Title: Understanding the molecular mechanism of antibiotic resistance gene transfer in bacteria
Funder: The Academy of Medical Sciences

Abstract: Resistance to last resort antibiotics among pathogenic bacteria is on the rise causing global public health concerns. Bacteria can rapidly evolve against antibiotics via horizontal exchange of antibiotic resistance genes (ARG), which can be acquired by conjugation, a process ubiquitous in all bacteria. In this proposal we aim to reveal the molecular mechanism underlying the conjugative process to develop therapeutic methods towards tackling ARG transfer. The conjugative process requires a type-4 secretion system (T4SS), a pilus and a DNA processing complex called relaxosome. These components enable the transport of the substrate, a protein-DNA complex effectively transporting ARGs. Essential to the process of conjugation is an hexameric AAA+ ATPase called coupling protein that links the relaxosome and the T4SS. The coupling protein and the relaxosome recruit the relaxase, a bi-fuctional enzyme central to the conjugative process. The relaxase then covalently attaches to the DNA and pilots the transfer of DNA. So far, how the coupling protein engages with and translocates a protein-nucleic acid complex is not known. Furthermore how the relaxosome – coupling protein complex enable relaxase mediated DNA processing is also not known. In this proposed work we aim to elucidate how the coupling protein engages with the protein component of the substrate, relaxase and study its interaction using single particle cryo-EM. We will also assemble a relaxosome – coupling protein complex, biophysically characterize this complex and perform structural studies using cryo-EM subsequently. With this proposed work we aim to unravel the molecular mechanism behind conjugative substrate processing and transport.

Lay abstract: The discovery of antibiotics in the earlier part of the 20th century has a significant impact on agriculture, veterinary and humans, saving millions of lives over the years. However, our current arsenal of antibiotics is becoming obsolete due to a dramatic rise of drug-resistant bacterial infections. Currently, drug resistance infections claim 50,000 lives in Europe and 700,000 lives globally per year, numbers which are projected to rise. A major reason for this increase in antibiotic resistance is because of the bacteria’s ability to acquire resistance genes from other bacteria. Bacteria use sophisticated molecular machines to acquire these resistance genes and conjugation is one such process. Even though bacterial conjugation was discovered several decades ago, the molecular mechanism underlying this process is still not known. By gaining an in depth understanding into these processes, we can develop methods to stop them and hence prevent the spread of resistance. We aim to address the issue on the spread of resistance by studying the molecular mechanism behind the transfer of genetic material harbouring antibiotic resistance causing genes. We will capture these molecular machines in action using an innovative protein engineering approach. Using cutting edge cryo electron microscopy technique we will obtain the structure of these molecular machines to near-atomic detail, which will be exploited for structure based drug discovery and development of therapeutic methods to stop the spread of these resistance causing genes. Ultimately our work will contribute towards tackling the spread of antibiotic resistance in bacteria.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: INFECTION