The increasing threat of antibiotic resistance calls for the development of new treatment methods. Bacteriophages are interesting candidates since they can lyse bacteria with great efficiency. Bacteriophages produce enzymes called endolysins which break down the peptidoglycan in the cell wall at the end of the infection cycle. The endolysins are also of great interest to use against bacteria since they can lyse cells from the outside, when the peptidoglycan is accessible.  When using bacteriophages and endolysins as therapeutics there is a risk that the human immune system will react to them since they are foreign particles. The lysate from the bacteria can cause the immune system to react with a massive release of cytokines. The plasma half-life can also become short since the protein is cleared from the blood stream. With protein engineering it is possible to combine functional domains from different proteins to construct new chimeric proteins, these domains can also be optimized for new functions through modification.  

In project 1 a chimeric protein was created that contained a cell wall binding domain from an endolysin and a domain from another protein that binds to IgG. Assays were made to see if the chimeric protein could attach non-specific IgG to bacteria and if this could induce binding of phagocytes to the bacteria. Induction of phagocytosis can potentially help clear the infection with lower risk of cytokine release, because the bacterial lysate will not be released. 

In project 2 the endolysin SAL-1 and the enzyme dispersin B were fused respectively with the spider silk protein 4RepCT to create antibacterial coatings. The ability of SAL-1-4RepCT to break down bacteria in the liquid surrounding the surface was measured. The dispersin B-4RepCt was examined for its ability to prevent biofilm formation.

Project 3 characterized the bacteriophage SU57. Both host interaction parameters and the genome were examined. One challenge that phage treatment faces is that bacteriophages can only be added in low concentrations and must thus multiply in situ. This requires that the bacteriophage has a large burst size while having a high adsorption rate and short latency period. To assess promising bacteriophages it is important to be able to decipher their genome. 

The goal of project 4 was to find the tertiary structure and active site of the endolysin SU57e. Bioinformatics were used to predict properties of the protein, the tertiary structure and the active site. Attempts were made to produce and purify the protein in order to enable crystallization for X-ray crystallography. 

Overall the projects in this thesis aim to increases the knowledge of the use of bacteriophages and phage derived proteins as antibacterials.

Bacteriophages, or as they are most commonly referred to as phages, are viruses which are capable of infecting bacteria. They are environmentally plenty, found everywhere from the ground we walk on to contributing to our microbiome. They are highly diverse, coming in various sizes, shapes, and genomic compositions. Compared to animal viruses, phages have larger genomes that are highly mosaic, with different segments having diverse evolutionary backgrounds. Around 96% of all phages found are tailed phages with double stranded DNA genomes, belonging to the order Caudovirales.

Phage infection can be categorized into three stages; adsorption whereby the phage attaches to its host bacterial cell wall followed by the maturation and lysis stages, whereby a phage primarily replicates either via the lytic (i.e. progressively take over bacterial transcription machinery and materials in favor of its own and results in bacterial lysis) or the lysogenic cycle (i.e. integration of its genome into the host genome). As phages are ubiquitous and require a bacterial host for replication, it is not surprising that they interact with their surrounding environments and other phages (e.g. influencing global nutrient cycling as well as affecting bacterial pathogenicity), constantly coevolving. With the continuing rise in antibiotic resistance and dawning of the “post-antibiotic” era, there has been renewed interest in phage research and their therapeutic potential. However, there are many obstacles that must be overcome before phages can reach clinical settings and be widely applied (e.g. regulatory issues and phage pharmacology). The main element behind these obstacles is a phage’s inherent biology. As such, this thesis aimed to improve the understanding of phage biology by addressing specific components such as infection kinetics, phylogenetics, and host interactions.

In Papers I and II, phages vB_EcoD_SU57 (SU57) and vB_EcoP_SU7 (SU7) were characterized based on their infection kinetics and phylogenetics. SU57 was determined to be a T1-like Drexlerviridae phage with a relatively fast infection kinetics (short latent time of 14 minutes and small burst size) whereas SU7 was determined to be a Podoviridae phage belonging to the Kuravirus genus, with the rare C3 morphology of an elongated capsid. It was also shown to be a slower infecting phage (long latent time of 30 minutes and small burst size).  Paper III delved into the evolutionary origins of phages with the rare C3 morphotype making up the Kuravirus genus. These phages were found to be monophyletic in origins, closely related to marine Vibrio phages. Interestingly, these phages have a unique genomic end comprising of 33 genes which encode for hypothetical proteins of unknown function and a tRNA. In Paper IV, the population dynamics between two phages (SU10 and SU57) and one bacterial host (ECOR57) were studied in terms of population structure, size, and cell viability (i.e. resistance and/or susceptibility).

From the papers presented in this thesis, one thing is certain: phages are highly complex organisms. They come in a variety of morphologies with highly mosaic genomes of various sizes, which often hampers bioinformatics and phylogenetic analyses. Phages also have unique infection kinetics which influences how they interact and coevolve with other phages and bacteria.