Therapeutic targeting of the small RNA GcvB in Actinobacillus pleuropneumoniae

Student thesis: Doctoral Thesis

Abstract

Actinobacillus pleuropneumoniae (APP) is a porcine respiratory Gram-negative pathogen, which has a profound negative impact on the farming economy, with serotype 8 being the most predominant in the UK. In anticipation of an antibacterial resistance era, there is increasing interest in the development of novel therapeutic strategies to combat this disease. Small non-coding RNAs (sRNAs), involved in post-transcriptional regulation of gene expression, are amongst some key components employed by bacteria to mediate virulence. sRNAs interact with their target mRNAs to tightly regulate the turnover of virulence factors, resulting in altered patterns of gene expression to rapidly adapt to environmental changes. Identifying, targeting and disrupting the production of virulence factors is a promising novel strategy to treat infectious diseases. Thus, sRNAs have been identified as potential antimicrobial targets. However, there are no therapeutic approaches targeting sRNAs being currently used at the moment.
This project aimed to understand and exploit the bacterial regulatory mechanism responsible for the control of virulence factor production by investigating and targeting regulatory sRNAs not yet characterised in APP. A combined approach of computational prediction and gene expression analysis using RNA sequencing data was designed, resulting in the establishment of a bioinformatic pipeline to identify novel trans-acting sRNAs from APP serotype 8 and predict their putative mRNA partners. Moreover, this research provides the first insights into the mechanisms of interaction of the sRNA GcvB and its predicted mRNA partners in APP. GcvB is a global regulatory sRNA involved in amino acid biosynthesis and peptide transport in various pathogenic Gram-negative bacteria. The expression levels of GcvB and its targets, quantified in different growth conditions, confirmed their presence in this species. The predicted interactions were then screened, validated and quantified using a novel RNA array technology developed at the University of Portsmouth with a customised protocol created to test APP molecules. After gaining a greater understanding of the mechanisms of sRNA-mRNA binding, a bespoke inhibitory nucleic acid mimic (NAM) was designed to specifically disrupt the interaction in vitro. The NAM molecule specifically disrupted the interaction between GcvB and its selected targets. Furthermore, the results showed that the novel high throughput RNA array technology can be applied and customised to identify and quantify interactions between sRNAs and their target transcripts. This work may have applications for novel therapeutics that target virulence pathways within the Pasteurellaceae family, and other bacterial species, that are mediated by sRNAs
Date of AwardMar 2021
Original languageEnglish
SupervisorJohn McGeehan (Supervisor), Anastasia Callaghan (Supervisor), Andrew Pickford (Supervisor) & Colin Sharpe (Supervisor)

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