Investigating and Circumventing Mechanisms of Resistance to the Antibacterial Candidate, Niclosamide

<p dir="ltr">The need for novel antibiotics is urgent, as mobilised resistance elements have emerged against even antibiotics of last resort and the productivity of the traditional drug development pipeline has severely waned. Repurposing existing approved pharmaceuticals with cryptic antibacterial properties offers a faster, cheaper route to novel antibacterials. Niclosamide, an anthelmintic developed in the 1950s, has been demonstrated to be a potent inhibitor of gram-positive bacteria, and no spontaneous resistance to it has been observed. However, the impact of mobilised resistance elements highlights the need for techniques that assess potential mechanisms of resistance more broadly. Functional screening of metagenomic libraries can reveal a diversity of potential resistance elements. However, libraries made using traditional methods are biased towards recovering mature resistance elements and will not identify genes that may only be one or two point mutations away from providing high-level resistance. The discovery of such genes can inform possible stewardship measures and provide fundamental insights to inform the synthesis of improved analogues. This thesis presents work aimed at identifying and characterising novel mechanisms of niclosamide resistance from a small insert, high-expression metagenomic library. To extend the utility of niclosamide, we also sought to generate niclosamide analogues with broad-spectrum activity. </p><p dir="ltr">Functional screening employed a bespoke high-expression metagenomic library, created using the restriction enzyme FatI (cuts CATG) to enrich for ATG start codons that could be precision-cloned downstream of a strong plasmid-borne promoter and ribosome binding sequence. Niclosamide selection recovered 88 unique resistance elements that varied in the strength of niclosamide resistance conferred. Three enzyme-mediated methods of detoxification were validated using mass spectrometry. Forty-four resistance elements were annotated as flavoenzymes and were found to detoxify niclosamide by nitroreduction. A further ten resistance genes were annotated as methyltransferases, and a final resistance element, recovered from a pilot library, was found to hydrolyse niclosamide. </p><p dir="ltr">To further assess the evolutionary potential of these resistance elements, directed evolution campaigns using error-prone PCR were conducted for representatives of these three mechanisms. Selected flavoenzymes were demonstrated to be easily evolvable, requiring one to two mutations to develop a mature resistance phenotype. The single hydrolysing resistance element was shown to be evolvable, albeit via the accrual of up to eight amino acid substitutions. In contrast, no improved methyltransferases or putative drug-sequestering proteins were recovered, suggesting that while other mechanisms might confer low levels of resistance, their evolutionary potential to become mature resistance elements is low. The collateral resistance and sensitivity profiles of improved enzymes were shown to be altered, suggesting potential strategies for combating the emergence of niclosamide resistance, including rational analogue design. While effective against gram-positive bacteria, gram-negative bacteria are intrinsically resistant to niclosamide due to TolC-mediated efflux. A parallel avenue of investigation sought to generate and evaluate niclosamide analogues effective against E. coli. A structure-activity relationship study revealed several positions on niclosamide that lead to increased antibacterial activity in TolC-intact E. coli and a panel of ESKAPE pathogens. Work is ongoing to determine if these analogues are effective in a murine abscess model.</p>