Novel molecular targets to combat antimicrobial resistance: probing the assembly dynamics of a bacterial genome segregation machine

Bacterial antimicrobial resistance is a growing burden on human health worldwide. However, the latter half of the twentieth century has witnessed a gap in research and development of novel antimicrobial drugs together with the emergence and dissemination of multidrug-resistant bacteria. The emergence of multidrug-resistant ‘superbugs’ among bacterial populations results either from mutations within the bacterial genome or from the horizontal transfer of resistance genes often present on mobile genetic elements such as plasmids, bacteriophages, transposons and pathogenicity islands. The mobility of these genetic entities is the key for intra-species and, more worryingly, inter-species dissemination of multidrug resistance.

Large, low copy number plasmids, such as those implicated in antibiotic resistance, have evolved sophisticated strategies to ensure their faithful distribution at cell division. These plasmids harbour their own survival system, a partition cassette, which ensures an accurate and equitable segregation of the plasmids from one generation to the next. When this system malfunctions, the plasmid is not stably inherited and is ultimately lost, resulting in bacterial populations vulnerable to antibiotics.

The TP228 plasmid replicates at low copy number in Escherichia coli. It specifies resistance to a range of antibiotics such as tetracycline, streptomycin, kanamycin, neomycin, spectinomycin and sulphonamides, and to mercuric ions. The plasmid partition cassette encodes two proteins, an ATPase and a DNA-binding protein, that form a complex responsible for maintaining the plasmid by shuttling it to specific cellular addresses. We have proposed a Venus flytrap model as a mechanism for plasmid segregation (1). This model predicts that the plasmid is retained by being entrapped within a matrix formed by one of the partition proteins.

This project will investigate the synergistic interaction between the two segregation proteins and the dynamics of complex formation and plasmid shuttling at single-molecule level in live cells. The work will involve molecular biology, genomic methodologies, ensemble and single-molecule biochemical and biophysical approaches (AFM) in parallel to super-resolution fluorescence microscopy (2) to visualize proteins and plasmid localization in bacterial cells.