Ovotransferrins are present in egg white and belong to an ancient family of iron-binding proteins which are autolytically processed (1). They are known to have antimicrobial activity against a range of different gram-negative and gram-positive bacteria and thereby protect offspring against infections. Although it is thought that this activity is mainly mediated by iron depletion, there is evidence that additional mechanisms are involved that directly lead to bacterial membrane damage (2). Due to their ubiquitous presence ovotransferrins have co-evolved in many species with their respective pathogens. Ovotransferrin can be used as the first replacement of antibiotics during fattening cycles of turkeys, it was shown to possess antimicrobial activity in animal models and was licenced as drug in humans. The antimicrobial activity of ovotransferrins is only very poorly characterized, and the precise mechanism as well as the activity spectrum mostly unknown. In preliminary experiments in the Auer lab using a propriety phage display library screening technique that detects potential peptide cleavage sites it has been found that chicken ovotransferrin binds and cleaves peptides that are present in membrane proteins of bacterial pathogens that affect chicken.
The aim of this project is to corroborate these findings and investigate the precise mechanisms involved using a novel method of substrate identification. The project also aims at predicting and confirming the target pathogens of ovotransferrins from different species and test whether ovotransferrins can be used to naturally replace antibiotics (3). The project will first focus on the characterization of the substrate specificities, binding and proteolytic activity, of ovotransferrins from different species using a M13 phage display library. Subsequently, the identified substrate specificities will be characterized by state-of-the-art bioinformatics to predict the proteolytic or binding activity of different ovotransferrin family members for different pathogens and the molecular target of each individual pathogen. In bacterial growth assays it will be tested whether growth of the predicted pathogen is inhibited, and the minimal inhibitory concentration (MIC) will be determined. Using a range of different gram-positive and gram-negative bacteria the specificity of the antimicrobial activity of the ovotransferrin from each species will be determined. By Western blot it will be tested whether the predicted bacterial target protein is cleaved by the ovotransferrin. The active proteolytic domain in ovotransferrin will be determined by deletion and alanine scanning mutants in ovotransferrin cDNA expression plasmids. By modelling the structure of this domain together with the target peptide the amino acids responsible for target specificity will be determined. Using new ovotransferrin mutants generated by side-directed mutagenesis (and synthetic DNA genes) novel synthetic ovotransferrins will be generated that specifically cleave proteins of new target bacteria. These ovotransferrins will subsequently be tested for their antimicrobial activity and their applicability as anti-microbial reagents. Finally, using a range of different eGFP-tagged reporter viruses and infection assays in cell culture we will test whether the ovotransferrins from different animal species and synthetic ovotransferrins also possess antiviral activity. In summary, a broad range of wet lab and bioinformatic technologies will be learnt and applied in this project.
1. H. R. Ibrahim, T. Haraguchi, T. Aoki, Ovotransferrin is a redox-dependent autoprocessing protein incorporating four consensus self-cleaving motifs flanking the two kringles. Biochimica et biophysica acta 1760, 347-355 (2006).
2. S. Jan et al., Biochemical and micrographic evidence of Escherichia coli membrane damage during incubation in egg white under bactericidal conditions. J Food Prot 76, 1523-1529 (2013).
3. C. Van Droogenbroeck et al., Use of ovotransferrin as an antimicrobial in turkeys naturally infected with Chlamydia psittaci, avian metapneumovirus and Ornithobacterium rhinotracheale. Vet Microbiol 153, 257-263 (2011).