Title Promoter Affiliations Abstract "Study and exploitation of bacteriophage encoded endolysins and expolysaccharide depolymerases." "Guido Volckaert" "Department of Biosystems (BIOSYST), Division Animal and Human Health Engineering" "Bacteriophages (or phages) are widespread viruses which infect bacteria. They use refined and highly adapted mechanisms to hijack the bacterial cell, which is subsequently used for the phage's replication. Unraveling the underlying fundamental processes and interactions between phage and host cell constitutes a corner stone in fundamental molecular research and served as a base for numerous applications. During the replication cycle, phages cross different barriers such as the peptidoglycan and biofilm matrix. Phages encode a diverse set of hydrolases to degrade these polymer structures. These enzymes are optimized by endless adaptive co-evolution between phage and host cell in order to function quickly, efficiently and with high specificity. Hence, these hydrolases are an attractive research topic to combat the different barriers posed by bacteria. Phages cross the peptidoglycan barrier twice, once during infection and once during lysis of the host cell. Therefore, they encode a distinct peptidoglycan hydrolase for each step, accompanied with its specific properties. Eight different peptidoglycan hydrolases encoded by Pseudomonas aeruginosa and Staphylococcus aureus have been extensively characterized on a biochemical, molecular and structural level. Furthermore, their potential as enzybiotics (the use of enzymes as antibiotics) to treat P. aeruginosa or S. aureus infections has been assessed. Different bacteria form a biofilm structure which constitute a possible obstruction for the successful application of peptidoglycan hydrolases as enzybiotics. Furthermore, biofilm formation causes problems in numerous other fields (e.g. silting up industrial pipes, blocking of catheters, increased resistance to antibiotics,..). The strategy used by a phage to circumvent this barrier is considered in this project. Aims: 1. General characterization and preclinical evaluation of P. aeruginosa and S. aureus bacteriophage encoded peptidoglycan hydrolases and their chimeric combinations as enzybiotics. 2. Identification of biofilm degrading P. aeruginosa phage proteins and peptidoglycan hydrolases originating from phages which infect Enterobacteriaceae or other Pseudomonads. The ultimate goals is to create a multi component system which combines the best of phage encoded hydrolases to combat bacterial pathogens." "Lytic bacteriophages and probiotic Bacillus spp. as novel biocontrol agents of Motile Aeromonad Septicaemia (MAS) in farmed Nile tilapia (Oreochromis niloticus): A holistic approach to disease management." "Daisy Vanrompay, Annelies Declercq, Peter Bossier" "Department of Animal Sciences and Aquatic Ecology, Busitema University" "Local bacteriophages and probiotic Bacillus spp. will be isolated and characterized. The isolates wil be tested against Aeromonas hydrophila, both in vivo and in vitro under lab conditions. Bacillus spp. will be combined together with selected bacteriophages to test for a synergistic effect under lab conditions. Semi-field trials will conducted on Nile tilapia (Oreochromis niloticus) for field verification." "Development of molecular tools for characterization and exploitation of a library of bacteriophages infecting Pseudomonas aeruginosa." "Rob Lavigne" "Division Animal and Human Health Engineering" "Bacterial viruses (bacteriophages) are the most abundant and genetically diverse biological entities on this planet. Billion years of coevolution with their bacterial host organisms enabled the development of various phage-encoded mechanisms to shut down the host metabolism and direct it towards phage production. My doctoral research focused on the genetic diversity of bacteriophages infecting Pseudomonas aeruginosa, an important multi-drug resistant opportunistic pathogen. The described research project aims initially at the completion of the genome and proteome analysis of two Pseudomonas phages, LIT1 and LUZ7. This will be followed by the start-up of three parallel research lines to identify and characterize phage-encoded antibacterial proteins. In collaboration with Dr. Bart Landuyt (Afdeling Dierenfysiologie en Neurobiologie, K.U.Leuven), all peptides of a phage-infected cell will be screened using the 'Peptidomics' approach, leading to the experimental identification of (un)annotated phage peptides. Secondly, the suitability of 'Tandem Affinity Purification' to identify interactions between phage proteins and different subunits of the bacterial RNA polymerase will be evaluated. Finally, the recombineering system, which catalyses in vivo homologous recombination between linear DNA substrates and their counterpart in the phage genome, will be optimized in Pseudomonas aeruginosa to create specific virulent phage mutants. These three research lines will be integrated into each other in a later stage of research, enabling future study of phage-encoded antibacterials." "Application of bacteriophages for treatment and prevention of avian pathogenic Escherichia coli (APEC) and antibiotic resistance: a basic study into the bacteriophage-bacterium interaction." "Patrick Butaye" "Department of Pathobiology, Pharmacology and Zoological Medicine" "CARTNET trains young researchers in Europe to address serious and global problems of antibiotic resistance. Diseasecausing bacteria are becoming resistant even to last resort antibiotics and transmission of resistance genes and resistant pathogens is widespread between human, animal and environmental reservoirs. Therefore, CARTNET will focus on alternative approaches to handle antibiotic resistance and treating resistant infections in both humans and animals. It wil provide competences to young researchers and future policymakers that will allow them to use knowledge at all relevant scientific levels and in collaboration with stakeholders in both human and veterinary medicine as well as in chemistry, structural biology and natural product chemistry to develop new antimicrobial solutions. CARTNET comprises researchers who are leading experts on antibiotic resistance and antimicrobial drug discovery. They will in cooperation with the recruited early stage research (ESR) trainees use structural biology to synthesize new antimicrobials, screen environmental microorganisms and microbiota for new active compounds and limit spread of resistance through knowledge of dissemination of mobile genetic elements. The research training is hosted by universities with a strong track record of graduate training and is composed of an ambitious scientific program flanked by essential generic training courses and with ample opportunity for networking at network meetings, conferences and shared secondments. In addition to training young researchers for the challenges of tomorrow CARTNET will from day one provide excellent research with impressive scientific and societal impact." "Direct deposition of therapeutic bacteriophages on orthopedic implant materials: development of an in vitro, preclinical proof-of-concept antibacterial strategy (PHAGE-DEPOSIT)" "Rob Lavigne" "Animal and Human Health Engineering (A2H), Surface and Interface Engineered Materials (SIEM)" "There is growing interest in phage therapy as an adjunct to antibiotic therapy, particularly for the treatment of multi-drug resistant device-associated biofilm infections, which suffer from high treatment failure rates. Therefore, optimal phage treatment administration protocols still need to be established further for these types of infections. In this project, we will develop a Staphylococcus aureus phage coating directly on the medical device surfaces, as a solution to avoid postoperative interventions to locally apply phages. A proof of concept for orthopedic implants of different materials including titanium, ceramics or plastics will be developed. Our consortium has key expertise in novel pretreatment procedures, that enable fixation of phages to the implant surface in two ways: via alternating current electrophoretic deposition and via phage display of biomaterial-binding peptides on the phage surface enabling spontaneous adsorption.Finally, the antibacterial activity of the different coatings will be evaluated in an implant-biofilm model in a continuous in vitro flow cell system. Moreover, naturally evolved phages as well as resistant bacterial mutants emerging during incubation will be tracked and analyzed using whole genome sequencing to ensure the sustainability and usefulness of the approach." "Bacteriophages and monolith-cryogels: A new robust tool in chromatography" "Hans Deckmyn" "Chemistry, Kulak Kortrijk Campus" "In view of the fast evolution in the biomedical research, there is an increasing need for quick, inexpensive and robust methods, including in the niche of protein chromatography. In this project, we want to focus on the use of monolith-cryogel as matrix and peptide-displaying phage as ligands in affinity chromatography. The cryogel matrix, with its specific large pore structure, is especially fit to purify components from crude materials such as e.g. milk, blood, and fermentor cultures without pretreatment. The affinity ligand, peptide-displaying phage, can be readily selected for high specificity and affinity and can be produced in large quantities in a fast and inexpensive manner, in contrast to the often tedious and labour intensive production of poly- and monoclonal antibodies. We already provided “proof of principle” and now intend to optimize the technology to improve the column capacity. To this end we will evaluate the use of spacers, specific (guided) coupling strategies and the possibilities to build larger columns, in order to define the optimal chromatographic conditions and purification yields." "Development and validation of an oral influenza A vaccine based on M2e-expressing bacteriophages" "Department of Biomedical molecular biology" "The aim is to generate an orally administrable influenza A vaccine based on the conserved ectodomain of M2 (M2e) and for veterniary purposes. The filamentous fc phage will be used as a carrier. The vaccine will be targeted to M cells in the gut by using a bioadhesine." "HavePhAIth: Human Phage therapy using AI to design phage cocktails against ESKAPE pathogens" "Rob Lavigne" "Animal and Human Health Engineering (A2H), Microbial and Plant Genetics (CMPG)" "Human Phage Therapy (PT) is a promising route for the treatment of increasingly drug-resistant bacterial infections. Belgium is leading the implementation of PT in Europe, and the technique is currently in operations at the Queen Astrid Military Hospital (QAMH), and was recently implemented at UZ Leuven. Indeed, a Multidisciplinary Phage Task Force (MPTF) has been set up within UZ Leuven to provide PT to patients with difficult-to-treat infections. However, the current design strategies of phage cocktails are blindfolded, relying on empirical rules that fail to leverage the rapidly expanding omics data to predict bacteria-phage interactions. In my doctoral research, I developed machine learning models of phage infectivity in Pseudomonas aeruginosa that predict which phages from a collection can infect given bacterial strains based on their genomic content. As a member of the MPTF and frequent collaborator of the QAMH, two entities that will generate big datasets of omics/clinical data on PT in vivo, I will translate these novel modeling approaches to the ESKAPE pathogens found in the patients. This will also put me in a unique position to assess the dynamics of bacteria-phage co-evolution in vivo, by inspecting longitunal isolates from given patients undergoing treatment. Importantly, these analyses will enable us to extract ground rules for the design of phage cocktail products, while productively translate our research towards 'sur-mesure' phage treatment of given patients. This effort will also result in the introduction of an 'AI doctor' that will rank therapeutic phages from our joint collections against ESKAPE, based on their predicted efficacy against the specific strain infecting the patient." "Management of hairy root disease in tomato cultivation using integrated biocontrol organisms (BioMan)" "Hans Rediers" "Microbial and Plant Genetics (CMPG), Animal and Human Health Engineering (A2H)" "“Hairy root disease” (HRD) (also known as “crazy roots”) is caused by rhizogenic agrobacteria, and is characterized by extensive root proliferation, which results in severe economic losses. Yield decreases of more than 10% in tomato horticulture have been observed. HRD is a rapidly spreading and highly persistent disease, which is not easily managed with conventional biocides. In this project, we propose an integrated and sustainable biocontrol approach that is based on two complementary strategies: the use of ‘antagonistic’ bacterial strains and ‘lytic’ bacteriophages.We patented on the one hand several bacterial strains showing antagonistic activity against rhizogenic agrobacteria. Preliminary experiments indicate that these biocontrol organisms (BCO’s) show high potential to prevent or at least reduce infection with rhizogenic agrobacteria. On the other hand, we isolated and characterized in an ongoing European H2020 project a number of bacterial viruses (phages) that very selectively kill rhizogenic agrobacteria, thus making them potentially applicable as biocontrol system. Moreover, we postulate that the integration of both biocontrol organisms (BCOs) potentially has a unique synergistic action, that could also be expanded to other pathosystems in the future.The BioMan consortium aims to optimize the use of these BCOs in tomato cultivation to reduce the economic losses associated with HRD." "The role of capsular exopolysaccharide depolymerases as host determinant for Klebsiella pneumoniae phages, and their enzymatic properties." "Yves Briers" "Department of Biotechnology, Department of Applied biosciences, Wroclaw University of Environmental and Life Sciences" "This doctoral research proposal investigates the unique relationship between the bacterial capsular serotype, bacteriophage-encoded depolymerases that degrade capsular exopolysaccharides and the bacteriophage host spectrum, specifically for Klebsiella pneumoniae bacteriophages. The enzymatic properties of these exopolysaccharide depolymerases will be analysed. A first-in-class phage engineering method will be introduced to study the role of these enzymes in the tail fibers of bacteriophages"