Engineering of Antibacterial Phage-Derived Proteins

Sammanfattning: 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.