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Transcript
Pseudomonas aeruginosa lytic bacteriophage oKMV receptor analysis. Andrew Chibeu Samenvatting van het onderzoek / Summary of Research Viruses that specifically infect bacteria (bacteriophages or phages) are obligate parasites and ubiquitous in nature. Since their discovery in the early twentieth century, research into the biological nature of phages has contributed immensely to their exploitation in providing biotechnology tools. In the recent past, there has been renewed interest in bacteriophages as potential antibacterial agents against the increasing number of antibiotic-resistant bacteria. Among the bacteria for which the potential of such phage therapy has found application is Pseudomonas aeruginosa, an opportunistic pathogen whose infections occur in patients with compromised host defences such as those with HIV, burn patients or with cystic fibrosis. These infections are difficult to treat with conventional antibiotic therapies and often result in morbidity and mortality. As the main drawback of using phages in treatment of bacterial infections is their narrow host range, there is the need to develop cocktails containing phages that lyse a wider variety of host strains. Any phage cocktail should contain phages that use a diversity of receptors, to ensure an expanded host range and also to suppress the emergence of phage-resistant strains. Hence, phage receptor studies are paramount where phage therapy is envisaged. BacteriophagephiKMV is a virulent phage of P. aeruginosa isolated from a water source in the Moscow suburbs. Its genome sequence and basic biology have been the focus of active research at the K.U. Leuven Laboratory of Gene Technology (LoGT) in the past decade. To date, the receptor and host range determinants of phage phiKMV had not been determined. This dissertation focuses on determining the receptor of bacteriophage phiKMV and developing tools for the identification of other virulent phage receptors for phage therapy applications. Host infectivity screens, adsorption tests as well as the classical mutant characterization approach were applied in identifying host receptor proteins/structures. These tests established that the growth of P. aeruginosa strains on solid surface was essential for phage phiKMV infectivity. As follows, the receptor structures are accessible to the phage when expressed under these growth conditions. These structures were identified to be type IV pili through functional complementation of the isolated spontaneous phage phiKMV-resistant mutant strain with the genomic library of the wild type phiKMV-sensitive strain. Further in depth analysis and characterization of the spontaneous phiKMV-resistant mutant strain established that the phage phiKMV resistance mechanism is a complex process, possibly involving post-transcriptional regulation of the type IV pili biogenesis genes. A second putative host receptor protein (PA4736) was identified by a library scale yeast two-hybrid screen. To our knowledge, this study was the first time the yeast two-hybrid system was used in determining phage-host receptor interactions. This P. aeruginosa PAO1 protein was found to interact with phage phiKMV gp48. PA4736 is co-transcribed with the outer membrane protein PA4735 and it is highly likely that these proteins interact. It is thus proposed that the P. aeruginosa protein PA4736, which had no previously annotated function, is the secondary receptor for phage phiKMV. When successfully verified by an in vitro technique, the interaction between phiKMV gp48 and P. aeruginosa PAO1 protein PA4736 will be ascertained to be a true positive interaction. Recombinant expression of the proteins followed by in vitro interaction assays would provide data which when combined with that from this study would lead to the functional annotation of the phage phiKMV co-receptor protein, PA4736. Type IV pili-dependent infectivity of phage phiKMV has profound implications in its use for phage therapy applications, such as its potential influence when the need for phage propagation in batch cultures arises. The role of the differential expression the type IV pili biogenesis genes in bacteriophage phiKMV-sensitivity would make the phage a valuable tool in the typing of P. aeruginosa strains with the intact type IV pili assembly complex. Hence, the phage can assume a role in experiments to determine the presence or absence of intact type IV pili assembly machinery and wild type twitching motility. Several tools for use in phage receptor studies were developed in the course of this research. These tools can be used in studies to identify other P. aeruginosa phage receptors for phage therapy applications.