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

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

Species-selective killing of bacteria by antimicrobial peptide-PNAs

This is an open-access article distributed under the terms of the Creative Commons Attribution License, CC BY 4.0 which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Broad-spectrum antimicrobials kill indiscriminately, a property that can lead to negative clinical consequences and an increase in the incidence of resistance. Species-specific antimicrobials that could selectively kill pathogenic bacteria without targeting other species in the microbiome could limit these problems. The pathogen genome presents an excellent target for the development of such antimicrobials. In this study we report the design and evaluation of species-selective peptide nucleic acid (PNA) antibacterials. Selective growth inhibition of B. subtilis, E. coli, K. pnuemoniae and S. enterica serovar Typhimurium in axenic or mixed culture could be achieved with PNAs that exploit species differences in the translation initiation region of essential genes. An S. Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials. Analysis of the genomes of E. coli and S. Typhimurium gave a conservative estimate of >150 PNA targets that could potentially discriminate between these two closely related species. This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.Peer reviewedFinal Published versio

<i>S</i>. Typhimurium-selective growth inhibition.

<p>Peptide-PNA Se0002 was designed to target the −5 to +5 region of the translational initiation region (TIR) of <i>ftsZ</i> in <i>S</i>. Typhimurium. Se0002 has 2 base pair mismatches in the TIR of <i>ftsZ</i> in <i>E. coli</i>. (A) Growth curve analysis of Se0002 in pure culture. <i>E. coli</i> growth in the presence of 1.25 µM Se0002 (solid line) was identical to that of untreated controls (not shown). <i>S</i>. Typhimurium growth was inhibited in the presence of 1.25 µM of Se0002 (dotted line) relative to the untreated control (dashed line). Growth in the treated samples after 10 hrs was not due to resistance (see text for details). (B) Mixed cultures of GFP-labeled <i>S</i>. Typhimurium AC02 and DsRed-labeled <i>E. coli</i> AC01 were treated with 1.25 µM Se0002; and imaged by fluorescence microscopy after 6 hrs of incubation. The filamentous growth phenotype was only observed in <i>S</i>. Typhimurium AC02 and is consistent with silencing of <i>ftsZ</i> expression.</p

Species-selective antibacterial peptide-PNAs in three-species mixed culture.

<p><i>B. subtilis</i> (dark grey), <i>K. pneumoniae</i> (white) and <i>S</i>. Typhimurium (light grey) in mixed culture were separately treated with Ec108 at 3.5 µM, Kp0001 or Se0001 at 4.5 µM or by combined treatment of Kp0001 and Se0001 both at 4.5 µM. All cultures were incubated for 16 hrs. Selective inhibition of either <i>K. pneumoniae</i> or <i>S</i>. Typhimurium individually or together, achieved with the peptide-PNAs, could not theoretically be achieved with any combination of the twenty known antimicrobial compounds tested in this study. Error bars as above.</p

Bacterial strains used in this study.

a<p>American Type Culture Collection.</p>b<p><i>Salmonella</i> Genetic Stock Center.</p

Species-selective antibacterial peptide-PNAs in axenic and two-species mixed culture.

<p><i>E. coli</i> (dark grey), <i>K. pneumoniae</i> (white) and <i>S</i>. Typhimurium (light grey). All cultures were incubated for 16 hrs. A) axenic cultures of the species were treated with <i>E. coli</i>- specific Ec1000 at 3.2 µM, <i>K. pneumoniae</i>-specific Kp0001 at 3.2 µM and <i>S</i>. Typhimurium-specific Se0001 at 2.0 µM. Asterisks indicate species-selective growth inhibition of <i>E. coli</i>, <i>K. pneumoniae</i> and <i>S</i>. Typhimurium respectively. B) Two-species mixed cultures treated with peptide-PNAs as above. The control cultures show the relative proportion of the two species without treatment, the two treatments to the left of the control represent the same mixed culture treated with a peptide-PNA. Black arrows indicate non species-selective growth inhibition of <i>S</i>. Typhimurium by Ec1000. Error bars are standard error for biological replicates (<i>n</i> = 3).</p