Bacteriophage: from exploration to exploitation

Over the past decades, bacteriophage research has revealed the abundance of phages in nature, their morphological and genomic diversity, their influence in the regulation of microbial balance in the ecosystem and their impact on the evolution of microbial diversity. Since the 1950s, phages have also played a central role in some of the most significant fundamental discoveries in biological sciences that have been crucial for the development of molecular biology. More recently, phage research has resulted in the development of genome editing tools, and it has generated the renewed interest of using phages and phage-related products as therapeutic agents. Although major progress has been made, basic understanding on phage biology is still lacking. The number of phage genes with unknown function still largely outnumber those with established roles. Therefore, further progress depends on a deeper understanding on phage biology. The present thesis aims at developing tools to support phage research, explores the use of phages for therapeutic purposes, and expands our insights into the biology of phages. A literature review on the molecular, structural and evolutionary determinants of phage-host interaction (Chapters 1 and 2) underlines the relatively poor understanding of the subject. A great variety of structures and mechanisms of infection are being revealed, but no correlations have yet been established between these and host interaction. Furthermore, so far no evolutionary model accurately describes the coevolution of phages and bacteria. A particular interest of evolutionary studies concerns the understanding of the prevalence of broad-host range phages in natural environments, since these are rarely isolated using standard laboratory isolation procedures. Indeed, we have tried to isolate broad host range phages targeting the Escherichia coli reference collection (Chapter 3), but found narrow-host range phages to be more prevalent. Only one phage of relatively broad host range was found (S2-36s), being able to infect 14 of the 72 strains. Proteins of interest for further exploration were found, such as depolymerases and colanic acid-degrading proteins, both with potential anti-biofilm activity. The isolation procedures against the ECOR collection proved to be challenging due to the amount of strains and samples to be evaluated. Consequently, a high-throughput methodology was developed to simplify these isolation procedures (Chapter 4). By automated monitoring of cell growth in 96-well plates it is possible to use differences in optical densities (plotted as heatmaps) between cells subjected to the samples and in control conditions to screen for the presence of phages. The method revealed an accuracy of 98% and reduced the workload by 90%. The method developed can also be used to screen for broad-host range phages or to screen collections of phages for variants or cocktails that are suitable for treating bacterial infections. A discussion is provided of the advantages and limitations of phages for therapeutic applications (Chapter 5). It is suggested that phages in their natural state cannot be used in therapeutic applications. The future of phage therapy may possibly be genome engineering for tailoring of phage properties. Subsequently, the genetic modification of phage T7 was shown to improve (2-log) the capacity of the phage to resist to the strongly acidic conditions and enzymatic challenges of the gastrointestinal tract (Chapter 6). This was achieved by modifying the phage to express a signal peptide on its capsid to which phospholipids attach forming a protective coating. The removal of the phospholipid coating using phospholipase caused reversion to the pH-sensitive phenotype of the wild-type phage. In case of orally-delivered phages, this may improve the efficacy of phage therapy. Engineering of phage genomes can also support evolutionary studies and basic phage research, e.g. analyzing if a certain gene is essential. A strategy developed for the random recombination of phage genomes (Chapter 7) demonstrated that it is possible to create novel productive phages by combining elements of different phage families. The findings reveal an unexpected level of flexibility and adaptability of phage genomes to accommodate and re-arrange genetic information, reflecting the pre-existing evolutionary compatibility of genes from different phages. The method is further expected to serve as a platform for improving our understanding of phage gene function and importance, where the random recombination of a single phage genome may be the preferred approach. A different approach for the therapeutic application of phages was explored. Using phage display it was possible to identify peptides targeting claudin-low breast cancer cells (Chapter 8) and osteoarthritis cells (Chapter 9) with high levels of specificity. The peptides identified may contribute to an early detection of claudin-low breast carcinomas, and to develop more individualized therapies for both breast cancer and osteoarthritis. In summary, the work developed in this thesis has resulted in new methodologies and biological data, thereby contributing to an increased understanding of phage biology and of the opportunities for the use of phages for diagnosis and therapy.