The application of antisense silencing for the characterisation of essential gene stringency and for the development of species-specific antimicrobials

Abstract

PhD ThesisThe emergence of multiple-antibiotic resistance among clinical pathogens has created an urgent requirement for the development of new antibiotics. The current lack of new antibiotics has not only renewed interest in traditional natural product screening approaches, but also prompted efforts to develop alternate antimicrobial strategies. Antisense RNA based silencing provides a strategy for developing whole cell screening assays, whereby antisense RNA induction leads to target protein depletion and subsequently the increased sensitivity of test organisms to target specific inhibitors. The development of synthetic derivatives to expressed antisense RNA such as peptide Nucleic Acids (PNA), has also been explored for use as bacterial inhibitors. This thesis aims to examine two novel antimicrobial strategies, firstly by comparing mRNA and protein based techniques to evaluate essential gene requirement in bacteria, to identify novel targets for antibiotic screening assays. Secondly, to evaluate the potential use of peptide peptide-PNA’s as antimicrobials capable of targeting individual bacterial species. To successfully develop either approach requires the identification and validation of suitable gene encoded molecular targets. Essential genes may provide potential candidates, yet a suitable system is necessary for characterisation to enable genes to be ranked, so that the most suitable targets can be prioritized. A disproportionate growth requirement (stringency) is known to exist among essential genes, which provides a means to delineate between essentially required targets, yet is based upon the measurement of mRNA abundance. Due to post-transcription and translation mechanisms, mRNA does not provide a reliable indicator of expressed protein, which represents the ultimate output of gene expression. This study demonstrates the use of a quantitative proteomics strategy for evaluating essential gene stringency at the protein level, using the E.coli gene fabI. Using expressed antisense RNA silencing to deplete target protein concentration and to reduce normal growth rate to 50%, absolute protein determinations were used to define a Minimum Protein Level (MPL50), for the quantitative characterisation of essential gene stringency. To support the justification of evaluating gene stringency using expressed protein abundance, the stringency of operon based genes fusA and rplE using antisense RNA silencing was investigated and revealed transcript profiles that contradict the use of Minimum Transcript Level (MTL50) previously used to define gene stringency. Finally, to demonstrate a potential application that would benefit from the characterisation of essential gene stringency, the species-specificity of a peptide-PNA targeting the essential gene ftsZ was evaluated. Exposing a mixed culture of S.typhimurium and E.coli to a peptide-PNA conjugate, incorporating a 2 base pair mismatch demonstrated the capacity to inhibit translation of ftsZ in S.typhimurium but not E.coli. This study highlights how characterising essential genes using the MPL50 can be used to delineate stringently required gene targets to support antimicrobial screening and the development of species specific antimicrobials. Furthermore the applications of evaluating gene stringency may be extended further, to provide a tool for standardising genetic components in synthetic biology approaches.BBSRC