AbstractThe bacterial pathogen Neisseria gonorrhoeae (Ng) has a proficient system for acquiring novel genetic material via natural transformation, which is a major factor underlying the rising ubiquity of drug-resistant gonorrhoea infections. The mechanism by which DNA is taken up from the extracellular milieu by Ng cells during transformation is not well-understood, but is known to rely on 12 bp DNA uptake sequence (DUS) motifs in donor DNA and retractile filaments termed type IV pili (T4P) on the surface of recipient cells. In this project, single-cell and single-molecule microscopy were employed to clarify the role of T4P in DNA binding and uptake during natural transformation. This approach enabled direct visualisation of DUS-dependent binding of single DNA molecules to Ng T4P fibres in situ for the first time, as well as measuring DUS-T4P unbinding forces indicative of a high affinity interaction. Collectively, the data support a model in which DNA binding during uptake is mediated primarily by sequence-specific interactions between DUSs and bona fide T4P fibres, with little involvement of nonspecific DUS-independent binding. By demonstrating DUS-specific uptake of double- stranded oligonucleotides <25 bp, these results also pave the way for a novel ‘Trojan horse’ drug delivery strategy exploiting the DNA uptake apparatus for improved intracellular accumulation of therapeutic compounds.
Most research into gene transfer in Ng has focussed on natural transformation, but chromosomal mobile genetic elements, which provide an alternative means of disseminating resistance determinants, remain relatively poorly studied. Here, a novel bioinformatic method based on genomic distributions of DUS motifs is developed to detect exogenous chromosomal regions typically acquired through mechanisms other than transformation. Using this technique, two novel integrative and conjugative element (ICE) families were identified: one found primarily in pathogenic Neisseria, constituting the first reported gonococcal ICE, and the other belonging to a group of Neisseria mucosa-like commensals.
|Date of Award||29 Sep 2020|
|Supervisor||Sean A Davis (Supervisor) & Darryl J Hill (Supervisor)|