Study of virulence factors of Staphylococcus aureus.
PhD thesis, University of Glasgow.
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In vivo expression technology (IVET) is a promoter-trap strategy deigned to identify genes whose expression in induced in a specific environment, typically that encountered in a host. Signature-tagged mutagenesis (STM) uses comparative hybridisation to isolate mutants unable to survive specified environmental conditions and has been used to identify genes critical for survival in the host. Both methods have been used to identify virulence genes in S. aureus. The main aim of this project was to find any probable new genes of S. aureus that are essential for biofilm formation and infection mouse model by STM.
A library of tagged insertion mutants of S. aureus and a series of selected tags in plasmids of S. aureus strain RN6390 were used. Most of the experiments with both the library and selected tags had problems with cross-hybridisation. All the selected tags were therefore sequenced and 33 tags with less than 50% identity were chosen for future experiments. A library of 825 mutants was made with the 33 selected tags. The mutants were arrayed in 25 pools of 33 mutants. Different tests were done to determine that the new library was reliable for a cross-hybridisation free screening. The library was then used in an infection model in mouse and biofilm formation. A total of 12 mutants with significantly reduced signals were sequenced. 7 out of 12 attenuated mutants showed homology to different genes in S. aureus and other bacteria. Tetrahydrodipicolinate succinylase homolog, opp-2F, acetoin utilization AcuC protein, phosphate ABC transporter, dapD, branched-chain-amino-acid transporter, pepF, and flaR genes were identified. This work was the first STM screening in a biofilm system, and the dapD gene was identified in a biofilm for the first time. 2 out of 12 genes had also been identified in previous STM screens. 5 out of 12 attenuated mutants showed homology to some hypothetical proteins. A hypothetical protein of the same locus was identified in two mutants.
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