Engineering of Antibacterial Phage-Derived Proteins

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

Structure prediction of endolysin SU57e

Endolysins are used by bacteriophages to break down the cell wall of the bacterial host towards the end of the infection cycle. Endolysins are interesting options for the creation of antibacterial substances and domains from endolysins can be combined with domains from other proteins to create antibacterial constructs. This article aims to find the tertiary structure and the enzymatically active site of the endolysin SU57e. This is done through the use of bioinformatics software, comparative analysis with other endolysins, and attempts at producing and purifying the protein for crystallization. SU57e is encoded by the phage vB_EcoD_SU57 (SU57) previously characterized by Koonjan et al. Bioinformatics analysis indicates that the structure is similar to that of endolysin R21 presented by Sun et al. SU57e also appears to have a signal-anchor-release-domain similar to that of R21.

Structure prediction of endolysin SU57e

Endolysins are used by bacteriophages to break down the cell wall of the bacterial host towards the end of the infection cycle. Endolysins are interesting options for the creation of antibacterial substances and domains from endolysins can be combined with domains from other proteins to create antibacterial constructs. This article aims to find the tertiary structure and the enzymatically active site of the endolysin SU57e. This is done through the use of bioinformatics software, comparative analysis with other endolysins, and attempts at producing and purifying the protein for crystallization. SU57e is encoded by the phage vB_EcoD_SU57 (SU57) previously characterized by Koonjan et al. Bioinformatics analysis indicates that the structure is similar to that of endolysin R21 presented by Sun et al. SU57e also appears to have a signal-anchor-release-domain similar to that of R21.

The rational aspects of an irrational whim. A qualitative study exploring the motivations behind consumer impulse buying in a socio-cultural context.

Purpose The purpose of the research is to analyse the consumer’s underlying motivations for behaviour and decision-making in an impulse buying context, in order to gain a deeper understanding of the drivers behind impulse buying. Method The study is conducted with an explorative approach. A combination of self-administered diaries and semi-structured interviews were chosen to collect the empirical data. Theory The theoretical framework of the study is mainly based on socio-cultural theories within consumer research that illustrate the symbolic meaning of possessions, as well as describe how the consumer’s self-identity and life situation can be linked to her buying behaviour and brand perceptions. Analysis In the analysis the participants of the study are presented as well as the dominant themes within the empirical material. The first theme indicates that the consumer is well aware of her incapability to resist an urge as it occurs and therefore tries to control and plan when to allow herself to engage in impulsive buying. The consumer tends to have expectations on the impulse buying activity that needs to be satisfied, otherwise the consumer will develop negative feelings towards the event. An additional theme in the empirics concerns the way the consumer uses impulse buying to express the self. The empirical material indicates that the consumer’s impulse buying behaviour can be seen as rational in relation to the individual consumer as it helps her create meaning to her life. The final theme stresses the influence of brand preferences on the consumer’s impulse buying as it limits the scope of the impulsiveness and directs the consumer to choose from an evoked set. Conclusion The study concludes that impulsive buying involves far more rational aspects than suggested by previous research. By putting the impulse purchase in a wider context and viewing it as one sequence in a larger pattern of sequences evidence is found that suggests that the impulsiveness is dependent on the consumer’s perception of the self and the life situation

Improved bacteria-phagocyte interaction by means of a fusion protein binding Staphylococcus peptidoglycan and Immunoglobulin G

The immune system plays an important role in the body´s defense against bacterial infections. Here, we hypothesize that adding an artificial opsonin that binds to antigens on the surface of infecting bacteria as well as to endogenous IgG can increase the interaction between bacteria and immune cells. A chimeric protein was made by fusion of the Src homology domain 3b (SH3b) from the Staphylococcus phage K endolysin (LysK) and the IgG-binding C2 domain from Streptococcus Protein G. SH3b binds to the bacterial cell wall of staphylococci and the C2 domain to the Fab region of the IgG which in turn binds to the Fc receptors of the phagocytes, facilitating interaction between phagocytes and bacteria. Comparative experiments with and without the chimeric protein showed that it both increased the amount of Staphylococcus carnosus cells bound by a humanized monoclonal IgG1 with unrelated specificity and increased the interaction of phagocytes with bacteria. The results justify development of chimeric proteins with the ability to act as artificial opsonins since these possibly can become an addition to future treatments of infections caused by antibiotic resistant bacteria

Virituell Tillit - Hur konsumenter uppfattar att förtroende skapas genom tredjepartsrecensioner på internet

Syfte: Uppsatsens syfte är att ur ett konsumentperspektiv undersöka hur tillit uppfattas på internet. Metod och Empiri: Genom en kvalitativ studie med ett iterativt förhållningssätt har vi genomfört 13 intervjuer. De semistrukturerade intervjuerna är det empiriska material som ligger till grund för analysen med hjälp av litteratur och akademiska artiklar. Teoretiska perspektiv: Teori som behandlar tillit är grunden för undersökningen, samt kompletterande teori om köpbeslutsprocessen används. Slutsatser: Vi fann att konsumenter bedömer andra konsumenter initialt genom texten i omdömen. De letar efter vem som skrivit omdömet, i syfte att kunna relatera till och identifiera sig med dem för att uppnå tillit. Konsumenter utvärderar trovärdigheten hos användaren och följer gärna en mängd omdömen i ett flocklikt beteende. De väger det relativa antalet positiva omdömen mot negativa för att ta ett köpbeslut

Improved bacteria-phagocyte interaction by means of a fusion protein binding Staphylococcus peptidoglycan and Immunoglobulin G

The immune system plays an important role in the body´s defense against bacterial infections. Here, we hypothesize that adding an artificial opsonin that binds to antigens on the surface of infecting bacteria as well as to endogenous IgG can increase the interaction between bacteria and immune cells. A chimeric protein was made by fusion of the Src homology domain 3b (SH3b) from the Staphylococcus phage K endolysin (LysK) and the IgG-binding C2 domain from Streptococcus Protein G. SH3b binds to the bacterial cell wall of staphylococci and the C2 domain to the Fab region of the IgG which in turn binds to the Fc receptors of the phagocytes, facilitating interaction between phagocytes and bacteria. Comparative experiments with and without the chimeric protein showed that it both increased the amount of Staphylococcus carnosus cells bound by a humanized monoclonal IgG1 with unrelated specificity and increased the interaction of phagocytes with bacteria. The results justify development of chimeric proteins with the ability to act as artificial opsonins since these possibly can become an addition to future treatments of infections caused by antibiotic resistant bacteria

Infection kinetics and phylogenetic analysis of vB_EcoD_SU57, a virulent T1-like Drexlerviridae coliphage

The morphology, infection kinetics, genome sequence and phylogenetic characterization of the previously isolated bacteriophage vB_EcoD_SU57 are presented. The phage vB_EcoD_SU57 was isolated on Escherichia coli strain ECOR57 from the E. coli reference collection and was shown to produce four mm clear plaques with halos. Infection kinetics, as assessed by one-step growth analyses, suggest that vB_EcoD_SU57 is a virulent phage with an adsorption rate of 8.5 × 10-10 mL×min-1, a latency period of 14 minutes, and a burst size of 13 PFU per bacterium. Transmission electron microscopy confirmed vB_EcoD_SU57 to be a phage that used to be classified as a Siphoviridae phage. Bioinformatic analyses showed that the genome was 46,150 base pairs long, contained 29 genes with predicted protein functions, and 51 open reading frames encoding proteins with unknown function, many of which were gathered in clusters. A putative tRNA gene was also identified. Phylogenetic analyses showed that vB_EcoD_SU57 is a Braunvirinae phage of the newly formed Drexlerviridae family and closely related to T1-like E. coli phages vB_EcoS_ACG-M12 (Guelphvirus) and Rtp (Rtpvirus) as well as the unclassified phages vB_EcoS_CEB_EC3a and ECH1