Study of phages for phage therapy : An Escherichia coli experience

University dissertation from Stockholm : Stockholm University

Abstract: The world is rapidly moving toward a post-antibiotic era as effective antibiotics are becoming fewer. phage therapy is a potentially possible method for treating infectious diseases and counts as an alternative method for antibiotic therapy. Phages' number on earth is estimated to be 1031 which ensures a great variety for these entities, and a never ending source for new phages. However, phages vary in their morphology, life cycle and virulent ability, and thus phage therapy will be unsuccessful without enough knowledge in phage biology. Only phages with a good virulence and broad host ranges are fitted for phage therapy applications, host range being one the most important factors that needs a careful study. There is no common method for studying the host range of phages and host range measurements are consequently dependent on the method that was used to study it. In our study, six polyvalent phages were selected from thirty virulent phages isolated using strains from E. coli reference collection (ECOR) as hostes. The selected phages were tested for their host range across 234 strains of E.coli (both sensetive and resistant to antibiotics) and Salmonella (reference collection SARA and SARB) using both spot test and efficiency of plating (EOP) method. Spot test results show no correlation to results of EOP analyses. One way to explain why spot test results are uncorrelated to EOP is the effects of resistant mechanisms of the host. In spot test, phages of a high concentration lystae might absorb, degrade the cell wall and kill the bacteria but the intrinsic resistant mechanisms of the host can recognize and degrade the genome, and stop phage replication and reproduction, which is visualized in the EOP analysis. Based on our studies, it is possible that prophages can contribute to bacterial defence against phages. Phages cannot in most cases produce a high EOP on those E. coli strains that have a P2 prophage in their genome.The phage SU10 had the highest EOP/spot test ratio among six selected phages. Transmission electron microscopy images revealed a very rare morphology for SU10, a short tail of 19 nm long and a 137 elongated head, and it was classified as a member of Podoviridae family. Genomic analysis of the phage show a genome size of 77,327 kb, 123 ORF encodes 38 proteins with known function. Twenty-two of theses proteins were identified using a mass-spectrometry based proteomics. The size of the tail was measured to be longer, 41 nm, in ultra-thin sectioning transmission electron microscopy images. A honeycomb structure is formed by the phage capsids during structural assembly, which is very rare for bacteriophages. The honeycomb structure of SU10 capsids might work as an aid or and external scaffolding protein during morphogenesis. The SU10 capsid is elongated before its genome is packed into the head according to the ultra-thin sectioning transmission electrographs which indicates that the genome packaging process has no role in elongation of the head. Based on our phylogenetic analysis, the scaffolding protein and major head protein have coevolved and the scaffolding protein in SU10 is not a recent attachment to the major head protein. According to the phylogenetic analysis of two proteins, the C1 and C3 morphotypes diverged 280 million years.

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