigatus. These proteogenomic strategies have involved fungal growth under specific conditions, comparative quantitative proteomics to identify altered proteins/pathways/systems, and subsequent gene deletion of cognate genes to elucidate discrete protein functionality.

Coordinated by Professor Sean Doyle, the BSc (Biotechnology) degree (CAO code: MH202) at Maynooth University was introduced in 1996 by Head of Department, Professor Peter Whittaker, and the first students graduated in 2000. Since then, over 470 students have graduated with a BSc (Biotechnology) from Maynooth University. Numerous teaching methodologies are utilized for the programme combining didactic, self-directed and active learning, and include lectures and practical classes, allied to experiential learning in terms of Industry-based educational outplacement (3 months in final year), and acquisition of presentation skills and site visits to biotechnology companies. The degree is of four-year duration, requires simultaneous study of Chemistry for 3 of the 4 years, and is one of the most popular and highest points courses in the Faculty of Science and Engineering. The course has been modularised and semesterised since 2003. Annually, it is actively promoted by course co-ordinator Professor Doyle, in association with colleagues in the University Admissions Office. In academic year 2018-19, there are 95 students taking Biotechnology at Maynooth University (years 1-4). Students take 4 subjects in 1st year, three in 2nd year, two in 3rd year and only Biotechnology in 4th year. Indeed, in 4th year, all lecture modules are completed in Semester 1 and students go on work placement during Semester 2- with modules being examined at the end of both Semester 1 and 2, respectively. It was the first Maynooth University degree programme to offer an industrial placement, which served as a predicate for many of the industrial placement modules now found in other Maynooth University degrees (2018). Professor Doyle managed the industrial outplacement programme until 2015, and the BSc (Biotechnology) was used as a basis to establish the BSc (Biomedical and Pharmaceutical Chemistry) degree at Maynooth University, indeed both courses still share high percentage identity for years 1 to 3. Dr David Fitzpatrick and Dr Paul Dowling now lead the industrial placement of 4th year Biotechnology students, with support from Professor Doyle. The course has evolved since its inception and now places a strong emphasis on both molecular biotechnology and chemical biology, in particular the identification, study and use of biomolecules as diagnostic and therapeutic agents. Students taking the BSc (Biotechnology) programme experience both subject-unique modules and shared modules with other Departmental undergraduates, and have found employment in the biopharmaceutical, pharmaceutical, diagnostic and food industries. Many BSc (Biotechnology) graduates have undertaken PhD degrees at Maynooth University and elsewhere, and are now in both academic and industrial leadership positions.