| Popis: |
DNA gyrase is an essential bacterial topoisomerase with a central role in many cellular processes. Its lack of homology to eukaryotic topoisomerases has been the basis of its role as an important drug target. The quinolone antibacterials have previously been shown to "poison" DNA gyrase of Escherichia coli, resulting in rapid bacterial cell death by a pathway which has been a subject of controversy. Following on from the finding that the complex of quinolone and gyrase on DNA results in truncated mRNAs in vitro, the results presented in this thesis show that in vivo, the addition of quinolone drugs results in the production of truncated mRNAs which are presumably translated into truncated proteins. This is expected to cause total deregulation of cellular processes and result in cell death. It has also been shown that the newer more potent quinolone drugs such as ciprofloxacin and ofloxacin have an additional effect on the bacterial cells in that the chromosome is broken down into fragments of DNA, some of which are estimated to be as small as 4kb in length. This process appears similar to the degradation of chromosomal DNA that occurs during apoptosis of some eukaryotic cells. The acquisition of quinolone-resistance in E. coli and Salmonella has also been investigated. Firstly, part of gyrA of S. typghimurium NCTC5710 was sequenced and found to be 94% homologous at the nucleotide level to the corresponding sequence of E.coli. Following on from this, clinical isolates were screened by DNA sequencing and amino acid mutations in the "quinolone resistance determining region" (QRDR) of gyrA identified. Mutations in gyrA such as serine-83 to leucine, serine-83 to phenylalanine, aspartate-87 to tyrosine, aspartate-87 to asparagine and aspartate-87 to glycine were found to have arisen in the QRDR of gyrA in common with other quinolone resistance mutations previously found. Cloning of gyrA into a vector in which the copy number could be altered confirmed that high level expression of gyrA is detrimental to the cell. |
